Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv.

Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv. Ann Microbiol (2016) 66:765–775 DOI 10.1007/s13213-015-1162-6 ORIGINAL ARTICLE Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv. 1 1 2 Aretusa Cristina Felber & Ravely Casarotti Orlandelli & Sandro Augusto Rhoden & 1 1 3 Adriana Garcia & Alessandra Tenório Costa & João Lúcio Azevedo & João Alencar Pamphile Received: 26 May 2015 /Accepted: 28 September 2015 /Published online: 10 October 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract Endophytic fungi colonize the interior of plant tis- identity of the endophytes. The biotechnological potential of sues and organs, establishing an intimate mutualistic associa- endophytes was tested in vitro for the control of pathogenic tion with no visible symptoms. The fungi may help protect the fungi of grapevines including Alternaria sp., Sphaceloma sp. plant against herbivores and pathogens, making them potential- and Glomerella sp. Inhibition percentages above 50 % as dem- onstrated by some isolates demonstrate their potential for bio- ly useful endophytes in the biological control of diseases and agricultural pests. The biotechnological interest in these organ- logical control. isms has stimulated research related to the bioprospecting of . . . endophytic fungi. Grapevine is among the oldest of plants cul- Keywords Endophytes Grapevine Biological control tivated by man, with the grape being one of the most highly Sequencing of rDNA Phylogenetic analysis consumed fruits in the world. Diseases cause significant dam- age to grape cultures, making their integrated control important to reduce the use of pesticides and, consequently, environmen- Introduction tal and human contamination. The rustic species Vitis labrusca L. (Vitaceae), used in the preparation of juices and wines, is The grape is one of the most important fruits grown in the highly resistant to fungal diseases. We isolated leaf endophytic world, and its worldwide production surpasses 67 million tons fungi of the Bordô and Concord cultivars (V. labrusca L.), annually (Faostat 2012). The grapevine belongs to the which were ordered into 68 and 62 morpho-groups of the Vitaceae family, consisting of approximately 14 genera and Bordô and Concord cultivars, respectively. We used scanning 900 species (Soejima and Wen 2006). The Vitis genus is the electron microscopy to confirm the presence of endophytes in most important economically because its species are used the leaves. Endophytic diversity was analyzed based on se- most commonly in agriculture. The Bordô and Concord cul- quencing the ITS1-5.8S-ITS2 region of rDNA, allowing the tivars of the species Vitis labrusca L. are hardy grapes that are identification of fungi belonging to genera including resistant to fungal diseases. These two cultivars are used for in Cochliobolus, Bipolaris, Fusarium, Alternaria, Diaporthe, natura consumption and primarily for the production of juices Phoma and Phomopsis. Phylogenetic analysis confirmed the and wines (Sousa 1996). Among the most serious problem faced in vine cultivation are fungal diseases that cause losses in production and fruit * João Alencar Pamphile quality (Fan et al. 2008). Endophytic microorganisms may be prof.pamphile@gmail.com promising alternatives to the use of pesticides in controlling fungal threats. Endophytes are microorganisms that inhabit the interior of plant tissues during all or part of their life cycle, Department of Cell Biology and Genetics, Universidade Estadual de Maringá, CEP 87020-900 Maringá, Paraná, Brazil without causing any visible symptoms (Petrini 1991;Azevedo et al. 2000). However, endophytes are relatively unexplored Câmpus de São Francisco do Sul, Federal Institute Catarinense (FIC), São Francisco do Sul, Brazil with regards to their production of useful natural compounds for agriculture, industry and medicine (Strobel et al. 2004). College of Agriculture BLuiz de Queiroz^ (ESALQ), Universidade de São Paulo, CEP 13400-970 Piracicaba, São Paulo, Brazil The ability of endophytes to produce substances in vitro that 766 Ann Microbiol (2016) 66:765–775 inhibit the growth of other species of microorganisms has Materials and methods stimulated research on the bioprospecting of endophytic mi- croorganisms and their use in biological control (Arnold Leaf sampling 2008). The mechanisms underlying the endophyte–host rela- Mature and healthy leaves of Bordô and Concord cultivars tionship are not well understood (Kogel et al. 2006). (V. labrusca) were selected randomly and used immediately Schulz and Boyle (2005) suggest that these are not neutral after collection. Two leaves from four different plants of each interactions, with the asymptomatic colonization requiring cultivar were used. The material was collected at Iguatemi a balance of antagonisms between the fungal endophyte Experimental Farm (IEF) belonging to Universidade and the host. According to the review of Rodriguez and Estadual de Maringá (UEM), planted in 0.048 ha located in Redman (2008), all plants in natural ecosystems are the Iguatemi District, city of Maringá, Paraná State, Brazil thought to be symbiotic with mycorrhizal and/or endophyt- (23°21′22^S, 52°4′18^W). The grapevines themselves were ic fungi. Collectively, fungi express several different sym- planted in the system known as espalier in a certifiably organic biotic lifestyles that are defined by fitness benefits to plant area. On the day of collection (10 August 2010), the majority hosts and symbionts. The range of symbiotic lifestyle ex- of berries carried by the grapevines were classified as pellet- pression from mutualism to parasitism has been described like according to the phenological stages of the grapevine as the symbiotic continuum. Within each group of fungal described by Eichhorn and Lorenz (1984). The temperature symbionts there are isolates and/or species that span the in the month prior to collection ranged from 12.1 °C to symbiotic continuum by expressing different lifestyles. 37.4 °C with an average temperature of 23.5 °C and average Several studies focusing on the isolation of endophytes relative humidity of 64.8 %. from asymptomatic plant tissues indicate that individual species express either mutualistic, commensal, or parasitic lifestyles when re-inoculated back onto the original host Isolation of endophytic fungi of Vitis labrusca species (Rodriguez and Redman 2008). Advances in the identification of fungi followed the de- Leaves were washed under running water, 0.01 % Tween velopment of sensitive and specific techniques of molecu- 80 aqueous solution (Synth; http://www.splabor.com.br) lar biology employed in the differentiation of species, such and two rinses in sterile, distilled water to remove as amplification of the internal transcribed spacer (ITS) of residues. They were then surface-disinfected to suppress ribosomal DNA (rDNA) using the polymerase chain reac- epiphytic microorganisms according to Vaz et al. (2009). tion (PCR); rDNA-ITS sequences can be sequenced and Washing was performed in series with 70 % ethanol for compared for homology with sequences available in data- 1 min, sodium hypochlorite (2 % available Cl )for bases (Magnani et al. 2005). 3min,70%ethanol for30sandtwo washes in sterile, Another important tool to assist our understanding of distilled water. The effectiveness of this method was veri- endophyte–host interactions is the use of scanning electron fied by spreading 100 μL of the final water used on Petri microscopy. This technique is advantageous for its high dishes containing potato dextrose agar (PDA) culture me- resolution and the possibility of in-depth analysis of vari- dium (Smith and Onions 1983) pH 6.6, supplemented with −1 ous materials (Pamphile et al. 2008a). tetracycline (Sigma, St. Louis, MO) (50 μgmL in 50 % Many studies have focused on endophytes in plants be- ethanol), to prevent bacterial growth. longing to the angiosperms and conifers (Arnold 2007). The Disinfected leaves were cut into 5-mm fragments and de- occurrence of endophytic fungi has been reported in the genus posited (five fragments per plate, with a total of 50 plates for Vitis. However, most studies have concentrated on fungi asso- each plant) on plates with PDA containing tetracycline. The ciated with V. vinifera (Mostert et al. 2000; Burruano et al. plates were incubated at 28 °C for 7 days. The colonization 2008; Casieri et al. 2009; Gonzáles and Tello 2011;Pancher frequency (CF) was determined by Hata and Futai (1995): CF et al. 2012). Studies related to the bioprospecting of endophyt- (%)=(number of fragments colonized by fungi / total number ic fungi in V. labrusca species are less common (Lima 2010; of fragments)×100. Brum et al. 2012). In the purification process, the fungal isolates were trans- Considering the potential of endophytic microorganisms as ferred to PDA plates and grown for 7 days. Then, fragments biological controllers (Azevedo et al. 2000), the aim of this (5 mm ) weresquashedin 1 mL0.01% Tween80 aqueous study was to isolate and characterize foliar endophytic fungi solution, and an aliquot of 100 μL solution was then spread on of Bordô and Concord grapevine cultivars (V. labrusca L.). plates containing PDA and incubated for 24 h. Single colonies Their biotechnological potential in the in vitro control of path- were transferred immediately to new plates with PDA and ogenic fungi of grapevine Alternaria sp., Sphaceloma sp. and incubated for 7 days. If necessary, the process was repeated Glomerella sp. was determined. untilpurecolonieswereobtained. Ann Microbiol (2016) 66:765–775 767 Molecular identification of isolated endophytic fungi RS. Fungi were: Alternaria sp. (CNPUV 674), responsible for leaf blight; Glomerella sp. (CNPUV 378), responsible for ripe Genomic DNAwas extracted as described by Raeder and Broda rot of grapes; and Sphaceloma sp. (CNPUV 102), which (1985) and modified according to Pamphile and Azevedo causes anthracnose. (2002), except that endophytes were previously grown for A paired culture technique described by Campanile et al. 7 days on plates with potato dextrose broth (PDB) medium (2007) was used. Endophytes and phytopathogenic fungal (Smith and Onions 1983) at 28 °C under stationary conditions. mycelial-disks (5 mm) were inoculated at a distance of 2 cm The concentration and purity of the genomic DNA were deter- on opposite sides of Petri dishes (9 cm) containing PDA. mined using a GENESYS 10S UV–vis spectrophotometer (OD Negative control plates had each pathogen disk inoculated in 260/280 nm). DNA integrity was analyzed by electrophoresis dual culture with a disk of PDA medium. The tests were per- in 1 % agarose gels, using the High DNA Mass Ladder formed in triplicate and all plates were incubated at 28 °C (Invitrogen, Carlsbad, CA) as the molecular weight standard. for 7 days. The inhibition percentages were calculated −1 Thefinal concentrationofDNAwasadjustedto10ngmL . according to Reyes Chilpa et al. (1997) as cited by Quiroga PCR amplification of the ITS1-5.8S-ITS2 of rDNA region et al. (2001): IP (%)=(average diameter of the pathogen was performed according to Magnani et al. (2005), using the colony in control − average diameter of the pathogen colony primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)and in the treatment / average diameter of the pathogen colony in ITS4 (5′-TCCCCGCTTATTGATATGC-3′)(Whiteetal. control)×100. 