Characterization of cellulases and antagonistic activities in fungal endophytes isolated from Espeletia spp
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(2) 1. Abstract. 2. Endophytes are microorganisms that asymptomatically invade plant tissue. They can. 3. stimulate plant growth and provide resistance to pathogen attacks by the synthesis of. 4. various secondary metabolites. Many of these endophytes are unknown and may have. 5. several applications in industry, agriculture and medicine, rendering their study. 6. essential to evaluate their metabolic capabilities. In order to discern these activities, we. 7. evaluated 100 endophytes isolates from Espeletia spp., a plant specie that is unique to. 8. the paramo ecosystem in the Andean mountain range. We determined their in vitro. 9. antimicrobial activity using the dual confrontation technique and culture filtrates of. 10. these isolates with the plant pathogen Phytophthora infestans. In addition, we evaluated. 11. the cellulolytic potential of these endophytes in saccharification of oil palm empty fruit. 12. bunch (OPEFB). This was determined by measuring the activity of total activity in filter. 13. paper (FPA), carboxymetilcelulase, exogluconases and β-glucosidase. Results showed. 14. that few (6%) of the endophytes showed a significant anti-oomycete activity against P.. 15. infestans. Among the isolates tested, Penicillium glabrum showed maximum in vitro. 16. inhibition of mycelia growth of P. infestans. In this study we showed for the first time. 17. the role of this endophyte in controlling P. infestans. Furthermore we determined four. 18. fungi that were positive for cellulases. The highest cellulolytic activity after partial. 19. purification was shown by Penicilium globrum, with maximum enzyme activity,. 20. CMCase, FPase, exogluconases and β-glucosidase of 44,5, 1,2, 48,3 and 0,45 U/ml. 21. respectively. Our data shows a highly metabolic activity of Penicillium glabrum and. 22. suggest that this fungus is a potential antagonistic of P. infestans and source for biofuel. 23. production.. 24. Keywords: Endophytes, antimicrobial activity, Phytophthora infestans, cellulases,. 25. OPEFB, Penicillium 2.
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(4) 1. Introduction. 2. Endophytes are microorganisms that live inside the tissues of living plants without. 3. causing apparent harm to their host (Schulz, Römmert et al. 1999; Tian, Cao et al. 2004;. 4. Kogel, Franken et al. 2006; Schulz and Boyle 2006; Naik, Shashikala et al. 2009;. 5. Rodriguez, White Jr et al. 2009; Lahlali and Hijri 2010; Zhao, Penttinen et al. 2011).. 6. Endophytic fungi within plants have been known to produce plant-growth regulatory,. 7. antimicrobial, antiviral or insecticidal substances to enhance the growth and. 8. competitiveness of the host in nature (Park, Park et al. 2003; Kim, Choi et al. 2007;. 9. Verma, Gond et al. 2009). They have been recognized as a repository of novel. 10. metabolites of agricultural, pharmaceutical and industrial importance. Thus, endophytes. 11. are expected to be potential sources of new bioactive molecules and to be useful as. 12. agents of biotechnological processes (Schulz, Boyle et al. 2002; Tian, Cao et al. 2004;. 13. Yu, Zhang et al. 2010; Zhao, Penttinen et al. 2011). These features render endophytes a. 14. bioprospection objective that through the use of biotechnological tools systematically. 15. search for genes, compounds, designs and organisms that may lead to the development. 16. of products with economic potential (Strobel and Daisy 2003; van den Burg 2003;. 17. Suryanarayanan, Thirunavukkarasu et al. 2009).. 18. Enzyme complexes produced by endophytes have been the target of multiple. 19. investigations, mainly those complexes that can provide a useful and economically. 20. feasible energy source (Karboune, Geraert et al. 2008). This is the case of cellulases. 21. which allow the bioconversion of biomass to biofuels and mitigate dependence on. 22. depleting fossil oil. Lignocellulosic biomass is the most abundant renewable source of. 23. sugars that can be fermented to biofuels like etanol (Sun and Cheng 2002; Jiang, Geng. 24. et al. 2011). Bioconversion of lignocellulolytic compounds to fuel requires biomass. 25. transformation into fermentable sugars by enzymatic hydrolysis and this requires a 4.