1990) with an initial denaturation at 92 °C for 4 min, followed The competitive interactions between endophytic and by 35 denaturation cycles at 92 °C for 40 s, annealing at 52 °C pathogenic fungi were characterized based on the scale of for 1 min and 30 s, extension at 72 °C for 2 min and a final Badalyan et al. (2002) where three types (A, B and C) and extensionat72°Cfor 5min. four subtypes (CA1, CA2, CB1 and CB2) of interaction are PCR products were purified with the GFX PCR DNA kit possible: A=deadlock with mycelial contact; B=deadlock at a and Gel Band Purification (Amersham Biosciences, distance; C=replacement, overgrowth without initial dead- Piscataway, NJ), according to the manufacturer’s instructions, lock; CA1 and CA2=partial and complete replacement after prepared for second sequencing reactions according to initial deadlock with mycelial contact; and CB1 and CB2= Magnani et al. (2005) using the primer ITS4. PCR reactions partial and complete replacement after initial deadlock at a were carried out in a thermocycler programmed to perform an distance. initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for Scanning electron microscopy 1 min, extension at 60 °C for 1 min and a final extension at 60 °C for 5 min. Sequencing was performed in a MegaBACE During the process of isolation of endophytic fungi, each fo- TM 1000 sequencer (Amersham Biosciences), with injection liar sample of V. labrusca was cut into two portions: one for and electrophoresis conditions of 1 kV/90 s and 7 kV/ isolation test and the other for microscopic observation. The 240 min, respectively. latter portions were incubated for 3 days and ruptured by the The nucleotide sequences were analyzed and edited, and freeze-fracture process then subjected to scanning electron compared to those deposited in GenBank (http://www.ncbi. microscopy (SEM). Sample preparation was modified from nlm.nih.gov). For research into genera or species, the that described by Pamphile et al. (2008b). A gradient of BLASTN program was used. Determination was based on 30 %, 50 %, 70 %, 90 % and 100 % alcohol was used for the best result obtained for identity. the dehydration of vegetable material, and the resultant frag- For the phylogenetic analyses, a dendrogram was construct- ments were dried to critical point in a Bal-tec-CPD030 (Bal- ed with the sequences obtained along with those available in tec, Los Angeles, CA), for seven cycles. Samples were GenBank. Sequences were aligned using Clustal X (Thompson mounted on stubs with the vertical position of the fragments et al. 1997), and the dendrogram was constructed using MEGA in a conductive tape for SEM. The fragments received a thin program version 5 (Tamura et al. 2011) with grouping by the layer of gold on Shimadzu-unit IC 50 (260 s, 50 mA, at 27 °C) neighbor-joining (NJ) method (Saitou and Nei 1987), using p- for three cycles. The fragment covered with metal was ob- distance for nucleotides with the option of pairwise gap dele- served in a field emission scanning electron microscope tion and a bootstrap with 10,000 repetitions. (SHIMADZU-SS550; Shimadzu, Tokyo, Japan) at 15 kV and 7 mm away. In vitro antagonistic activity of endophytic isolates against pathogenic fungi Statistical analysis Tests were performed with pathogenic fungi of grapevine pro- Experiments were conducted in a completely randomized de- vided by EMBRAPA Grape and Vine of Bento Gonçalves – sign, and evaluated statistically by analysis of variance 768 Ann Microbiol (2016) 66:765–775 (ANOVA) and means compared by Scott-Knott test (P<0.05) with Sordariomycetes (JX174146.1) in BLAST analysis], using the statistical program SISVAR 5.3 (Ferreira 2008). C40-59 [99 % identity with Sordariomycetes (JX174146.1) in BLAST analysis] and B35-107 [99 % identity with Sordariomycetes (JX174146.1) in BLAST analysis], were Results clustered with the Diaporthe sp. (anamorph=Phomopsis) group with 100 % bootstrapping (BP) analysis. Isolate C25- Isolation and molecular identification of endophytic fungi 19 [99 % identity with Diaporthe longicolla (JQ753971.1) in of V. labrusca BLAST analysis] was grouped with the Diaporthe sp. sub- clade with 100 % BP analysis. In that case, the species was The frequency of colonization of the 250 leaf fragments sam- identified as Diaporthe sp. instead of D. longicolla. The iso- pled from the Bordô cultivar was 74 %. A sample of 140 fungi late B43-48 [99 % identity with Phomopsis sp. (GQ461582.1) was isolated and grouped randomly into 68 morpho-groups in BLAST analysis] and the isolate B30-122 [99 % identity based on macroscopic characteristics including morphology with Phomopsis sp. (GQ461582.1) in BLAST analysis] and characteristics when grown on the culture media PDA: grouped with the Diaporthe/Phomopsis sub-clade with sporulation, mycelium aspect, mycelium coloration, colora- 100 % BP, showing that they are both Diaporthe sp. The tion of the reverse of the Petri dish, pigmentation of the culture isolates B15-03 [98 % identity with Phomopsis sp. medium and average diameter of colony growth. Of the 250 (KF159989.1) in BLAST analysis], B40-119 [98 % identity leaf fragments sampled from the Concord cultivar, we obtain- with Diaporthe sp. (EF423554.2) in BLAST analysis], B28- ed a colonization frequency of 64 % and 145 fungi were iso- 47 [99 % identity with Phomopsis sp. (JN153054.1) in lated and divided randomly into 62 morpho-groups. A fungus BLAST analysis] and B45-62 [99 % identity with from each morpho-group was chosen randomly and purified Phomopsis sp. (KF153062.1) in BLAST analysis] clustered for other experiments. with the sub-clade of Diaporthe/Phomopsis with 100 % BP, Sequence analyses of ITS1-5.8S-ITS2 regions allowed confirming the Diaporthe sp. identity. identification of endophytic fungi (Table 1) belonging to sev- In the second sub-clade of the first clade of en genera: Cochliobolus, Bipolaris, Fusarium, Alternaria, Sordariomycetes, isolate C22-22 [99 % identity with Diaporthe, Phoma and Phomopsis (the latter two genera being Fusarium oxysporum (FJ867931.1) in BLAST analysis] was the most common). The percentages of identity between the grouped in the Fusarium sp./Gibberella sp. with 100 % BP, sequences obtained and those available in GenBank varied confirming its identity as Fusarium sp. Isolate C03-45 [99 % between 97 % and 100 %. identity with Fusarium culmorum (KC311482.1) in BLAST analysis] was grouped with Fusarium culmorum with 8 % BP, Phylogenetic analyses of the endophytic isolates indicating its identitiy as Fusarium sp. of V. labrusca based on rDNA sequence data In the second clade, the isolates B31-38 [99 % identity with Phoma herbarum (KJ767079.1) in BLAST analysis], B61-72 Phylogenetic analysis (Fig. 1) resulted in the grouping of iso- [99 % identity with Phoma herbarum (JX867222.1) in lated endophytes, along with fungal sequences obtained from BLAST analysis], B46-95 [100 % identity with Phoma sp. GenBank (NCBI), in three clades belonging to the phylum (KJ572232.1) in BLAST analysis] and C18-40 [99 % identity Ascomycota. Clade I belongs to the Sordariomycetes class, with Phoma herbarum (KJ767079.1) in BLAST analysis] Diaporthales order, with representatives of the Phomopsis were grouped together in the Phoma sp. clade with 100 % and Diaporthe genera. In Clade II, the Sordariomycetes class BP. The isolate C07-137 [99 % identity with Phoma exigua is also represented by the Hypocreales order, including the (AY531684.1) in BLAST analysis] was grouped with Phoma fungi Fusarium and Giberella genera. Clade III is represented exigua with 70 % BP, indicating that C07-137 belongs to by the Dothideomycetes class and Pleosporales order, includ- Phoma exigua species. ing fungal Phoma, Cochliobolus, Bipolaris and Alternaria The third clade of the Pleosporales group was divided into genera. three sub-clades. The two initial sub-clades clustered with the The first Clade was subdivided into two sub-clades. In the Cochliobolus (anamorph=Bipolaris) species. Isolates B33-61 first sub-clade, isolates B17-49 [99 % identity with [98 % identity with Bipolaris sp. (GU017499.1) in BLAST Sordariomycetes (JX174146.1) in BLAST analysis], B27- analysis] and B60-132 [99 % identity with Bipolaris sp. 116 [98 % identity with Sordariomycetes (JX174146.1) in (GU017499.1) in BLAST analysis] were grouped in the first BLAST analysis], C53-134 [99 % identity with sub-clade with 100 % BP, confirming their Cochliobolus/ Sordariomycetes (JX174146.1) in BLAST analysis], C27-07 Bipolaris identity. Isolates C19-144 [98 % identity with [98 % identity with Sordariomycetes (JX174146.1) in BLAST Cochliobolus sp. (JQ754043.1) in BLAST analysis] and analysis], B63-34 [97 % identity with Sordariomycetes B14-69 [99 % identity with Cochliobolus sp. (JQ936204.1) (JX174146.1) in BLAST analysis], B64-46 [100 % identity in BLASTanalysis] also grouped with Cochliobolus sativus in Ann Microbiol (2016) 66:765–775 769 Table 1 Identification of 28 endophytic fungi of Vitis labrusca based on rRNA sequencing and inhibition percentages (IP) and competitive interactions (CI) with three phytopathogenic fungi in dual culture Endophytic Closely related fungal sequences Maximal Phytopathogenic fungi fungi identity Alternaria Sphaceloma Glomerella IP* CI** IPCI IPCI B14-69 Uncultured fungus (GU053859.1) 99 % 22.80 f A 27.64 d A 44.44 c A Cochliobolus sativus (JQ936204.1) 99 % B15-03 Fungal endophyte (JX155973.1) 99 % 37.42 c A 31.82 c A 37.94 d A Phomopsis sp. (KF159989.1) 98 % B17-49 Sordariomycetes (JX174146.1) 99 % 39.34 c A 42.79 b A 63.95 a A B27-116 Sordariomycetes (JX174146.1) 98 % 22.94 f A 11.44 f A 15.19 f A B28-47 Phomopsis sp. (JN153054.1) 99 % 25.93 e B 26.33 d B 32.33 e A B30-122 Fungal endophyte (KF435373.1) 99 % 31.46 d A 32.60 c A 47.40 c A Phomopsis sp. (GQ461582.1) 99 % B31-38 Phoma herbarum (KJ767079.1) 99 % 37.63 c C 17.45 e A 36.47 d A A1 B33-61 Bipolaris sp. (GU017499.1) 98 % 21.03 g A 25.55 d A 37.65 d A B35-107 Sordariomycetes (JX174146.1) 99 % 20.39 g A 33.65 c A 37.65 d A B40-119 Fungal endophyte (KF435617.1) 97 % 23.37 f A 34.69 c A 30.85 e A Diaporthe sp. (EF423554.2) 98 % B43-48 Phomopsis sp. (GQ461582.1) 99 % 25.93 e A 38.61 b A 45.92 c A B45-62 Phomopsis sp. (JN153062.1) 99 % 35.29 c A 14.05 f A 15.48 f A B46-95 Phoma sp. (KJ572232.1) 100 % 47.85 b A 33.65 c C 53.01 b A A1 B60-132 Bipolaris sp. (GU017499.1) 99 % 22.73 f A 33.13 c A 42.97 c A B61-72 Fungal endophyte (JN163857.1) 97 % 24.01 f C 12.79 f A 45.33 c A A1 Phoma herbarum (JX867222.1) 99 % B63-34 Sordariomycetes (JX174146.1) 97 % 35.72 c C 29.21 d A 65.72 a A A1 B64-46 Sordariomycetes (JX174146.1) 100 % 31.67 d A 43.57 b A 47.70 c A C03-45 Fusarium culmorum (KC311482.1) 99 % 26.78 c C 44.36 b C 60.70 b C A1 A1 A1 C07-137 Phoma exigua (AY531684.1) 99 % 21.88 e B 10.66 d A 37.06 e A C16-83 Uncultured soil fungus (EU479850.1) 98 % 18.69 e C 16.93 d A 37.94 e A A1 Alternaria sp. (KJ935016.1) 97 % C18-40 Phoma herbarum (KJ767079.1) 99 % 21.03 e C 9.61 d A 49.47 d A A1 C19-144 Uncultured fungus (GU053874.1) 99 % 19.75 e C 14.31 d A 39.12 e A A1 Cochliobolus sp. (JQ754043.1) 98 % C21-69 Alternaria alternata (KJ410038.1) 98 % 19.54 e A 11.71 d A 37.95 e A C22-22 Fusarium oxysporum (FJ867936.1) 99 % 24.44 d C 12.23 d A 53.01 c A A1 C25-19 Diaporthe longicolla (JQ753971.1) 99 % 31.20 b A 17.4 n A 52.13 c A C27-07 Sordariomycetes (JX174146.1) 98 % 30.82 b A 33.65 c C 63.65 b A A1 C40-59 Sordariomycetes (JX174146.1) 99 % 26.56 c A 39.92 b C 58.33 b A A1 C53-134 Sordariomycetes (JX174146.1) 99 % 27.20 c C 25.55 c A 36.17 e A A1 *Means of triplicate. Means in the same column followed by different letters indicate that intervals of IP are significantly different according to the Scott- Knott test (P<0.05) **Classification by Badalyan scale (Badalyan et al. 2002): A=deadlock with mycelial contact; B=deadlock at a distance; CA1=partial replacement after initial deadlock the second sub-clade with 99 % BP, showing their C. sativus In vitro evaluation of antagonism of endophytic fungi identity. Isolates C16-83 [97 % identity with Alternaria sp. against pathogenic fungi (KJ935016.1) in BLAST analysis] and C21-69 [98 % identity with Alternaria alternata (KJ410038.1) in BLAST analysis] According to the Badalyan scale, competitive interactions be- were grouped in the third sub-clade of Alternaria sp. with tween endophytes and pathogens were defined as follws: A= 100 % BP. deadlock with mycelial