(5) 1. specific coktail of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-. 2. glucosidase) (Cianchetta, Galletti et al. 2010; de Castro, de Albuquerque de Carvalho et. 3. al. 2010). Cellulase production is a very expensive process and increases the costs of. 4. ethanol production from cellulosic biomass, in that it accounts for approximately 40%. 5. of the total cost. A significant cost reduction is required in order to enhance the. 6. commercial viability of cellulase production technology. For that reason it is important. 7. to search microorganisms with a high rate of cellulase production and use a cheap. 8. lignocellulosic material (e.g. agricultural waste) in order to reduce the costs of cellulase. 9. production (Zhang, Himmel et al. 2006; Gao, Weng et al. 2008). Colombia has. 10. developed a cultivated important area dedicated to oil palm. This industry produces. 11. large amounts of biomass, more than half of the processed product is oil palm empty. 12. fruit bunch (OPEFB) (Fedepalma 2007). This large volume of agricultural waste has not. 13. been effectively used for its lignocellulosic materials and appears to be a viable. 14. alternative as a cheap source of substrate for ethanol production (Umikalsom, Ariff et. 15. al. 1997; Umikalsom, Ariff et al. 1997; Umikalsom, Ariff et al. 1998).. 16. Another application of cellulases is their use for biocontrol of plant diseases (Picard,. 17. Tirilly et al. 2000) caused by Oomycetes like Phytophthora infestans with a high. 18. content of cellulose in their cell walls. P. infestans is a foliar pathogen that causes. 19. serious losses of potato and tomato crops worlwide. The disease caused by P. infestans. 20. is difficult to control by chemical means beacuse of the high virulence of the pathogen. 21. and increasing resistance to fungicides (Fry 2008). As mentioned, the cell wall of P.. 22. infestans is composed primarily of cellulose so this could be a target for endophytic. 23. fungi that produce cellulolytic enzymes contributing to the biological control as an. 24. alternative to synthetic fungicides for protection of the plants for economic importance. 25. against late blight (Attard, Gourgues et al. 2008; Grenville-Briggs, Anderson et al. 5.
(6) 1. 2008; Richter, Ivors et al. 2011). Therefore the objective of this work was to find fungal. 2. endophytes that produce active cellulases that can be used in the process of. 3. saccharification of oil palm empty fruit bunch (OPEFB) and antagonistic activity. 4. against P. infestans.. 5. 6. Materials and Methods. 7 8. Fungal collection and Taxonomic identification. 9. Fungi were selected from the fungal collection of the Mycology and Plant Pathology. 10. Laboratory at the Universidad de los Andes. In a previous study, Avila et al., (in. 11. preparation), isolated 609 endophytes from Espeletia sp. plants in the Paramo Cruz. 12. Verde, Choachi Cundinamarca. From this collection, 100 fungi were selected and. 13. activated in potato dextrose agar (PDA) for 4 weeks. Morphological characterizations. 14. were performed with microscopic observation of preparations in water, lactophenol blue. 15. and lactophenol. Identifications were made following the traditional taxonomy keys. 16. (Barnett 1960 , Domsch 1980 and Hanlin 1998), and by macroscopic descriptions of the. 17. colonies.. 18 19. Antimicrobial assays. 20. Endophytes were preliminary screened for their ability to suppress the mycelial growth. 21. of Phytophthora infestans strain P8012 by in vitro dual culture assays on potato. 22. dextrose agar (PDA). The pathogen was cultivated one week earlier that endophytes.. 23. Strain P8012 was isolated from a tree tomato (Solanum betaceum) field near Colon,. 24. Putumayo.. 6.
(7) 1. Fungal isolates were screened for their ability to inhibit the mycelial growth of P.. 2. infestans by in vitro dual culture assays on potato dextrose agar (PDA). Each. 3. combination was replicated five times and plates were placed in darkness and incubated. 4. at 19ºC until the plate was covered with pathogen mycelia. Every replicate contained a. 5. control with P. infestans growing alone. The mycelial growth inhibitions were. 6. calculated with the formula :. 7. Equation 1 (Eq.-1). 8. (Rc-Ri)/Rc*100. (1). 9. (Lahlali and Hijri 2010) where Ri is the radial mycelial growth of P. infestans towards. 10. the antagonistic fungus and Rc is the growth of P. infestans in the control plate.. 11. Statistical analyses were performed with ANOVA using R 2.9.2 software (http://R-. 12. project.org).The antagonistic effects were compared with Tukey’s HSD Test (P < 0.05).. 13 14. Microscopic analysis of antagonistic effect on P. infestans. 15. Fungal endophytes that showed inhibition of P. infestans growth were prepared for. 16. inverted phase contrast microscopy using PDA agar plugs containing mycelia of both. 17. strains. Thin sections were made in the area of interaction between the endophyte and. 18. the pathogen and mounted on slides for direct microscopic observation to 40x. Three. 19. replicates for each combination pathogen/endophyte were examined.. 20 21. Growth of P. infestans using antagonistic culture filtrates. 22. We used the protocol described by (Campanile, Ruscelli et al. 2007) for the evaluation. 23. of inhibition of P. infestans with culture filtrates. Radial growth was recorded by. 24. measuring mean colony diameter at 1-day intervals for the time required to reach the. 25. margin of the dish in controls. The mycelial growth inhibitions were calculated with the. 7.