contact; B=deadlock at a distance and 770 Ann Microbiol (2016) 66:765–775 A B Cl C ade I Dia D porthales Clade III Pleosporales Dothideo omycetes Sordario omycetes Clade II Hypocreales Fig. 1 Phylogenetic tree constructed with endophytic sequences from of each branch in bootstrap analyses of 10,000 repetitions. All clades Vitis labrusca L. (in black) and sequences from GenBank (indicated by comprise fungi from the phylum Ascomycota. Clade I includes the database code), using the neighbor-joining (NJ) method and the p- Sordariomycetes class and Diaporthales order. Clade II comprises the distance matrix for nucleotides, with the pairwise gap deletion option. Sordariomycetes class and Hipocreales order. Clade III contains the The numbers above and beneath each knot indicate the frequency (%) Dothideomycetes class and Pleosporales order CA1=partial growth of the antagonist after deadlock with all endophytic fungi demonstrated some antagonistic activity. mycelial contact (Fig. 2). We analyzed 68 of the endophytes In Table 1, we can see the IP value of diverse endophytes. from the Bordô cultivar. Interactions with Alternaria sp. Endophytes B55-50 associated with the Bordô cultivar were (CNPUV 674) were A (58 of endophytes), B (6) and CA1 the most efficient in the control of Alternaria sp. (CNPUV (4). The same interactions were observed regarding 674) with an IP of 58.71 %. The endophyte B45-62, identified Sphaceloma sp. (CNPUV 102), representing 62, 3 and 3 en- molecularly as Phomopsis sp., exhibited an IP of 35.29 % dophytes, respectively. Most endophytes (67) from the Bordô against this pathogen. The isolates B63-34 (Table 1; cultivar showed inhibition per mycelial contact (type A) with Fig. 4b), B25-86 and B17-49 showed IPs between 65.72 % Glomerella sp. (378 CNPUV), while one isolate presented and 63.95 % in relation to the pathogen Glomerella sp. interaction deadlock at a distance (type B). (CNPUV 378). Endophytes B46-95 (Phoma sp.) and B43- A sample of 62 endophytes from the Concord cultivar was 48 (Phomopsis sp.) presented IPs of 53.01 and 45.92 %, re- analyzed. Interactions with Alternaria sp. (CNPUV 674) were spectively, against Glomerella sp. (CNPUV 378). Isolate B25- classified as A (45 of the endophytes), B (6) and CA1 (11). 86, which was promising in the control of Glomerella sp. Type A and CA1 interactions were observed with Sphaceloma (CNPUV 378), was the best antagonist to Sphaceloma sp. sp. (CNPUV 102) (57 and 5 endophytes, respectively) and (CNPUV 102) showing an IP of 52.98 %. with Glomerella sp. (CNPUV 378) (61 and 1, respectively). Isolates C13-98, C52-63 and C11-65 from the Concord Figure 3 shows the statistical groups distribution based on cultivar displayed the best inhibition percentages against the inhibition percentage (IP) of each phytopathogen by en- Alternaria sp. (CNPUV 674), varying between 51.04 % and dophytes, i.e., the number of endophytes that belongs to a 45.93 %. The C13-98 isolate also showed the best results in specific range of IP. So, the IP of mycelial growth of the three antagonism tests against Glomerella sp. (CNPUV 378) and pathogens analyzed in relation to the 68 Bordô cultivar iso- Sphaceloma sp. (CNPUV 102) with IPs of 75.18 % and lates and 62 Concord cultivar isolates showed variations, but 57.94 %, respectively. Isolate C27-07 isolate also Ascomycota Ascomycota Ann Microbiol (2016) 66:765–775 771 Fig. 2 Competitive interactions (CI) between endophytes of V. labrusca and phytopathogenic fungi in dual culture. Badalyan rating scale (Badalyan et al. 2002): A=deadlock with mycelial contact; B=deadlock at a distance; CA1=partial replacement after initial deadlock demonstrated promising in vitro control of Glomerella sp. 2007). This was observed by Burruano et al. (2008) who ver- (CNPUV 378) with an IP of 63.65 % (Table 1; Fig. 4c). ified that Acremonium byssoides fungus—an isolated endo- Endophyte isolate C03-45 identified as Fusarium culmorum phyte of V. vinifera cv. Regina Bianca—was found regularly demonstrated an IP of 60.70 % in relation to Glomerella sp. in samples collected in the fall. In summer, with dry weather (CNPUV 378) and an IP of 44.36 % to Sphaceloma sp. and high temperatures, the physiological state of the vineyards (CNPUV 102). was affected and, consequently, the colonization levels of this fungus were reduced. In our study, the samples were collected Scanning electron microscopy during mild spring temperatures, and the frequencies of fungal colonization of the Bordô and Concord cultivars were 74 % Fungal hyphae intensely colonizing the leaf mesophyll (Fig. 5a) and 64 %, respectively. were visualized by SEM. A hypha enveloping the leaf meso- Organic cultivars probably maintain endophytic communi- phyll cell (Fig. 5b) was also observed, indicating the possibility ties better than cultivars treated with phytosanitary products. of intercellular colonization of leaves by endophytic fungi. According to Azevedo et al. (2000), the use of insecticides and fungicides to control pests and phytopathogens also eliminates important species such as endophytes. This was verified by Discussion Gonzáles and Tello (2011) for endophytic fungi isolated from grapevine cultivars (V. vinifera), where less diverse samples The composition of the endophytic community associated were obtained from a cultivar planted in an experimental farm with plants may be influenced both by the identity of the host that was subjected to phytosanitary treatments. (Arnold et al. 2007) and by environmental factors such as Pancher et al. (2012) carried out an extensive comparison temperature and annual precipitation (Arnold and Lutzoni of communities of endophytic fungi between Merlot and Fig. 3 Inhibition percentage (IP) between 130 endophytic fungi of Vitis labrusca and pathogenic fungi in dual culture. IP indicates the reduction (%) in growth of mycelia of the pathogen. *Means of triplicate compared by the Scott-Knott test (P<0.05), in which different letters indicate that IP intervals are significantly different 772 Ann Microbiol (2016) 66:765–775 AB C B 63-34 C 27-07 Glomerella sp. Glomerella sp. Glomerella sp. Fig. 4 Inhibition of pathogen Glomerella sp. (CNPUV 378) growth. a mycelia contact (type A)]: b fungal isolate B63-34 from Bordô cv. (IP= Control plate with only the pathogen. b, c Antagonism mediated by 65.72 %), c fungal isolate C27-07 from Concord cv. (IP=63.65 %) V. labrusca endophytes [both show the interaction deadlock with Chardonnay cultivars (V. vinifera) in vineyards under integrat- isolates of endophytic fungi of V. vinifera most frequently ed pest management and organic management. Their results found by Gonzáles and Tello (2011) and Musetti et al. (2006). indicated that mycota present in grapevines of organic farms Alternaria was the dominant genus in studies of grapevines form significantly different communities than those in grape- reported by Mostert et al. (2000) and Pancher et al. (2012); vine farms under integrated pest management. these authors also reported the presence of Phoma sp. In an Traditional approaches to identifying endophytic fungi in- additional study, Alternaria sp. and Fusarium sp. were report- volve the microscopic analysis of morphological characteris- ed as dominant in five sampled grapevine cultivars along with tics. However, significant portions of the isolated endophyte the less frequent Diaporthe sp., Phoma sp. and Phomopsis consist of sterile mycelium and consequently could not be iden- viticola (Casieri et al. 2009). The latter species were also re- tified by this method (Rivera-Orduña et al. 2011). Data on the ported in studies by Mostert et al. (2000) and Gonzáles and ITS region available in databases increase the chances of accu- Tello (2011). In the isolation of endophytes of V. labrusca, rately identifying taxa because of the possibility of obtaining a Brum et al. (2012) observed the presence of the general fungi taxonomically correct correspondence (Albrectsen et al. 2010). Diaporthe and Fusarium. Our genomic sequence analysis of ITS1-5.8S-ITS2 regions Fungi belonging to the genera Cochliobolus and Bipolaris verified the higher general frequency of Phoma and identified in our study were not observed previously in grape- Phomopsis fungi, as well as the presence of isolates of the vines; however, they and other genera identified in our study genera Cochliobolus, Bipolaris, Fusarium, Alternaria and are present in several other plants. In the isolation of endo- Diaporthe. Mostofthese fungi havealsobeenidentifiedin phytic fungi of Taxus globosa by Rivera-Orduña et al. (2011), previous research into endophytic microbiota in grapevines. Cochliobolus and Alternaria were among the genera most The genera Alternaria, Fusarium and Phoma were among the frequently identified. Fig. 5a,b Scanning electron A B microscopy (SEM) image of leaves of Concord cv. (V. labrusca) incubated for 3 days. a Hyphae intensely colonizing the leaf mesophyll. b Leaf mesophyll with arrow indicating fungal hyphae enveloping mesophyll cell Ann Microbiol (2016) 66:765–775 773 Isolates of Bordô and Concord cultivars identified in in a dual culture experiment, all the endophytes showed some this study were grouped into three clades belonging to degree of inhibition against phytopathogens. Among the fungi the phylum Ascomycota: Clade I belongs to the identified molecularly, highlighted are B46-95 (Phoma sp. Sordariomycetes class, Diaporthales order; Clade II also KJ572232.1) with 47.85 % inhibition of growth of the phyto- belongs to the Sordariomycetes class, Hypocreales order; pathogen Alternaria sp., C03-45 (Fusarium culmorum and Clade III belongs to the class Dothideomycetes and KC311482.1) with 44.36 % inhibition of Sphaceloma sp. Pleosporales order. This predominance of endophytic fungi and B63-34 (Sordariomycetes JX174146.1) with 65.72 % in- belonging to the phylum Ascomycota was also reported in hibition of Glomerella sp. many other recent studies (Albrectsen et al. 2010;Vega Analyzing 46 endophytic fungi isolated from Luehea et al. 2010; Rocha et al. 2011; Rhoden et al. 2012;Garcia divaricata in a dual culture experiment against the grapevine et al. 2012;Orlandelli etal. 2012; Aharwal et al. 2014). phytopathogen Alternaria alternata, Bernardi-Wenzel et al. Gonzáles and Tello (2011) obtained 91 % of ascomycetes (2013) observed that antagonism rates ranged from 3.7 % to while investigating the endophytic mycota in nine grapevine 62.7 %, and competitive interactions classified as B and CA1 cultivars (V. vinifera). Seven main orders of this phylum were observed. The same interactions were observed in our showed a similar distribution among the six cultivars most study, but antagonism rates among our endophytes and this sampled, and the Hyporeales and Pleosporales orders were phytopathogen ranged from 13.79 % to 58.71 %. the most abundant of all cultivars. In addition, analyzing the This is the first study using SEM to demonstrate the colo- fungal communities of five grapevine cultivars (V. vinifera)in nization of leaf endophytes in V. labrusca species. The tech- Switzerland, Casieri et al. (2009) showed that the vast major- nique employed was effective enough to view endophytes in ity of isolates were ascomycetes (87.5 %), including leaf tissues incubated for up to 3 days, confirming the pres- Sordariomycetes as the most represented class comprising ence of endophytic fungi in leaf mesophyll cells. Their inter- 55.1 % of isolates. Brum et al. (2012) examined the diversity cellular colonization could also be seen as fungal hyphae of endophytic fungi in leaves from the Niagara Rosada grape- clearly emerging from inside a cell. According to Stone vine (V. labrusca) and observed that 77 % of the species iden- et al. (2000), the colonization of endophytes may be intracel- tified belonged to the phylum Ascomycota. lular and limited to a single cell, intercellular and located in an Biological control is based