(8) 1. formula (Rc-Ri)/Rc*100 (1) (Lahlali and Hijri 2010). Significance of the main effects and. 2. interaction effects of the antagonistic fungi was determined by ANOVA and Tukey’s. 3. HSD Test (R 2.9.2 software).. 4 5. Enzymatic assay. 6. In a first step, the cellulolytic capacity of fungi was screened on solid agar with CMC as. 7. the substrate and using congo red as an indicator. The selected fungi were grown on. 8. modified Fries medium with the following composition: 1% w/v NH4NO2, 1% w/v,. 9. K2HPO4, 0.5% w/v, MgSO4, 0.1% w/v, NaCl, 0.13% w/v, CaCl2, 0.01% w/v, MnSO4,. 10. 0.01% w/v, boric acid, 0.001% w/v, CuSO4, 0.2% w/v, FeSO4 and 0.001% w/v, ZnSO4,. 11. 10 g/L oil palm empty fruit bunch (OPEFB), 5 g / L glucose and 6 g/L peptone. Three. 12. plugs of the fungus were added on 30 mL of the modified Fries’ basal medium (100 ml. 13. Erlenmeyer flask) and incubated at room temperature with agitation (150 rpm) for 20. 14. days. Each experiment was carried out in duplicate. Two ml samples were withdrawn at. 15. regular time intervals (3 times a week), centrifuged at 13000 rpm at 4 ºC for 20 min and. 16. the supernatant was filtered using a Whatman Nº 1 filter paper. The filtered raw enzyme. 17. extracts was used to determine the total activity in filter paper (FPA) and for other. 18. enzymatic assays.. 19. For the endophytes showing cellulolytic activity, we measured the activity of the three. 20. main components of cellulase (CMCase, β-glucosidase and Cellobiohydrolase), and total. 21. cellulolytic activity (Filter paper activity FPA) using the procedures described by (Ghose. 22. 1987) , (Miller 1959) and (Zhang, Himmel et al. 2006). The protein content was. 23. measured with the Lowry method, using bovine serum albumin as a standard (Waterborg. 24. and Matthews 1996), and also by measuring the absorbance at 280 nm.. 25. 8.
(9) 1. Partial purification of cellulases. 2. In order to carry out the celullase purification process we selected the fungal extract that. 3. showed the highest enzyme activity.. 4. Raw extract clarification: the raw enzyme extract was precipitated with ammonium. 5. sulfate (90%). The preparation was kept overnight at 4°C to allow precipitated protein to. 6. sediment and then recovered by centrifugation at 10000 rpm for 30 min at 4 ºC and. 7. dissolved in buffer A (two times the volume of the precipitate).. 8. Desalting: The enzyme solution was desalted using dialysis. The proteins obtained in the. 9. desalinated sample were quantified by Folin-Lowry methods. Total cellulolytic activity. 10. (FPA) was quantified.. 11. Sample purification: Desalted enzyme solution was placed on a DAE-shepadex A-50. 12. column previously equilibrated with buffer A. The fractions were withdrawn with a. 13. linear NaCl gradient and analyzed for protein concentration by measuring the. 14. absorbance at 280 nm using a Spectrophotometer Nanodrop® ND-1000. Total. 15. cellulolytic activity (FPA) and proteins of the fractions were also measured.. 16. Fraction concentration: the fractions with cellulolytic activity were pooled and then. 17. concentrated by ultrafiltration with a centriprep (Amicon) having a 10 kDa molecular. 18. mass cut-off.. 19. Enzyme storage: The concentrated enzyme was dried in a freeze-dryer and then the. 20. solution was dissolved in buffer A.. 21. Concentrated samples were analyzed to quantify proteins by the Folin-Lowry and. 22. cellulolytic activity.. 23 24 25. 9.
(10) 1. Determination of molecular weight. 2. Sodium dodecyl sulfate–polyacylamide gel electrophoresis (SDS–PAGE) was used to. 3. determine the molecular weight of the enzymes under denaturing conditions, as. 4. described by Laemmli (Laemmli 1970) using BIORAD (161-0318) as weight marker.. 5 6. Molecular identification. 7. Fungal strains that showed a significant antagonistic or cellulolytic activity were. 8. identified by sequence analysis. For these strains total DNA was obtained by extraction. 9. using protocol described by (Gardes and Bruns 1993) and then these samples were. 10. amplified by polymerase chain reaction (PCR) using ITS5 and ITS4 primers (White,. 11. Bruns et al. 1990). Amplification reactions were performed in a final volume of 25µL. 12. reaction mix at 1X Green GoTaq® Flexi buffer, 2.5mM MgCl2, 10mM each dNTP,. 13. 0.2µM forward and reverse primers, 1.25U GoTaq® DNA Polymerase (Promega,. 14. Madison, WI) and 1µL of total DNA. PCR products were sequenced on an ABI Prism. 15. 3730XL (Applied Biosystems, Carlsbad, CA) automated sequencer. Sequence analyses. 16. were performed using the GenBank BLAST online program (Blastn software using. 17. Blosum62 matrix, e-value near 0 and similarity percentage higher to 90%.).. 18. http://www.ncbi.nlm.nih.gov/BLAST (Altschul, Gish et al. 1990). 19. 20. Results. 21. Morphological and Molecular characterization of the isolates. 22. Most fungal isolates were identified using traditional taxonomy keys and macroscopic. 23. observations. We found 24 sterile mycelia and a large diversity of fungal endophytes.. 24. Fungal endophytes showing significant inhibition of Phytophthora infestans or. 10.
(11) 1. cellulolytic activities were identified using the ITS sequence. All endophytes showed 98-. 2. 100% sequence identity with the species determined by morphology (Supplementary. 3. tables 1 and 2).. 4 5. Antagonistic activity against P. infestans. 6. The dual cultures between Phytophthora infestans and each of the isolates showed. 7. different inhibitions rates (Table 1). Penicillium glabrum had the maximum inhibitory. 8. effect on mycelial growth of P. infestans, with a reduction of 67,9% compared to. 9. control, followed by Trichoderma sp, Aspergillus niger with a reduction of pathogen. 10. growth of 54,4 and 39,9% respectively. These strains showed fast growth and covered. 11. the plate completely including the mycelium of the pathogen. Epiccocum nigrum did not. 12. show the highest rates of growth inhibition (24,3%) of P. infestans but was the only. 13. isolate that showed an inhibition halo (Supplementary figure 1).. 14. Microscopic characterization showed different patterns of interaction between the. 15. endophytes and P. infestans (Figure 1). Penicillium glabrum hyphae and conidia. 16. completely covered P. infestans hyphae and caused reduced growth and sporulation. 17. (Fig. 1a). Trichoderma sp. hyphae established close contact with P. infestans and their. 18. conidia invade the pathogen hyphae (Fig 1b). The hyphal density of A. niger, was higher. 19. than P. infestans, but no evidence of any hyphal penetration was observed (Fig 1c).. 20. Epicoccum nigrum grew alongside of P. infestans hyphae and did not show any evidence. 21. of penetration, although inhibition zones were observed and Epicoccum hyphae invade. 22. and surround the pathogen mycelia (Fig 1d). The pathogen mycelium was easily. 23. distinguished from the antagonistic by its hyphal morphology (Fig 1e).. 24. The greatest reduction of P. infestans growth was observed with Penicillium glabrum. 25. and Epicoccum nigrum. culture filtrates with a reduction of growth of 73 and 70,2%. 11.
(12) 1. respectively. Culture filtrates of Trichoderma sp., Chaetomium sp and Aspergillus niger. 2. reduced the growth of P. infestans by 28-23% (Table 1). Statistical analysis showed that. 3. growth of P. infestans was significantly reduced by all endophytes tested except. 4. Beauveria sp., and Fusarium oxysporum.. 5 6. Cellulolytic activity of the isolates. 7. Cellulolytic evaluation with Congo red showed 28 positive fungal endophytes, which. 8. were evaluated for total cellulolytic activity on filter paper (FPA). Of these isolates, 17. 9. endophytes showed an FPA greater than 0.05 U/ml (Supplementary table 3), and four. 10. were selected for quantification of the different cellulolytic activities (B-glucosidase,. 11. endoglucanase and cellobiohydrolase). These profiles for Aspergillus sp 2796,. 12. Penicillium glabrum 2399, Strain 2830 (Sterile mycelium) and Chaetomium sp 2779,. 13. showed an increased cellulolytic activity over time, but there were no clear trends for the. 14. four endophytes (Fig. 2). Aspergillus sp. showed maximum activity the first few days,. 15. and then a decrease in the last days (Fig. 2d). Penicillium glabrum showed the highest. 16. cellulolytic activity, attaining a maximum CMCase activity of 0.41, 0.035 U/ml for FPA,. 17. 0.06 U/ml for β-glucosidase and 0.068 U/ml for exoglucanases (Fig. 2c). Surprisingly,. 18. this isolate showed mycelial growth from the third day, the samples reported total. 19. cellulolytic activity only from the eighth day with a very low value. However, after ten. 20. days this endophyte had the highest total activity in filter paper (FPA) and cellulolytic. 21. complex.. 22. A partial purification of the cellulolytic enzyme complex from Penicillium. 23. glabrum.2399 was performed using ammonium sulfate precipitation and desalting.. 24. Fourteen-day-old cultures showed the highest total and specific cellulolytic activity.. 25. Fractions determined in each process were then grouped according to experimental plots. 12.