on the beneficial interactions intra- and intercellular systemic fashion. resulting from competition, antibiotic activity and hyperpara- The results obtained in this study demonstrate the presence sitism of microorganisms against pathogens, insects and of endophytic fungi in V. labrusca as well as the biotechno- weeds (Mathre et al. 1999). It is an important alternative strat- logical potential of this organism to control grapevine patho- egy for reducing or eliminating the use of chemicals in agri- gens. Future studies should focus on developing efficient tech- niques for applying these endophytes in biological control culture (Azevedo et al. 2000). Several studies have demonstrated a reduction in the strategies against fungal diseases of grapevine. growth of pathogens resulting from interactions between en- dophytic fungi isolates of various plants and different phyto- pathogens (Živković et al. 2010; Rehman et al. 2011). Most Acknowledgments We thank the Complexo de Centrais de Apoio à studies related to the characterization of endophytic Pesquisa (COMCAP/UEM) for sequencing the ITS1-5.8S-ITS2 regions communities and applications in viticulture pathogen and for the production of electronic scanning micrographs. We thank the EMBRAPA Uva e Vinho for the phytopathogen strains. We thank the biocontrol are focused on the cause of the disease known as Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) downy mildew, caused by the fungus Plasmopara viticola. for the Master’s scholarship for the first author and CAPES/PNPD-UEM Musetti et al. (2006) confirmed the inhibition of sporulation postdoctoral scholarship. We thank CNPq (311534/2014-7; 447265/ of the pathogen by the endophyte Alternaria alternata isolated 2014-8) and Fundação Araucária – FA (276/2014) for financial support. from grapevine leaves (V. vinifera). Burruano et al. (2008)also reported the inhibition of sporulation of the P. viticola patho- gen by crude extracts of the endophyte Acremonium byssoides References isolated from grapevine leaves (V. vinifera). However, Brum et al. (2012) tested endophytic fungi iso- Albrectsen BR, Björkén L, Varad A, Hagner A, Wedin M, Karlsson J, lated from the Niagara Rosada grapevine (V. labrusca)inan Jansson S (2010) Endophytic fungi in European aspen (Populus experiment with a dual culture against the pathogenic grape- tremula)leaves—diversity, detection, and a suggested correlation with herbivory resistance. Fungal Divers 41:17–28 vine fungus Fusarium oxysporum. In this experiment, 52 % of Arnold AE (2007) Understanding the diversity of foliar endophytic fungi: endophytes were able to inhibit pathogen growth, and the progress, challenges, and frontiers. Fungal Biol Rev 21:51–66 largest halo of inhibition was from the isolates identified as Arnold AE (2008) Endophytic fungi: hidden components of tropical Colletotrichum gloeosporioides and Flavodon flavus.Inour community ecology. In: Carson WP, Schnitzer SA (eds) Tropical study, with fungi isolated from the same plant species and also Forest Community Ecology. Wiley-Blackwell, Hoboken 774 Ann Microbiol (2016) 66:765–775 Arnold AE, Lutzoni F (2007) Diversity and host range of foliar fungal Mostert L, Crous PW, Petrini O (2000) Endophytic fungi associated with shoots and leaves of Vitis vinifera, with specific reference to the endophytes: are tropical leaves biodiversity hotspots? Ecology 88: 541–549 Phomopsis viticola coplex. Sydowia 52:46–48 Arnold AE, Henk DA, Eells RL, Lutzoni F, Vilgalys R (2007) Diversity Musetti R, Vecchione A, Stringher L, Borselli S, Zulini L, Marzani C, and phylogenetic affinities of foliar fungal endophytes in loblolly Dambrosio M, Sanità Di Toppi L, Pertot I (2006) Inhibition of spor- pine inferred by culturing and environmental PCR. Mycologia 99: ulation and ultrastructural alterations of grapevine downy mildew by 185–206 the endophytic fungus Alternaria alternata. Phytopathology 96: 689–698 Azevedo JL, Maccheroni JW, Pereira JO, Araújo WL (2000) Endophytic microrganisms: a review on insect control and recent advances on Orlandelli RC, Alberto RN, Rubin Filho CJ, Pamphile JA (2012) tropical plants. Electron J Biotechnol 3:40–65 Diversity of endophytic fungal community associated with Badalyan SM, Innocenti G, Garibyan NG (2002) Antagonistic activity of Piper hispidum Sw. (Piperaceae) leaves. Genet Mol Res 11: xylotrophic mushrooms against pathogenic fungi of cereals in dual 1575–1585 culture. Phytopathol Mediterr 41:200–225 Pamphile JA, Azevedo JL (2002) Molecular characterization of endo- phytic strains of Fusarium verticillioides (Fusarium moniliforme) Bernardi-Wenzel J, Garcia A, Azevedo JL, Pamphile JA (2013) from maize (Zea mays.L). World J Microbiol Biotechnol 18:391– Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi Pamphile JA, Gai CS, Pileggi M, Rocha CLMSC, Pileggi SAV (2008a) and pathogenic bacteria. Genet Mol Res 12(4):5072–5084 Plant-microbe interactions between host and endophytes observed Brum MCP, Araújo WL, Maki CS, Azevedo JL (2012) Endophytic fungi by scanning electron microscopy (SEM). In: Sorvari S, Pirttilä AM from Vitis labrusca L. (‘Niagara Rosada’) and its potential for the (eds) Prospects and Applications for Plant-Associated Microbes. A biological control of Fusarium oxysporum. Genet Mol Res 11: Laboratory Manual, part A: Bacteria. BBI (BioBien Innovations), 4187–4197 Finland, pp 184–189 Pamphile JA, Pileggi M, Gai CS, Rocha CLMSC, Pileggi SAV (2008b) Burruano S, Alfonzo A, Lo Piccolo S, Conigliaro G, Mondello V, Torta L, Scanning electron microscopy (SEM). In: Sorvari S, Pirttilä AM Moretti M, Assante G (2008) Interaction between Acremonium (eds) Prospects and Applications for Plant-Associated Microbes. A byssoides and Plasmopara viticola in Vitis vinifera. Phytopathol Laboratory Manual, part A: Bacteria. BBI (BioBien Innovations), Mediterr 47:122–131 Finland, pp 9–13 Campanile G, Ruscelli A, Luisi N (2007) Antagonistic activity of endo- phytic fungi towards Diplodia corticola assessed by in vitro and in Pancher M, Ceol M, Corneo PE, Longa CMO, Yousaf S, Pertot I, planta tests. Eur J Plant Pathol 117:237–246 Campisano A (2012) Fungal endophytic communities in Grapevines (Vitis vinifera L.) respond to crop management. Appl Casieri L, Hofstetter V, Viret O, Gindro K (2009) Fungal communities Environ Microbiol 78:4308–4317 living in the wood of different cultivars of young Vitis vinifera plants. Phytopathol Mediterr 48:73–83 Petrini O (1991) Fungal endophyte of tree leaves. In: Andrews J, Hirano Eichhorn KW, Lorenz DH (1984) Phaenologische entwicklungsstadien SS (eds) Microbial Ecology of Leaves. Spring, New York, pp 179– der rebe. EPPO 14:295–298 197 Quiroga EN, Sampietro AR, Vattuone MA (2001) Screening antifungal FanC,PuN,WangX,WangY,FangL,Xu W,Zhang J(2008) activities of selected medicinal plants. J Ethnopharmacol 74:89–96 Agrobacterium mediated genetic transformation of grapevine (Vitis vinifera L.) with a novel stilbene synthase gene from Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous Chinese wild Vitis pseudoreticulata. Plant Cell Tissue Organ fungi. Lett Appl Microbiol 1:17–20 Cult 92:197–206 Rehman S, Mir T, Kour A, Qazi PH, Sultan P, Shawl AS (2011) In vitro Faostat—Food and Agriculture Organization of the United Nations antimicrobial studies of Nodulisporium specie: an endophytic fun- (2012) Available from: http://faostat.fao.org/site/339/default.aspx. gus. J Yeast Fungal Res 2:53–58 Accessed: 20 july 2014 Reyes Chilpa R, Quiroz Vásquez RI, Jiménez Estrada M, Navarro-Ocaña Ferreira DF (2008) SISVAR: um programa para análise e ensino de A, Cassini Hernández J (1997) Antifungal activity of selected plant estatística. Rev Científica Symp 6:36–41 secondary metabolites against Coriolus versicolor. J Trop Forest Garcia A, Rhoden SA, Rubin Filho CJ, Nakamura CV, Pamphile JA Prod 3:110–113 (2012) Diversity of foliar endophytic fungi from the medicinal plant Rhoden SA, Garcia A, Rubin Filho CJ, Azevedo JL, Pamphile JA (2012) Sapindus saponaria L. and their localization by scanning electron Phylogenetic diversity of endophytic leaf fungus isolates from the microscopy. Biol Res 45:139–148 medicinal tree Trichilia elegans (Meliaceae). Genet Mol Res 11: Gonzáles V, Tello ML (2011) The endophytic mycota associated with 2513–2522 Vitis vinifera in central Spain. Fungal Divers 47:29–42 Rivera-Orduña FN, Suarez-Sanchez RA, Flores-Bustamante ZA, Hata K, Futai K (1995) Endophytic fungi associated with healthy pine Gracida-Rodriguez JN, Flores-Cotera LB (2011) Diversity of endo- needles and needles infestes by the pine needle gall midge, phytic fungi of (Mexican yew). Fungal Divers 47:65–74 Thecodiplosis japonensis. Can J Bot 73:384–390 Rocha ACS, Garcia D, Uetanabaro APT, Carneiro RTO, Araújo IS, Kogel KH, Franken P, Huckelhoven R (2006) Endophyte or parasite— Mattos CRR, Góes-Neto A (2011) Foliar endophytic fungi from what decides? Curr Opin Plant Biol 9:358–363 Hevea brasiliensis and their antagonism on Microcyclus ulei. Lima TEF (2010) Micobiota endofitica de Vitis labrusca L. CV Isabel no Fungal Divers 47:75–84 vale do Siriji, Pernambuco, Brasil. Thesis, Universidade Federal de Rodriguez R, Redman R (2008) More than 400 million years of evolution Pernambuco and some plants still can’t make it on their own: plant stress toler- ance via fungal symbiosis. J Exp Bot 59:1109–1114 Magnani M, Fernandes T, Prete CEC, Homechim M, Ono EYS, Vilas- Boas LA, Sartori D, Furlaneto MC, Fungaro MHP (2005) Molecular Saitou N, Nei M (1987) The neighbor-joining method: a new meth- identification of Aspergillus spp. isolated from coffee beans. Sci od for reconstructing phylogenetic trees. Mol Biol Evol 4:406– Agric 62:45–49 425 Mathre DE, Cook RJ, Callan NW (1999) From discovery to use: travers- Sandhu SS, Aharwal RP, Kumar S (2014) Isolation and antibacterial ing the world of commercializing biocontrol agents for plant disease property of endophytic fungi isolated from Indian medicinal plant control. Plant Dis 83:972–983 Calotropis procera (Linn.). World J Pharm Pharm Sci 3:678–691 Ann Microbiol (2016) 66:765–775 775 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882 109:661–686 Smith D, Onions AHS (1983) The preservation and maintenance of living Vaz ABM, Mota RC, Bomfim MRQ, Vieira MLA, Zani CL, Rosa fungi. Page, Norwick CA, Rosa LH (2009) Antimicrobial activity of endophytic fun- Soejima A, Wen J (2006) Phylogenetic analysis of the grape family gi associated with Orchidaceae in Brazil. Can J Microbiol 55: (Vitaceae) based on three chloroplast markers. Am J Bot 93:278– 1381–1391 287 Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Sousa JSI (1996) Uvas para o Brasil, 2nd edn. FEALQ, Piracicaba Infante F, Castillo A, Arnold AE (2010) Fungal endophyte diversity Stone JK, Bacon CW, White JF (2000) An overview of endophytic mi- in coffee plants from Colombia, Hawai’i, Mexico and Puerto Rico. crobes: endophytism defined. In: Bacon CW, White JF (eds) Fungal Ecol 3:122–138 Microbial Endophytes. Dekker, New York, pp 3–30 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from sequencing of fungal ribosomal RNA genes for phylogenetics. In: endophytic microorganisms. J Nat Prod 67:257–268 Innis MA, Gelfland DH, Sninsky JJ, White TJ (eds) PCR Protocols. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) A Guide to Methods and Applications. Academic, New York, MEGA5: molecular evolutionary genetics analysis using maximum pp 315–322 likelihood, evolutionary distance, and maximum parsimony Živković S, Stojanović S, IvanovićŽ,Gavrilović V, Popović T, Balaž J methods.MolBiolEvol28:2731–2739 (2010) Screening of antagonistic activity of microorganisms against Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Colletotrichum acutatum and Colletotrichum gloeosporioides. Arch (1997) The ClustalX windows interface: flexible strategies for Biol Sci 62:611–623 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv.

Download PDF
11 pages

Loading next page...