(13) 1. obtained (Supplementary fig. 2). Each purification step showed a decrease in total. 2. protein and total activity. The greatest loss occurred in protein precipitation with. 3. ammonium sulfate, compared to the loss in the following steps on DEAE-sephadex. 4. column and fraction concentration (Table 2). However the total activity and cellulolytic. 5. complex increased in each purification step. Carboxymethylcellulose activity had a. 6. significant increase compared to the other two enzymes of 0,35 U / ml to 44.5 U / ml in. 7. the fraction concentration (Table 3).. 8. Molecular weight of the purified enzyme showed that the extract (B lane, Supplementary. 9. Fig. 3) was highly diluted, and thus the molecular weight of the enzyme could not be. 10. determined. The dialysis product (C lane), sample column (D lane) and the purified. 11. extract (E lane) revealed a high concentration of proteins with a weight range from 30 to. 12. 100 kDa (Supplementary figure 3). This high concentration and weight of the purified. 13. protein sample may indicate the presence of the three enzymes of the cellulolytic. 14. complex.. 15 16. Discussion. 17. Endophytic fungi are ubiquitous in higher plants and produce a vast repertoire of natural. 18. bioactive compounds that constitutes a reservoir for bioprospection (Verma, Gond et al.. 19. 2009). Our results support these findings by showing that some endophytic fungi. 20. isolated from Espeletia sp, had antagonistic activity against P. infestans and or showed. 21. cellulolytic potential for saccharification of oil palm empty fruit bunch (OPEFB) as a. 22. source of second generation biofuels.. 23. However, although very few (6%) of the endophytes showed a significant antimicrobial. 24. activity against P. infestans, we showed that Penicillium glabrum and Trichoderma sp.. 25. were the best antagonists. Few authors report the genus Penicilliun for its antagonistic. 13.
(14) 1. activity, with reports focusing mainly on its celullolytic activity and its role as a. 2. pathogen (de Castro, de Albuquerque de Carvalho et al. 2010). Naik et al. (2009) and. 3. Kim et al. (2007) reported that endophytic Penicillium species have antagonistic activity. 4. against different pathogens of plants, such as Rhizoctonia solani, which might be due to. 5. the production of biologically active compounds. On the other hand, Trichoderma has. 6. been widely used in biological control studies against Rhizoctonia solani (Lahlali and. 7. Hijri 2010), (Anees, Tronsmo et al. 2010) and Diplodia corticola (Campanile, Ruscelli. 8. et al. 2007), among others.. 9. The microscopic characterization of the antagonism trials and the growth of P. infestans. 10. with antagonistic culture filtrates suggest that these endophytes inhibit the pathogen. 11. growth mostly by competition and not by production of metabolites or degradation of its. 12. cell wall. However, the inhibitory effect of Penicillium glabrum suggests the production. 13. of cellulolytic enzymes or metabolites that limit the pathogen development. This. 14. efficacy has never been evaluated against P. infestans and is the first investigation. 15. showing the role of this fungus controlling P. infestans. In contrast, Epicoccum nigrum.. 16. was the only fungus that showed an inhibition area, which could indicate the production. 17. of secondary metabolites, as reported by Lahlali (2010) for Epicoccum nigrum against. 18. Rizoctonia solani. These antimicrobial compounds produced by fungal endophytes have. 19. been widely reported in previous studies ((Tian, Cao et al. 2004; Campanile, Ruscelli et. 20. al. 2007; Kim, Choi et al. 2007; Naik, Shashikala et al. 2009; Lahlali and Hijri 2010).. 21. The bioprospection potential of Espeletia spp. endophytes was also evidenced by the. 22. celulolytic activity observed in seventeen of the isolates tested, some of them significant.. 23. Penicillium glabrum, showed the highest cellulolytic activity, consistent with previous. 24. literature reports (Sukumaran, Singhania et al. 2005), (Karboune, Geraert et al. 2008),. 25. (de Castro, de Albuquerque de Carvalho et al. 2010). However, compared to these. 14.
(15) 1. reports, the FPA, exoglucanase and β-glucosidase activities determined for the isolate in. 2. this study were low. It has been proposed that the reason for the low secretion of β-. 3. glucosidase is that the major part of this enzyme is tightly bound to the cell walls of the. 4. fungus during culture and some parts of the enzyme may be found inside the cells (Jiang,. 5. Geng et al. 2011). Another reason for the low activity of cellulases is that the. 6. cellobiohydrolase do not act synergistically with endoglucanase to hydrolyse crystalline. 7. cellulose and produce cellobiose that β- glucosidase convert into fermentable sugars. 8. (Umikalsom, Ariff et al. 1997; Karboune, Geraert et al. 2008). Therefore, one way to. 9. alleviate the effect of cellobiose inhibition on the overall hydrolysis is to supplement. 10. with exogenous β- glucosidase. In addition it is recommend optimization of the medium. 11. with the complementation of cellulase inducers and may be using other modes of. 12. fermentation (Barros, Oliveira et al. 2010; Cianchetta, Galletti et al. 2010; Jiang, Geng et. 13. al. 2011). Our results suggest that strain complementation in combination with medium. 14. optimization may greatly enhance the cellulolytic enzyme yield and its biomass. 15. hydrolysis capacity. Finally we found that the molecular weights obtained are in a range. 16. between 60 – 100 kDa, which according to previous reports is within the molecular. 17. weight of endoglucanase, cellobiohydrolase and β-glucosidase reported with 83-94 kDa,. 18. 66 kDa and 85 kDa respectively (Zeikus 1981; Wei, Kirimura et al. 1996; Zhang, Liu et. 19. al. 1998).. 20. In this study we showed that a some fungal endophytes tested were effective for. 21. controlling P. infestans and produce fermentable sugars. Penicillium glabrum 2399, was. 22. the best isolate, showing significant antagonistic and cellulolytic activity. Screening. 23. these fungal resources for novel metabolites and enzymes and their application are major. 24. goals. 25. development.. of. current. research. to. accomplish. environment-friendly. technological. 15.
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(24) 1. Table 1. Antagonistic effect of fungal endophytes on the mycelial growth of. 2. Phytophthora infestans. Growth (% inhibition growth over control) Fungal Endophytes. Dual culture*. Culture filtrate**. Penicillium glabrum. 2399. 67,96 ± 8,77A. 73,06±4,39A. Trichoderma sp. 2807. 54,4±12,38AB. 28,61±1,84B. T. asperellum 2721. 39,07±11,07BC. 24,67±7,76B. Aspergillus niger 2783. 39,94±15,29BC. 23,31±3,96B. Epicoccum nigrum. 2368. 24,30±5,81CD. 70,21±4,33A. Fusarium oxysporum 2763. 27,5±7,57CD. 14,42±8.81BC. Chaetomium sp.2779. 20,7±6,36CD. 23,47±2,46B. Beauveria sp. 2820. 7,49±6,51DE. 4,14±3,05CD. 0,0±0E. 0,0±0D. Control ∆ 3 4. *Mean of 5 replicates, values having the same letter are not statiscally different (P<. 5. 0,05), according to the Tukey test.. 6. ∆ Growth of P. infestans in the absence of endophyte.. 7. ** Mean of three replicates, values having the same letter are not statiscally different. 8. (P< 0,05), according to the Tukey test.. 9. ± Standard deviation 24.
(25) 1 2. Table 2. Partial Protein purification and quantification using the Folin-Lowry method. 3. for Penicillium glabrum. Total. Total. Specific. Volume FPA. Yield Protein. activity. activity. (ml). Raw extract Ammonium sulfate. (%) (mg). (U). (U/mg). 980. 1852,544. 39,246. 0,021. 100. 24. 148,874. 3,993. 0,026. 10,174. 10. 85,398. 2,996. 0,035. 7,636. 1,5. 40,947. 1,808. 0,044. 4,608. precipitation (80%) DEAE-sephadex A-50 Fraction concentration 4. 5. 25.
(26) 1. 2. Table 3. Total cellulolytic activity in filter paper (FPA), carboxymetilcelulose, β-. 3. glucosidase and exoglucanase activities for the extract and the purified enzyme. Activity (U/ml). Total activity (U). Fraction Extract. Fraction Extract. concentration. concentration. FPA. 0,040. 1,205. 39,246. 1,808. CMC. 0,356. 44,520. 349,288. 66,780. β-Glucosidase. 0,090. 0,451. 89,044. 0,677. Exoglucanase. 1,1275. 48,358. 1104,98. 72,537. 4. 5. 26.