 
/lp/springer-journals/bioprospecting-foliar-endophytic-fungi-of-vitis-labrusca-linnaeus-bord-J9hP4J0kfL

References (50)

Publisher
Springer Journals
Copyright
Copyright © 2015 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-015-1162-6
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2016) 66:765–775 DOI 10.1007/s13213-015-1162-6 ORIGINAL ARTICLE Bioprospecting foliar endophytic fungi of Vitis labrusca Linnaeus, Bordô and Concord cv. 1 1 2 Aretusa Cristina Felber & Ravely Casarotti Orlandelli & Sandro Augusto Rhoden & 1 1 3 Adriana Garcia & Alessandra Tenório Costa & João Lúcio Azevedo & João Alencar Pamphile Received: 26 May 2015 /Accepted: 28 September 2015 /Published online: 10 October 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract Endophytic fungi colonize the interior of plant tis- identity of the endophytes. The biotechnological potential of sues and organs, establishing an intimate mutualistic associa- endophytes was tested in vitro for the control of pathogenic tion with no visible symptoms. The fungi may help protect the fungi of grapevines including Alternaria sp., Sphaceloma sp. plant against herbivores and pathogens, making them potential- and Glomerella sp. Inhibition percentages above 50 % as dem- onstrated by some isolates demonstrate their potential for bio- ly useful endophytes in the biological control of diseases and agricultural pests. The biotechnological interest in these organ- logical control. isms has stimulated research related to the bioprospecting of . . . endophytic fungi. Grapevine is among the oldest of plants cul- Keywords Endophytes Grapevine Biological control tivated by man, with the grape being one of the most highly Sequencing of rDNA Phylogenetic analysis consumed fruits in the world. Diseases cause significant dam- age to grape cultures, making their integrated control important to reduce the use of pesticides and, consequently, environmen- Introduction tal and human contamination. The rustic species Vitis labrusca L. (Vitaceae), used in the preparation of juices and wines, is The grape is one of the most important fruits grown in the highly resistant to fungal diseases. We isolated leaf endophytic world, and its worldwide production surpasses 67 million tons fungi of the Bordô and Concord cultivars (V. labrusca L.), annually (Faostat 2012). The grapevine belongs to the which were ordered into 68 and 62 morpho-groups of the Vitaceae family, consisting of approximately 14 genera and Bordô and Concord cultivars, respectively. We used scanning 900 species (Soejima and Wen 2006). The Vitis genus is the electron microscopy to confirm the presence of endophytes in most important economically because its species are used the leaves. Endophytic diversity was analyzed based on se- most commonly in agriculture. The Bordô and Concord cul- quencing the ITS1-5.8S-ITS2 region of rDNA, allowing the tivars of the species Vitis labrusca L. are hardy grapes that are identification of fungi belonging to genera including resistant to fungal diseases. These two cultivars are used for in Cochliobolus, Bipolaris, Fusarium, Alternaria, Diaporthe, natura consumption and primarily for the production of juices Phoma and Phomopsis. Phylogenetic analysis confirmed the and wines (Sousa 1996). Among the most serious problem faced in vine cultivation are fungal diseases that cause losses in production and fruit * João Alencar Pamphile quality (Fan et al. 2008). Endophytic microorganisms may be prof.pamphile@gmail.com promising alternatives to the use of pesticides in controlling fungal threats. Endophytes are microorganisms that inhabit the interior of plant tissues during all or part of their life cycle, Department of Cell Biology and Genetics, Universidade Estadual de Maringá, CEP 87020-900 Maringá, Paraná, Brazil without causing any visible symptoms (Petrini 1991;Azevedo et al. 2000). However, endophytes are relatively unexplored Câmpus de São Francisco do Sul, Federal Institute Catarinense (FIC), São Francisco do Sul, Brazil with regards to their production of useful natural compounds for agriculture, industry and medicine (Strobel et al. 2004). College of Agriculture BLuiz de Queiroz^ (ESALQ), Universidade de São Paulo, CEP 13400-970 Piracicaba, São Paulo, Brazil The ability of endophytes to produce substances in vitro that 766 Ann Microbiol (2016) 66:765–775 inhibit the growth of other species of microorganisms has Materials and methods stimulated research on the bioprospecting of endophytic mi- croorganisms and their use in biological control (Arnold Leaf sampling 2008). The mechanisms underlying the endophyte–host rela- Mature and healthy leaves of Bordô and Concord cultivars tionship are not well understood (Kogel et al. 2006). (V. labrusca) were selected randomly and used immediately Schulz and Boyle (2005) suggest that these are not neutral after collection. Two leaves from four different plants of each interactions, with the asymptomatic colonization requiring cultivar were used. The material was collected at Iguatemi a balance of antagonisms between the fungal endophyte Experimental Farm (IEF) belonging to Universidade and the host. According to the review of Rodriguez and Estadual de Maringá (UEM), planted in 0.048 ha located in Redman (2008), all plants in natural ecosystems are the Iguatemi District, city of Maringá, Paraná State, Brazil thought to be symbiotic with mycorrhizal and/or endophyt- (23°21′22^S, 52°4′18^W). The grapevines themselves were ic fungi. Collectively, fungi express several different sym- planted in the system known as espalier in a certifiably organic biotic lifestyles that are defined by fitness benefits to plant area. On the day of collection (10 August 2010), the majority hosts and symbionts. The range of symbiotic lifestyle ex- of berries carried by the grapevines were classified as pellet- pression from mutualism to parasitism has been described like according to the phenological stages of the grapevine as the symbiotic continuum. Within each group of fungal described by Eichhorn and Lorenz (1984). The temperature symbionts there are isolates and/or species that span the in the month prior to collection ranged from 12.1 °C to symbiotic continuum by expressing different lifestyles. 37.4 °C with an average temperature of 23.5 °C and average Several studies focusing on the isolation of endophytes relative humidity of 64.8 %. from asymptomatic plant tissues indicate that individual species express either mutualistic, commensal, or parasitic lifestyles when re-inoculated back onto the original host Isolation of endophytic fungi of Vitis labrusca species (Rodriguez and Redman 2008). Advances in the identification of fungi followed the de- Leaves were washed under running water, 0.01 % Tween velopment of sensitive and specific techniques of molecu- 80 aqueous solution (Synth; http://www.splabor.com.br) lar biology employed in the differentiation of species, such and two rinses in sterile, distilled water to remove as amplification of the internal transcribed spacer (ITS) of residues. They were then surface-disinfected to suppress ribosomal DNA (rDNA) using the polymerase chain reac- epiphytic microorganisms according to Vaz et al. (2009). tion (PCR); rDNA-ITS sequences can be sequenced and Washing was performed in series with 70 % ethanol for compared for homology with sequences available in data- 1 min, sodium hypochlorite (2 % available Cl )for bases (Magnani et al. 2005). 3min,70%ethanol for30sandtwo washes in sterile, Another important tool to assist our understanding of distilled water. The effectiveness of this method was veri- endophyte–host interactions is the use of scanning electron fied by spreading 100 μL of the final water used on Petri microscopy. This technique is advantageous for its high dishes containing potato dextrose agar (PDA) culture me- resolution and the possibility of in-depth analysis of vari- dium (Smith and Onions 1983) pH 6.6, supplemented with −1 ous materials (Pamphile et al. 2008a). tetracycline (Sigma, St. Louis, MO) (50 μgmL in 50 % Many studies have focused on endophytes in plants be- ethanol), to prevent bacterial growth. longing to the angiosperms and conifers (Arnold 2007). The Disinfected leaves were cut into 5-mm fragments and de- occurrence of endophytic fungi has been reported in the genus posited (five fragments per plate, with a total of 50 plates for Vitis. However, most studies have concentrated on fungi asso- each plant) on plates with PDA containing tetracycline. The ciated with V. vinifera (Mostert et al. 2000; Burruano et al. plates were incubated at 28 °C for 7 days. The colonization 2008; Casieri et al. 2009; Gonzáles and Tello 2011;Pancher frequency (CF) was determined by Hata and Futai (1995): CF et al. 2012). Studies related to the bioprospecting of endophyt- (%)=(number of fragments colonized by fungi / total number ic fungi in V. labrusca species are less common (Lima 2010; of fragments)×100. Brum et al. 2012). In the purification process, the fungal isolates were trans- Considering the potential of endophytic microorganisms as ferred to PDA plates and grown for 7 days. Then, fragments biological controllers (Azevedo et al. 2000), the aim of this (5 mm ) weresquashedin 1 mL0.01% Tween80 aqueous study was to isolate and characterize foliar endophytic fungi solution, and an aliquot of 100 μL solution was then spread on of Bordô and Concord grapevine cultivars (V. labrusca L.). plates containing PDA and incubated for 24 h. Single colonies Their biotechnological potential in the in vitro control of path- were transferred immediately to new plates with PDA and ogenic fungi of grapevine Alternaria sp., Sphaceloma sp. and incubated for 7 days. If necessary, the process was repeated Glomerella sp. was determined. untilpurecolonieswereobtained. Ann Microbiol (2016) 66:765–775 767 Molecular identification of isolated endophytic fungi RS. Fungi were: Alternaria sp. (CNPUV 674), responsible for leaf blight; Glomerella sp. (CNPUV 378), responsible for ripe Genomic DNAwas extracted as described by Raeder and Broda rot of grapes; and Sphaceloma sp. (CNPUV 102), which (1985) and modified according to Pamphile and Azevedo causes anthracnose. (2002), except that endophytes were previously grown for A paired culture technique described by Campanile et al. 7 days on plates with potato dextrose broth (PDB) medium (2007) was used. Endophytes and phytopathogenic fungal (Smith and Onions 1983) at 28 °C under stationary conditions. mycelial-disks (5 mm) were inoculated at a distance of 2 cm The concentration and purity of the genomic DNA were deter- on opposite sides of Petri dishes (9 cm) containing PDA. mined using a GENESYS 10S UV–vis spectrophotometer (OD Negative control plates had each pathogen disk inoculated in 260/280 nm). DNA integrity was analyzed by electrophoresis dual culture with a disk of PDA medium. The tests were per- in 1 % agarose gels, using the High DNA Mass Ladder formed in triplicate and all plates were incubated at 28 °C (Invitrogen, Carlsbad, CA) as the molecular weight standard. for 7 days. The inhibition percentages were calculated −1 Thefinal concentrationofDNAwasadjustedto10ngmL . according to Reyes Chilpa et al. (1997) as cited by Quiroga PCR amplification of the ITS1-5.8S-ITS2 of rDNA region et al. (2001): IP (%)=(average diameter of the pathogen was performed according to Magnani et al. (2005), using the colony in control − average diameter of the pathogen colony primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)and in the treatment / average diameter of the pathogen colony in ITS4 (5′-TCCCCGCTTATTGATATGC-3′)(Whiteetal. control)×100. 