(27) 1. 2. Figure 1. Inverted microscopical observations of the interaction zones between the. 3. antagonistic fungal isolates and Phytophthora infestans taken during dual culture. 4. assays. a. P. infestans hyphae covered by mycelium and spores of Penicillium glabrum.. 5. b. Interaction between Trichoderma and P. infestans. c. hyphae of P. infestans. 6. surrounded by Aspergillus niger. d. Growth of P. infestans in presence of Epicoccum. 7. nigrum. e. P. infestans mycelia growth without antagonistic.. 8. Figure 2. Cellulase production by fungal endophytes. Total cellulolytic activity on. 9. filter paper (FPA), β-glucosidase activity, Carboximetylcelulase activity (CMCase) and. 10. exoglucanase activity in the time. A. Chaetomium sp. B. Strain 2830 C. Penicillium. 11. glabrum. D. Aspergillus sp.. 12. 27.
(28) 1 2. 3. a.. 4. c.. b.. d.. e.. 5. 6. 7. Figure 1. 8. 28.
(29) 1. FPA (U/ml). B-glucosidase (U/ml). 0,045. 0,9. FPA. 0,04. 0,8. CMC. 0,035. 0,7. beta. 0,03. 0,6. exo. 0,025. 0,5. 0,02. 0,4. 0,015. 0,3. 0,01. 0,2. 0,005. 0,1. 0. 0 0. 5. 10. 20. Time (days). 3. B.. 0,035. 0,9. FPA (U/ml). B-glucosidase (U/ml). FPA 0,03. BETA. 0,025. CMC. 0,8 0,7 0,6. exo 0,02. 0,5. 0,015. 0,4 0,3. 0,01. 0,2 0,005. 0,1. 0. 0 0. 5. Exogluconase (U/ml). 4. 15. CMCase (U/ml) Exogluconase (U/ml). A.. CMC (U/ml). 2. 5. 10 Time (days). 15. 6. 7. 8. 29.
(30) 1. C.. B-glucosidase (U/ml) FPA (U/ml). BETA. FPA. CMC. exo. 1,8. 0,07 0,06. 1,6 1,4. 0,05. 1,2. 0,04. 1. 0,03. 0,8 0,6. 0,02 0,4 0,01. 0,2 0. 0 0. 5. 3. 10 Time (days). 15. 20. D.. 0,035. BETA. 0,03. FPA. 1,2 1. CMC. 0,025. 0,8. exo. 0,02 0,6. FPA (U/ml). 0,015 0,4. 0,01. 0,2. 0,005 0. 0 0. 5. 10. 15. 20. Time (days). 5 6. Exogluconase (U/ml). B-glucosidase (U/ml). 4. CMCase (U/ml) Exogluconoase (U/ml). 2. 0,08. CMCase (U/ml). 2. Figure 2. 7. 30.
(31) 1. 2. Supplementary Material. 3. Supplementary table 1. Molecular identification of fungal endophytes with. 4. antagonistic or cellulolytic activity using ITS5 and ITS4 primer.. Isolate number 2368 2399 2721 2763 2779 2783 2796 2807 P8012. Most closely related specie Epicoccum nigrum Penicilium glabrum Trichoderma asperellum Fusarium oxysporum Chaetomium sp Aspergillus niger Aspergillus sp. Trichoderma sp Phytophthora infestans. Accession number EU715663 HQ891544.1 EU272525. GU566301.1 EU035804.1 HQ891666.1 * HM124450.1 EU200294.1. Identity % 99.4% 98% 100% 98% 98% 98% * 99% 98%. 5. 6. 31.
(32) 1. 2. Supplementary table 2. Morphological identification of fungal endophytes. Strain. 3. collection code of the Laboratory of Mycology and Plant Pathology (LAMFU) Strain collection code (LAMFU) 2796 2347 2783 2367 2373 2359a 2350 2679 2353 2761 2767 2770 2804 2819 2820 2842 2844 2849 2850 2773 2356 2365 2779 2358 2362 2824 2359c 2715 2843 2716 2754 2755 2366 2834 2364 2764 2368. Endophyte isolate Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus sp Aspergillus sp Aspergillus sp Aureobasidium Aureobasidium pullulans Aureobasidium sp Beauveria bassiana Beauveria sp Beauveria sp Beauveria sp Beauveria sp Beauveria sp Botrytis sp Botrytis spp Botrytis spp Botrytis spp Chaetomium globosum Chaetomium sp Chaetomium sp Chaetomium sp Cladosporium sp Cladosporium sp Cladosporium sp Cladosporium sp Curvularia oryzae Deschlera sp. Diaporthe phaseolorum Diaporthe phaseolorum Diaporthe phaseolorum Dreschlera sp Dreschlera spp Eladia sacculum Epicoccum nigrum Epicoccum sp. 32.
(33) 2757 2769 2763 2751 2752 2766 2768 2771 2823 2826 2825 2821 2361 2778 2781 2797 2808 2809 2810 2839 2840 2846 2847 2393 2723 2822 2360 2396 2397 2398 2399 2776 2848 2756 2758 2811 2799 2800 2801 2802 2803 2812 2813 2814 2815 2816. Eutypella sp Eutypella sp Fusarium oxysporum Fusarium proliferatum Fusarium proliferatum Fusarium proliferatum Fusarium proliferatum Fusarium proliferatum Fusarium sp Fusarium sp Fusarium sp. Micelio esteril Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Nigrospora sp Paecilomyces Paecilomyces sinensis Paecilomyces sp Penicilium sp Penicilium sp Penicilium sp Penicilium sp Penicilium sp Pestalotiopsis sp Picnidio Scopulariopsis spp Scopulariopsis spp Scopulariopsis spp Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium 33.
(34) 2817 2818 2827 2828 2829 2830 2832 2833 2835 2836 2837 2838 2845 2721 2719 2788 2807. Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Sterile mycelium Trichoderma asperellum Trichoderma atroviride Trichoderma sp Trichoderma sp. 1 2 3. 34.
(35) 1. 2. Supplementary table 3. Total cellulolytic activity on filter paper (FPA) : Cellulase. 3. positive endophytes evaluated 7 and 14 days of growth in inducing medium.. 4. Strain. FPA day 7/ U/ml. FPA day 14/ U/ml. Sterile mycelium 2830. 0. 0,08408. 6. Diaphorthe phaseolorum 2716. 0. 0,01075. 7. Penicilium glabrum 2399. 0,01088. 0,09143. 8. Eladia sacculum 2364. 0,03457. 0. 9. Sterile mycelium 2815. 0,04883. 0. 0,01186. 0. Aspergillus sp 2796. 0,05817. 0,04278. Deschlera spp 2843. 0,03431. 0. Nigrospora sp 2846. 0,03472. 0,00624. Cladosporium spp 2358. 0,06847. 0,0181. Aspergillus niger 2783. 0,05328. 0,00566. Chaetomium sp 2779. 0,07343. 0,02051. Fusarium sp 2826. 0. 0,04347. 17. Penicillium sp 2396. 0,02673. 0,04793. 18. Fusarium proliferatum 2768. 0. 0,03045. 19. Fusarium proliferatum 2752. 0,01657. 0. 5. 10 11 12. Strain 2825. 13 14 15 16. 20 21. 35.
(36) 1 2. Supplementary figure 1. Antagonistic activity: dual culture between endophytic fungi. 3. and the pathogen Phytophthora infestans, a. Chaetomium sp., b. Fusarium oxysporum,. 4. c. Epicoccum nigrum, d. Trichoderma sp., e. Penicillium glabrum., f. Aspergillus niger.,. 5. g. Beauveria sp., h. Phytophthora infestans P8012.. 6. Supplementary figure 2. Cellulolytic activity and protein quantification in Sephadex. 7. column and Absorbance and total cellulolytic activity on filter paper (FPA) of. 8. Penicillium glabrum.. 9. Supplementary figure 3. Molecular weight determination of cellulolytic enzymes of. 10. Penicillium glabrum. Polyacrylamide gel electrophoresis (10%): (A) Molecular weight. 11. marker, (B) Extract, (C) Dialysis product, (D) Sample column, (E) Purified extract. 12. 36.
(37) 1. a.. b.. 3. c.. d.. 4. e.. f.. 5. g.. h.. 2. 6. .. Supplementary figure 1.. 37.
(38) 1. Absorbance FPA. 12. 0,4 0,35 0,3. 8. 0,25. 6. 0,2 0,15. 4. 0,1 2. 0,05. 0. 0 0. 10. 20. 30. 40. 50. 60. Fraction no.. 2 3. FPA (U/ml). Absorbance 280 nm. 10. Supplementary figure 2.. 4. 38.
(39) 1 2. Supplementary figure 3.. 3 4 5. 6. 39.
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