1990) with an initial denaturation at 92 °C for 4 min, followed The competitive interactions between endophytic and by 35 denaturation cycles at 92 °C for 40 s, annealing at 52 °C pathogenic fungi were characterized based on the scale of for 1 min and 30 s, extension at 72 °C for 2 min and a final Badalyan et al. (2002) where three types (A, B and C) and extensionat72°Cfor 5min. four subtypes (CA1, CA2, CB1 and CB2) of interaction are PCR products were purified with the GFX PCR DNA kit possible: A=deadlock with mycelial contact; B=deadlock at a and Gel Band Purification (Amersham Biosciences, distance; C=replacement, overgrowth without initial dead- Piscataway, NJ), according to the manufacturer’s instructions, lock; CA1 and CA2=partial and complete replacement after prepared for second sequencing reactions according to initial deadlock with mycelial contact; and CB1 and CB2= Magnani et al. (2005) using the primer ITS4. PCR reactions partial and complete replacement after initial deadlock at a were carried out in a thermocycler programmed to perform an distance. initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for Scanning electron microscopy 1 min, extension at 60 °C for 1 min and a final extension at 60 °C for 5 min. Sequencing was performed in a MegaBACE During the process of isolation of endophytic fungi, each fo- TM 1000 sequencer (Amersham Biosciences), with injection liar sample of V. labrusca was cut into two portions: one for and electrophoresis conditions of 1 kV/90 s and 7 kV/ isolation test and the other for microscopic observation. The 240 min, respectively. latter portions were incubated for 3 days and ruptured by the The nucleotide sequences were analyzed and edited, and freeze-fracture process then subjected to scanning electron compared to those deposited in GenBank (http://www.ncbi. microscopy (SEM). Sample preparation was modified from nlm.nih.gov). For research into genera or species, the that described by Pamphile et al. (2008b). A gradient of BLASTN program was used. Determination was based on 30 %, 50 %, 70 %, 90 % and 100 % alcohol was used for the best result obtained for identity. the dehydration of vegetable material, and the resultant frag- For the phylogenetic analyses, a dendrogram was construct- ments were dried to critical point in a Bal-tec-CPD030 (Bal- ed with the sequences obtained along with those available in tec, Los Angeles, CA), for seven cycles. Samples were GenBank. Sequences were aligned using Clustal X (Thompson mounted on stubs with the vertical position of the fragments et al. 1997), and the dendrogram was constructed using MEGA in a conductive tape for SEM. The fragments received a thin program version 5 (Tamura et al. 2011) with grouping by the layer of gold on Shimadzu-unit IC 50 (260 s, 50 mA, at 27 °C) neighbor-joining (NJ) method (Saitou and Nei 1987), using p- for three cycles. The fragment covered with metal was ob- distance for nucleotides with the option of pairwise gap dele- served in a field emission scanning electron microscope tion and a bootstrap with 10,000 repetitions. (SHIMADZU-SS550; Shimadzu, Tokyo, Japan) at 15 kV and 7 mm away. In vitro antagonistic activity of endophytic isolates against pathogenic fungi Statistical analysis Tests were performed with pathogenic fungi of grapevine pro- Experiments were conducted in a completely randomized de- vided by EMBRAPA Grape and Vine of Bento Gonçalves – sign, and evaluated statistically by analysis of variance 768 Ann Microbiol (2016) 66:765–775 (ANOVA) and means compared by Scott-Knott test (P<0.05) with Sordariomycetes (JX174146.1) in BLAST analysis], using the statistical program SISVAR 5.3 (Ferreira 2008). C40-59 [99 % identity with Sordariomycetes (JX174146.1) in BLAST analysis] and B35-107 [99 % identity with Sordariomycetes (JX174146.1) in BLAST analysis], were Results clustered with the Diaporthe sp. (anamorph=Phomopsis) group with 100 % bootstrapping (BP) analysis. Isolate C25- Isolation and molecular identification of endophytic fungi 19 [99 % identity with Diaporthe longicolla (JQ753971.1) in of V. labrusca BLAST analysis] was grouped with the Diaporthe sp. sub- clade with 100 % BP analysis. In that case, the species was The frequency of colonization of the 250 leaf fragments sam- identified as Diaporthe sp. instead of D. longicolla. The iso- pled from the Bordô cultivar was 74 %. A sample of 140 fungi late B43-48 [99 % identity with Phomopsis sp. (GQ461582.1) was isolated and grouped randomly into 68 morpho-groups in BLAST analysis] and the isolate B30-122 [99 % identity based on macroscopic characteristics including morphology with Phomopsis sp. (GQ461582.1) in BLAST analysis] and characteristics when grown on the culture media PDA: grouped with the Diaporthe/Phomopsis sub-clade with sporulation, mycelium aspect, mycelium coloration, colora- 100 % BP, showing that they are both Diaporthe sp. The tion of the reverse of the Petri dish, pigmentation of the culture isolates B15-03 [98 % identity with Phomopsis sp. medium and average diameter of colony growth. Of the 250 (KF159989.1) in BLAST analysis], B40-119 [98 % identity leaf fragments sampled from the Concord cultivar, we obtain- with Diaporthe sp. (EF423554.2) in BLAST analysis], B28- ed a colonization frequency of 64 % and 145 fungi were iso- 47 [99 % identity with Phomopsis sp. (JN153054.1) in lated and divided randomly into 62 morpho-groups. A fungus BLAST analysis] and B45-62 [99 % identity with from each morpho-group was chosen randomly and purified Phomopsis sp. (KF153062.1) in BLAST analysis] clustered for other experiments. with the sub-clade of Diaporthe/Phomopsis with 100 % BP, Sequence analyses of ITS1-5.8S-ITS2 regions allowed confirming the Diaporthe sp. identity. identification of endophytic fungi (Table 1) belonging to sev- In the second sub-clade of the first clade of en genera: Cochliobolus, Bipolaris, Fusarium, Alternaria, Sordariomycetes, isolate C22-22 [99 % identity with Diaporthe, Phoma and Phomopsis (the latter two genera being Fusarium oxysporum (FJ867931.1) in BLAST analysis] was the most common). The percentages of identity between the grouped in the Fusarium sp./Gibberella sp. with 100 % BP, sequences obtained and those available in GenBank varied confirming its identity as Fusarium sp. Isolate C03-45 [99 % between 97 % and 100 %. identity with Fusarium culmorum (KC311482.1) in BLAST analysis] was grouped with Fusarium culmorum with 8 % BP, Phylogenetic analyses of the endophytic isolates indicating its identitiy as Fusarium sp. of V. labrusca based on rDNA sequence data In the second clade, the isolates B31-38 [99 % identity with Phoma herbarum (KJ767079.1) in BLAST analysis], B61-72 Phylogenetic analysis (Fig. 1) resulted in the grouping of iso- [99 % identity with Phoma herbarum (JX867222.1) in lated endophytes, along with fungal sequences obtained from BLAST analysis], B46-95 [100 % identity with Phoma sp. GenBank (NCBI), in three clades belonging to the phylum (KJ572232.1) in BLAST analysis] and C18-40 [99 % identity Ascomycota. Clade I belongs to the Sordariomycetes class, with Phoma herbarum (KJ767079.1) in BLAST analysis] Diaporthales order, with representatives of the Phomopsis were grouped together in the Phoma sp. clade with 100 % and Diaporthe genera. In Clade II, the Sordariomycetes class BP. The isolate C07-137 [99 % identity with Phoma exigua is also represented by the Hypocreales order, including the (AY531684.1) in BLAST analysis] was grouped with Phoma fungi Fusarium and Giberella genera. Clade III is represented exigua with 70 % BP, indicating that C07-137 belongs to by the Dothideomycetes class and Pleosporales order, includ- Phoma exigua species. ing fungal Phoma, Cochliobolus, Bipolaris and Alternaria The third clade of the Pleosporales group was divided into genera. three sub-clades. The two initial sub-clades clustered with the The first Clade was subdivided into two sub-clades. In the Cochliobolus (anamorph=Bipolaris) species. Isolates B33-61 first sub-clade, isolates B17-49 [99 % identity with [98 % identity with Bipolaris sp. (GU017499.1) in BLAST Sordariomycetes (JX174146.1) in BLAST analysis], B27- analysis] and B60-132 [99 % identity with Bipolaris sp. 116 [98 % identity with Sordariomycetes (JX174146.1) in (GU017499.1) in BLAST analysis] were grouped in the first BLAST analysis], C53-134 [99 % identity with sub-clade with 100 % BP, confirming their Cochliobolus/ Sordariomycetes (JX174146.1) in BLAST analysis], C27-07 Bipolaris identity. Isolates C19-144 [98 % identity with [98 % identity with Sordariomycetes (JX174146.1) in BLAST Cochliobolus sp. (JQ754043.1) in BLAST analysis] and analysis], B63-34 [97 % identity with Sordariomycetes B14-69 [99 % identity with Cochliobolus sp. (JQ936204.1) (JX174146.1) in BLAST analysis], B64-46 [100 % identity in BLASTanalysis] also grouped with Cochliobolus sativus in Ann Microbiol (2016) 66:765–775 769 Table 1 Identification of 28 endophytic fungi of Vitis labrusca based on rRNA sequencing and inhibition percentages (IP) and competitive interactions (CI) with three phytopathogenic fungi in dual culture Endophytic Closely related fungal sequences Maximal Phytopathogenic fungi fungi identity Alternaria Sphaceloma Glomerella IP* CI** IPCI IPCI B14-69 Uncultured fungus (GU053859.1) 99 % 22.80 f A 27.64 d A 44.44 c A Cochliobolus sativus (JQ936204.1) 99 % B15-03 Fungal endophyte (JX155973.1) 99 % 37.42 c A 31.82 c A 37.94 d A Phomopsis sp. (KF159989.1) 98 % B17-49 Sordariomycetes (JX174146.1) 99 % 39.34 c A 42.79 b A 63.95 a A B27-116 Sordariomycetes (JX174146.1) 98 % 22.94 f A 11.44 f A 15.19 f A B28-47 Phomopsis sp. (JN153054.1) 99 % 25.93 e B 26.33 d B 32.33 e A B30-122 Fungal endophyte (KF435373.1) 99 % 31.46 d A 32.60 c A 47.40 c A Phomopsis sp. (GQ461582.1) 99 % B31-38 Phoma herbarum (KJ767079.1) 99 % 37.63 c C 17.45 e A 36.47 d A A1 B33-61 Bipolaris sp. (GU017499.1) 98 % 21.03 g A 25.55 d A 37.65 d A B35-107 Sordariomycetes (JX174146.1) 99 % 20.39 g A 33.65 c A 37.65 d A B40-119 Fungal endophyte (KF435617.1) 97 % 23.37 f A 34.69 c A 30.85 e A Diaporthe sp. (EF423554.2) 98 % B43-48 Phomopsis sp. (GQ461582.1) 99 % 25.93 e A 38.61 b A 45.92 c A B45-62 Phomopsis sp. (JN153062.1) 99 % 35.29 c A 14.05 f A 15.48 f A B46-95 Phoma sp. (KJ572232.1) 100 % 47.85 b A 33.65 c C 53.01 b A A1 B60-132 Bipolaris sp. (GU017499.1) 99 % 22.73 f A 33.13 c A 42.97 c A B61-72 Fungal endophyte (JN163857.1) 97 % 24.01 f C 12.79 f A 45.33 c A A1 Phoma herbarum (JX867222.1) 99 % B63-34 Sordariomycetes (JX174146.1) 97 % 35.72 c C 29.21 d A 65.72 a A A1 B64-46 Sordariomycetes (JX174146.1) 100 % 31.67 d A 43.57 b A 47.70 c A C03-45 Fusarium culmorum (KC311482.1) 99 % 26.78 c C 44.36 b C 60.70 b C A1 A1 A1 C07-137 Phoma exigua (AY531684.1) 99 % 21.88 e B 10.66 d A 37.06 e A C16-83 Uncultured soil fungus (EU479850.1) 98 % 18.69 e C 16.93 d A 37.94 e A A1 Alternaria sp. (KJ935016.1) 97 % C18-40 Phoma herbarum (KJ767079.1) 99 % 21.03 e C 9.61 d A 49.47 d A A1 C19-144 Uncultured fungus (GU053874.1) 99 % 19.75 e C 14.31 d A 39.12 e A A1 Cochliobolus sp. (JQ754043.1) 98 % C21-69 Alternaria alternata (KJ410038.1) 98 % 19.54 e A 11.71 d A 37.95 e A C22-22 Fusarium oxysporum (FJ867936.1) 99 % 24.44 d C 12.23 d A 53.01 c A A1 C25-19 Diaporthe longicolla (JQ753971.1) 99 % 31.20 b A 17.4 n A 52.13 c A C27-07 Sordariomycetes (JX174146.1) 98 % 30.82 b A 33.65 c C 63.65 b A A1 C40-59 Sordariomycetes (JX174146.1) 99 % 26.56 c A 39.92 b C 58.33 b A A1 C53-134 Sordariomycetes (JX174146.1) 99 % 27.20 c C 25.55 c A 36.17 e A A1 *Means of triplicate. Means in the same column followed by different letters indicate that intervals of IP are significantly different according to the Scott- Knott test (P<0.05) **Classification by Badalyan scale (Badalyan et al. 2002): A=deadlock with mycelial contact; B=deadlock at a distance; CA1=partial replacement after initial deadlock the second sub-clade with 99 % BP, showing their C. sativus In vitro evaluation of antagonism of endophytic fungi identity. Isolates C16-83 [97 % identity with Alternaria sp. against pathogenic fungi (KJ935016.1) in BLAST analysis] and C21-69 [98 % identity with Alternaria alternata (KJ410038.1) in BLAST analysis] According to the Badalyan scale, competitive interactions be- were grouped in the third sub-clade of Alternaria sp. with tween endophytes and pathogens were defined as follws: A= 100 % BP. deadlock with mycelial contact; B=deadlock at a distance and 770 Ann Microbiol (2016) 66:765–775 A B Cl C ade I Dia D porthales Clade III Pleosporales Dothideo omycetes Sordario omycetes Clade II Hypocreales Fig. 1 Phylogenetic tree constructed with endophytic sequences from of each branch in bootstrap analyses of 10,000 repetitions. All clades Vitis labrusca L. (in black) and sequences from GenBank (indicated by comprise fungi from the phylum Ascomycota. Clade I includes the database code), using the neighbor-joining (NJ) method and the p- Sordariomycetes class and Diaporthales order. Clade II comprises the distance matrix for nucleotides, with the pairwise gap deletion option. Sordariomycetes class and Hipocreales order. Clade III contains the The numbers above and beneath each knot indicate the frequency (%) Dothideomycetes class and Pleosporales order CA1=partial growth of the antagonist after deadlock with all endophytic fungi demonstrated some antagonistic activity. mycelial contact (Fig. 2). We analyzed 68 of the endophytes In Table 1, we can see the IP value of diverse endophytes. from the Bordô cultivar. Interactions with Alternaria sp. Endophytes B55-50 associated with the Bordô cultivar were (CNPUV 674) were A (58 of endophytes), B (6) and CA1 the most efficient in the control of Alternaria sp. (CNPUV (4). The same interactions were observed regarding 674) with an IP of 58.71 %. The endophyte B45-62, identified Sphaceloma sp. (CNPUV 102), representing 62, 3 and 3 en- molecularly as Phomopsis sp., exhibited an IP of 35.29 % dophytes, respectively. Most endophytes (67) from the Bordô against this pathogen. The isolates B63-34 (Table 1; cultivar showed inhibition per mycelial contact (type A) with Fig. 4b), B25-86 and B17-49 showed IPs between 65.72 % Glomerella sp. (378 CNPUV), while one isolate presented and 63.95 % in relation to the pathogen Glomerella sp. interaction deadlock at a distance (type B). (CNPUV 378). Endophytes B46-95 (Phoma sp.) and B43- A sample of 62 endophytes from the Concord cultivar was 48 (Phomopsis sp.) presented IPs of 53.01 and 45.92 %, re- analyzed. Interactions with Alternaria sp. (CNPUV 674) were spectively, against Glomerella sp. (CNPUV 378). Isolate B25- classified as A (45 of the endophytes), B (6) and CA1 (11). 86, which was promising in the control of Glomerella sp. Type A and CA1 interactions were observed with Sphaceloma (CNPUV 378), was the best antagonist to Sphaceloma sp. sp. (CNPUV 102) (57 and 5 endophytes, respectively) and (CNPUV 102) showing an IP of 52.98 %. with Glomerella sp. (CNPUV 378) (61 and 1, respectively). Isolates C13-98, C52-63 and C11-65 from the Concord Figure 3 shows the statistical groups distribution based on cultivar displayed the best inhibition percentages against the inhibition percentage (IP) of each phytopathogen by en- Alternaria sp. (CNPUV 674), varying between 51.04 % and dophytes, i.e., the number of endophytes that belongs to a 45.93 %. The C13-98 isolate also showed the best results in specific range of IP. So, the IP of mycelial growth of the three antagonism tests against Glomerella sp. (CNPUV 378) and pathogens analyzed in relation to the 68 Bordô cultivar iso- Sphaceloma sp. (CNPUV 102) with IPs of 75.18 % and lates and 62 Concord cultivar isolates showed variations, but 57.94 %, respectively. Isolate C27-07 isolate also Ascomycota Ascomycota Ann Microbiol (2016) 66:765–775 771 Fig. 2 Competitive interactions (CI) between endophytes of V. labrusca and phytopathogenic fungi in dual culture. Badalyan rating scale (Badalyan et al. 2002): A=deadlock with mycelial contact; B=deadlock at a distance; CA1=partial replacement after initial deadlock demonstrated promising in vitro control of Glomerella sp. 2007). This was observed by Burruano et al. (2008) who ver- (CNPUV 378) with an IP of 63.65 % (Table 1; Fig. 4c). ified that Acremonium byssoides fungus—an isolated endo- Endophyte isolate C03-45 identified as Fusarium culmorum phyte of V. vinifera cv. Regina Bianca—was found regularly demonstrated an IP of 60.70 % in relation to Glomerella sp. in samples collected in the fall. In summer, with dry weather (CNPUV 378) and an IP of 44.36 % to Sphaceloma sp. and high temperatures, the physiological state of the vineyards (CNPUV 102). was affected and, consequently, the colonization levels of this fungus were reduced. In our study, the samples were collected Scanning electron microscopy during mild spring temperatures, and the frequencies of fungal colonization of the Bordô and Concord cultivars were 74 % Fungal hyphae intensely colonizing the leaf mesophyll (Fig. 5a) and 64 %, respectively. were visualized by SEM. A hypha enveloping the leaf meso- Organic cultivars probably maintain endophytic communi- phyll cell (Fig. 5b) was also observed, indicating the possibility ties better than cultivars treated with phytosanitary products. of intercellular colonization of leaves by endophytic fungi. According to Azevedo et al. (2000), the use of insecticides and fungicides to control pests and phytopathogens also eliminates important species such as endophytes. This was verified by Discussion Gonzáles and Tello (2011) for endophytic fungi isolated from grapevine cultivars (V. vinifera), where less diverse samples The composition of the endophytic community associated were obtained from a cultivar planted in an experimental farm with plants may be influenced both by the identity of the host that was subjected to phytosanitary treatments. (Arnold et al. 2007) and by environmental factors such as Pancher et al. (2012) carried out an extensive comparison temperature and annual precipitation (Arnold and Lutzoni of communities of endophytic fungi between Merlot and Fig. 3 Inhibition percentage (IP) between 130 endophytic fungi of Vitis labrusca and pathogenic fungi in dual culture. IP indicates the reduction (%) in growth of mycelia of the pathogen. *Means of triplicate compared by the Scott-Knott test (P<0.05), in which different letters indicate that IP intervals are significantly different 772 Ann Microbiol (2016) 66:765–775 AB C B 63-34 C 27-07 Glomerella sp. Glomerella sp. Glomerella sp. Fig. 4 Inhibition of pathogen Glomerella sp. (CNPUV 378) growth. a mycelia contact (type A)]: b fungal isolate B63-34 from Bordô cv. (IP= Control plate with only the pathogen. b, c Antagonism mediated by 65.72 %), c fungal isolate C27-07 from Concord cv. (IP=63.65 %) V. labrusca endophytes [both show the interaction deadlock with Chardonnay cultivars (V. vinifera) in vineyards under integrat- isolates of endophytic fungi of V. vinifera most frequently ed pest management and organic management. Their results found by Gonzáles and Tello (2011) and Musetti et al. (2006). indicated that mycota present in grapevines of organic farms Alternaria was the dominant genus in studies of grapevines form significantly different communities than those in grape- reported by Mostert et al. (2000) and Pancher et al. (2012); vine farms under integrated pest management. these authors also reported the presence of Phoma sp. In an Traditional approaches to identifying endophytic fungi in- additional study, Alternaria sp. and Fusarium sp. were report- volve the microscopic analysis of morphological characteris- ed as dominant in five sampled grapevine cultivars along with tics. However, significant portions of the isolated endophyte the less frequent Diaporthe sp., Phoma sp. and Phomopsis consist of sterile mycelium and consequently could not be iden- viticola (Casieri et al. 2009). The latter species were also re- tified by this method (Rivera-Orduña et al. 2011). Data on the ported in studies by Mostert et al. (2000) and Gonzáles and ITS region available in databases increase the chances of accu- Tello (2011). In the isolation of endophytes of V. labrusca, rately identifying taxa because of the possibility of obtaining a Brum et al. (2012) observed the presence of the general fungi taxonomically correct correspondence (Albrectsen et al. 2010). Diaporthe and Fusarium. Our genomic sequence analysis of ITS1-5.8S-ITS2 regions Fungi belonging to the genera Cochliobolus and Bipolaris verified the higher general frequency of Phoma and identified in our study were not observed previously in grape- Phomopsis fungi, as well as the presence of isolates of the vines; however, they and other genera identified in our study genera Cochliobolus, Bipolaris, Fusarium, Alternaria and are present in several other plants. In the isolation of endo- Diaporthe. Mostofthese fungi havealsobeenidentifiedin phytic fungi of Taxus globosa by Rivera-Orduña et al. (2011), previous research into endophytic microbiota in grapevines. Cochliobolus and Alternaria were among the genera most The genera Alternaria, Fusarium and Phoma were among the frequently identified. Fig. 5a,b Scanning electron A B microscopy (SEM) image of leaves of Concord cv. (V. labrusca) incubated for 3 days. a Hyphae intensely colonizing the leaf mesophyll. b Leaf mesophyll with arrow indicating fungal hyphae enveloping mesophyll cell Ann Microbiol (2016) 66:765–775 773 Isolates of Bordô and Concord cultivars identified in in a dual culture experiment, all the endophytes showed some this study were grouped into three clades belonging to degree of inhibition against phytopathogens. Among the fungi the phylum Ascomycota: Clade I belongs to the identified molecularly, highlighted are B46-95 (Phoma sp. Sordariomycetes class, Diaporthales order; Clade II also KJ572232.1) with 47.85 % inhibition of growth of the phyto- belongs to the Sordariomycetes class, Hypocreales order; pathogen Alternaria sp., C03-45 (Fusarium culmorum and Clade III belongs to the class Dothideomycetes and KC311482.1) with 44.36 % inhibition of Sphaceloma sp. Pleosporales order. This predominance of endophytic fungi and B63-34 (Sordariomycetes JX174146.1) with 65.72 % in- belonging to the phylum Ascomycota was also reported in hibition of Glomerella sp. many other recent studies (Albrectsen et al. 2010;Vega Analyzing 46 endophytic fungi isolated from Luehea et al. 2010; Rocha et al. 2011; Rhoden et al. 2012;Garcia divaricata in a dual culture experiment against the grapevine et al. 2012;Orlandelli etal. 2012; Aharwal et al. 2014). phytopathogen Alternaria alternata, Bernardi-Wenzel et al. Gonzáles and Tello (2011) obtained 91 % of ascomycetes (2013) observed that antagonism rates ranged from 3.7 % to while investigating the endophytic mycota in nine grapevine 62.7 %, and competitive interactions classified as B and CA1 cultivars (V. vinifera). Seven main orders of this phylum were observed. The same interactions were observed in our showed a similar distribution among the six cultivars most study, but antagonism rates among our endophytes and this sampled, and the Hyporeales and Pleosporales orders were phytopathogen ranged from 13.79 % to 58.71 %. the most abundant of all cultivars. In addition, analyzing the This is the first study using SEM to demonstrate the colo- fungal communities of five grapevine cultivars (V. vinifera)in nization of leaf endophytes in V. labrusca species. The tech- Switzerland, Casieri et al. (2009) showed that the vast major- nique employed was effective enough to view endophytes in ity of isolates were ascomycetes (87.5 %), including leaf tissues incubated for up to 3 days, confirming the pres- Sordariomycetes as the most represented class comprising ence of endophytic fungi in leaf mesophyll cells. Their inter- 55.1 % of isolates. Brum et al. (2012) examined the diversity cellular colonization could also be seen as fungal hyphae of endophytic fungi in leaves from the Niagara Rosada grape- clearly emerging from inside a cell. According to Stone vine (V. labrusca) and observed that 77 % of the species iden- et al. (2000), the colonization of endophytes may be intracel- tified belonged to the phylum Ascomycota. lular and limited to a single cell, intercellular and located in an Biological control is based on the beneficial interactions intra- and intercellular systemic fashion. resulting from competition, antibiotic activity and hyperpara- The results obtained in this study demonstrate the presence sitism of microorganisms against pathogens, insects and of endophytic fungi in V. labrusca as well as the biotechno- weeds (Mathre et al. 1999). It is an important alternative strat- logical potential of this organism to control grapevine patho- egy for reducing or eliminating the use of chemicals in agri- gens. Future studies should focus on developing efficient tech- niques for applying these endophytes in biological control culture (Azevedo et al. 2000). Several studies have demonstrated a reduction in the strategies against fungal diseases of grapevine. growth of pathogens resulting from interactions between en- dophytic fungi isolates of various plants and different phyto- pathogens (Živković et al. 2010; Rehman et al. 2011). Most Acknowledgments We thank the Complexo de Centrais de Apoio à studies related to the characterization of endophytic Pesquisa (COMCAP/UEM) for sequencing the ITS1-5.8S-ITS2 regions communities and applications in viticulture pathogen and for the production of electronic scanning micrographs. We thank the EMBRAPA Uva e Vinho for the phytopathogen strains. We thank the biocontrol are focused on the cause of the disease known as Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) downy mildew, caused by the fungus Plasmopara viticola. for the Master’s scholarship for the first author and CAPES/PNPD-UEM Musetti et al. (2006) confirmed the inhibition of sporulation postdoctoral scholarship. We thank CNPq (311534/2014-7; 447265/ of the pathogen by the endophyte Alternaria alternata isolated 2014-8) and Fundação Araucária – FA (276/2014) for financial support. from grapevine leaves (V. vinifera). Burruano et al. (2008)also reported the inhibition of sporulation of the P. viticola patho- gen by crude extracts of the endophyte Acremonium byssoides References isolated from grapevine leaves (V. vinifera). However, Brum et al. (2012) tested endophytic fungi iso- Albrectsen BR, Björkén L, Varad A, Hagner A, Wedin M, Karlsson J, lated from the Niagara Rosada grapevine (V. labrusca)inan Jansson S (2010) Endophytic fungi in European aspen (Populus experiment with a dual culture against the pathogenic grape- tremula)leaves—diversity, detection, and a suggested correlation with herbivory resistance. Fungal Divers 41:17–28 vine fungus Fusarium oxysporum. In this experiment, 52 % of Arnold AE (2007) Understanding the diversity of foliar endophytic fungi: endophytes were able to inhibit pathogen growth, and the progress, challenges, and frontiers. Fungal Biol Rev 21:51–66 largest halo of inhibition was from the isolates identified as Arnold AE (2008) Endophytic fungi: hidden components of tropical Colletotrichum gloeosporioides and Flavodon flavus.Inour community ecology. In: Carson WP, Schnitzer SA (eds) Tropical study, with fungi isolated from the same plant species and also Forest Community Ecology. Wiley-Blackwell, Hoboken 774 Ann Microbiol (2016) 66:765–775 Arnold AE, Lutzoni F (2007) Diversity and host range of foliar fungal Mostert L, Crous PW, Petrini O (2000) Endophytic fungi associated with shoots and leaves of Vitis vinifera, with specific reference to the endophytes: are tropical leaves biodiversity hotspots? Ecology 88: 541–549 Phomopsis viticola coplex. Sydowia 52:46–48 Arnold AE, Henk DA, Eells RL, Lutzoni F, Vilgalys R (2007) Diversity Musetti R, Vecchione A, Stringher L, Borselli S, Zulini L, Marzani C, and phylogenetic affinities of foliar fungal endophytes in loblolly Dambrosio M, Sanità Di Toppi L, Pertot I (2006) Inhibition of spor- pine inferred by culturing and environmental PCR. Mycologia 99: ulation and ultrastructural alterations of grapevine downy mildew by 185–206 the endophytic fungus Alternaria alternata. Phytopathology 96: 689–698 Azevedo JL, Maccheroni JW, Pereira JO, Araújo WL (2000) Endophytic microrganisms: a review on insect control and recent advances on Orlandelli RC, Alberto RN, Rubin Filho CJ, Pamphile JA (2012) tropical plants. Electron J Biotechnol 3:40–65 Diversity of endophytic fungal community associated with Badalyan SM, Innocenti G, Garibyan NG (2002) Antagonistic activity of Piper hispidum Sw. (Piperaceae) leaves. Genet Mol Res 11: xylotrophic mushrooms against pathogenic fungi of cereals in dual 1575–1585 culture. Phytopathol Mediterr 41:200–225 Pamphile JA, Azevedo JL (2002) Molecular characterization of endo- phytic strains of Fusarium verticillioides (Fusarium moniliforme) Bernardi-Wenzel J, Garcia A, Azevedo JL, Pamphile JA (2013) from maize (Zea mays.L). World J Microbiol Biotechnol 18:391– Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi Pamphile JA, Gai CS, Pileggi M, Rocha CLMSC, Pileggi SAV (2008a) and pathogenic bacteria. Genet Mol Res 12(4):5072–5084 Plant-microbe interactions between host and endophytes observed Brum MCP, Araújo WL, Maki CS, Azevedo JL (2012) Endophytic fungi by scanning electron microscopy (SEM). In: Sorvari S, Pirttilä AM from Vitis labrusca L. (‘Niagara Rosada’) and its potential for the (eds) Prospects and Applications for Plant-Associated Microbes. A biological control of Fusarium oxysporum. Genet Mol Res 11: Laboratory Manual, part A: Bacteria. BBI (BioBien Innovations), 4187–4197 Finland, pp 184–189 Pamphile JA, Pileggi M, Gai CS, Rocha CLMSC, Pileggi SAV (2008b) Burruano S, Alfonzo A, Lo Piccolo S, Conigliaro G, Mondello V, Torta L, Scanning electron microscopy (SEM). In: Sorvari S, Pirttilä AM Moretti M, Assante G (2008) Interaction between Acremonium (eds) Prospects and Applications for Plant-Associated Microbes. A byssoides and Plasmopara viticola in Vitis vinifera. Phytopathol Laboratory Manual, part A: Bacteria. BBI (BioBien Innovations), Mediterr 47:122–131 Finland, pp 9–13 Campanile G, Ruscelli A, Luisi N (2007) Antagonistic activity of endo- phytic fungi towards Diplodia corticola assessed by in vitro and in Pancher M, Ceol M, Corneo PE, Longa CMO, Yousaf S, Pertot I, planta tests. Eur J Plant Pathol 117:237–246 Campisano A (2012) Fungal endophytic communities in Grapevines (Vitis vinifera L.) respond to crop management. Appl Casieri L, Hofstetter V, Viret O, Gindro K (2009) Fungal communities Environ Microbiol 78:4308–4317 living in the wood of different cultivars of young Vitis vinifera plants. Phytopathol Mediterr 48:73–83 Petrini O (1991) Fungal endophyte of tree leaves. In: Andrews J, Hirano Eichhorn KW, Lorenz DH (1984) Phaenologische entwicklungsstadien SS (eds) Microbial Ecology of Leaves. Spring, New York, pp 179– der rebe. EPPO 14:295–298 197 Quiroga EN, Sampietro AR, Vattuone MA (2001) Screening antifungal FanC,PuN,WangX,WangY,FangL,Xu W,Zhang J(2008) activities of selected medicinal plants. J Ethnopharmacol 74:89–96 Agrobacterium mediated genetic transformation of grapevine (Vitis vinifera L.) with a novel stilbene synthase gene from Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous Chinese wild Vitis pseudoreticulata. Plant Cell Tissue Organ fungi. Lett Appl Microbiol 1:17–20 Cult 92:197–206 Rehman S, Mir T, Kour A, Qazi PH, Sultan P, Shawl AS (2011) In vitro Faostat—Food and Agriculture Organization of the United Nations antimicrobial studies of Nodulisporium specie: an endophytic fun- (2012) Available from: http://faostat.fao.org/site/339/default.aspx. gus. J Yeast Fungal Res 2:53–58 Accessed: 20 july 2014 Reyes Chilpa R, Quiroz Vásquez RI, Jiménez Estrada M, Navarro-Ocaña Ferreira DF (2008) SISVAR: um programa para análise e ensino de A, Cassini Hernández J (1997) Antifungal activity of selected plant estatística. Rev Científica Symp 6:36–41 secondary metabolites against Coriolus versicolor. J Trop Forest Garcia A, Rhoden SA, Rubin Filho CJ, Nakamura CV, Pamphile JA Prod 3:110–113 (2012) Diversity of foliar endophytic fungi from the medicinal plant Rhoden SA, Garcia A, Rubin Filho CJ, Azevedo JL, Pamphile JA (2012) Sapindus saponaria L. and their localization by scanning electron Phylogenetic diversity of endophytic leaf fungus isolates from the microscopy. Biol Res 45:139–148 medicinal tree Trichilia elegans (Meliaceae). Genet Mol Res 11: Gonzáles V, Tello ML (2011) The endophytic mycota associated with 2513–2522 Vitis vinifera in central Spain. Fungal Divers 47:29–42 Rivera-Orduña FN, Suarez-Sanchez RA, Flores-Bustamante ZA, Hata K, Futai K (1995) Endophytic fungi associated with healthy pine Gracida-Rodriguez JN, Flores-Cotera LB (2011) Diversity of endo- needles and needles infestes by the pine needle gall midge, phytic fungi of (Mexican yew). Fungal Divers 47:65–74 Thecodiplosis japonensis. Can J Bot 73:384–390 Rocha ACS, Garcia D, Uetanabaro APT, Carneiro RTO, Araújo IS, Kogel KH, Franken P, Huckelhoven R (2006) Endophyte or parasite— Mattos CRR, Góes-Neto A (2011) Foliar endophytic fungi from what decides? Curr Opin Plant Biol 9:358–363 Hevea brasiliensis and their antagonism on Microcyclus ulei. Lima TEF (2010) Micobiota endofitica de Vitis labrusca L. CV Isabel no Fungal Divers 47:75–84 vale do Siriji, Pernambuco, Brasil. Thesis, Universidade Federal de Rodriguez R, Redman R (2008) More than 400 million years of evolution Pernambuco and some plants still can’t make it on their own: plant stress toler- ance via fungal symbiosis. J Exp Bot 59:1109–1114 Magnani M, Fernandes T, Prete CEC, Homechim M, Ono EYS, Vilas- Boas LA, Sartori D, Furlaneto MC, Fungaro MHP (2005) Molecular Saitou N, Nei M (1987) The neighbor-joining method: a new meth- identification of Aspergillus spp. isolated from coffee beans. Sci od for reconstructing phylogenetic trees. Mol Biol Evol 4:406– Agric 62:45–49 425 Mathre DE, Cook RJ, Callan NW (1999) From discovery to use: travers- Sandhu SS, Aharwal RP, Kumar S (2014) Isolation and antibacterial ing the world of commercializing biocontrol agents for plant disease property of endophytic fungi isolated from Indian medicinal plant control. Plant Dis 83:972–983 Calotropis procera (Linn.). World J Pharm Pharm Sci 3:678–691 Ann Microbiol (2016) 66:765–775 775 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882 109:661–686 Smith D, Onions AHS (1983) The preservation and maintenance of living Vaz ABM, Mota RC, Bomfim MRQ, Vieira MLA, Zani CL, Rosa fungi. Page, Norwick CA, Rosa LH (2009) Antimicrobial activity of endophytic fun- Soejima A, Wen J (2006) Phylogenetic analysis of the grape family gi associated with Orchidaceae in Brazil. Can J Microbiol 55: (Vitaceae) based on three chloroplast markers. Am J Bot 93:278– 1381–1391 287 Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Sousa JSI (1996) Uvas para o Brasil, 2nd edn. FEALQ, Piracicaba Infante F, Castillo A, Arnold AE (2010) Fungal endophyte diversity Stone JK, Bacon CW, White JF (2000) An overview of endophytic mi- in coffee plants from Colombia, Hawai’i, Mexico and Puerto Rico. crobes: endophytism defined. In: Bacon CW, White JF (eds) Fungal Ecol 3:122–138 Microbial Endophytes. Dekker, New York, pp 3–30 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from sequencing of fungal ribosomal RNA genes for phylogenetics. In: endophytic microorganisms. J Nat Prod 67:257–268 Innis MA, Gelfland DH, Sninsky JJ, White TJ (eds) PCR Protocols. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) A Guide to Methods and Applications. Academic, New York, MEGA5: molecular evolutionary genetics analysis using maximum pp 315–322 likelihood, evolutionary distance, and maximum parsimony Živković S, Stojanović S, IvanovićŽ,Gavrilović V, Popović T, Balaž J methods.MolBiolEvol28:2731–2739 (2010) Screening of antagonistic activity of microorganisms against Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Colletotrichum acutatum and Colletotrichum gloeosporioides. Arch (1997) The ClustalX windows interface: flexible strategies for Biol Sci 62:611–623

Journal

Annals of Microbiology – Springer Journals

Published: Oct 10, 2015

There are no references for this article.