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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.46 no.2 São Paulo Apr./June 2015

http://dx.doi.org/10.1590/S1517-838246220131042 

Environmental Microbiology

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Ravely Casarotti Orlandelli1 

Tiago Tognolli de Almeida1 

Raiani Nascimento Alberto1 

Julio Cesar Polonio1 

João Lúcio Azevedo1 

João Alencar Pamphile1 

1Laboratório de Biotecnologia Microbiana, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, PR, Brazil.

ABSTRACT

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Key words: antagonism; competitive interaction; dual culture; cup plate; protease

Introduction

Biological control through microorganisms that inhibit or antagonize plant pathogens and pests reduces or eliminates the use of chemical products. Fungal endophytes are effective antagonists (Azevedo et al., 2000) and constitute a taxonomically and metabolically diverse group of organisms that colonize internal plant tissues without causing apparent harm to the host plant (Wilson et al., 1991). Indeed, endophyte-mediated biological control has been investigated both in vivo and in vitro through screening experiments to verify the activity of endophytes against phytopathogens and pests (Andreote et al., 2009; Badalyan et al., 2002; Campanile et al., 2007; Flores et al., 2013; Mejía et al., 2008; Rocha et al., 2009; Rubini et al., 2005; Sánchez et al., 2007; Specian et al., 2012).

Endophytic and phytopathogenic fungi compete and interact within the same ecological niche through the action of hydrolytic enzymes such as proteases and chitinases, which degrade the hyphal cell walls of pathogenic microorganisms (Almeida et al., 2007; Guthrie and Castle, 2006; Sánchez et al., 2007). This enzymatic activity is closely associated with the fungus-host specificity: the fungal strains of a given species isolated from the same host plant are remarkably homogeneous with respect to enzymatic production (Leuchtmann et al., 1992; Petrini et al., 1992). To facilitate the entry of endophytes into host tissues through natural or artificial openings, hydrolytic enzymes including pectinases, cellulases and lipases are secreted (Polizeli et al., 1991).

Proteases or proteolytic enzymes have commercial importance (Rao et al., 1998), as these enzymes are used in bioremediation and waste treatment, detergents, cosmetics and leather manufacture, silk degumming, animal cell culture, contact lens cleaning, therapy and diagnosis and the pharmaceutical, photographic and food industries. In addition, proteases are considered as insecticidal agents because these enzymes are required for the complete digestion of complex insect cuticles (Anwar and Saleemuddin, 1998; Gupta et al., 2002; Harrison and Bonning, 2010; Hasan et al., 2013; Kumar and Takagi, 1999; Murthy and Naidu, 2010; Nielsen and Oxenboll, 1998).

The medicinal plant Piper hispidum Sw. (Piperaceae), commonly known as "bayuyo" (Cuba), "cordoncillo" (Mexico), "jaborandi" or "falso-jaborandi" (Brazil), harbors a diverse endophytic fungal community (Orlandelli et al., 2012a), including fungi presenting activity against human pathogenic bacteria (Orlandelli et al., 2012b). Considering the shortage of information concerning the antifungal and enzymatic activities of the endophytes from this plant, the aim of the present study was to evaluate the antagonism and competitive interactions between endophytic and phytopathogenic fungi in dual culture experiments and to detect the proteolytic activity of these endophytes using a cup plate assay and different growth substrates.

Materials and Methods

Endophytic and pathogenic fungi

A total of 98 endophytic fungi were isolated from the leaves of P. hispidum plants located in a forest remnant in southern Brazil (Orlandelli et al., 2012a) and belong to the fungal culture collection of the Laboratório de Biotecnologia Microbiana, Universidade Estadual de Maringá, Paraná, Brazil. These fungal strains were molecularly identified as Alternaria sp., Bipolaris sp., Colletotrichum sp., Colletotrichum gloeosporioides, Phyllosticta capitalensis, Lasiodiplodia theobromae, Marasmius cladophyllus, Phlebia sp., Phoma herbarum, Diaporthe sp., Schizophyllum commune and one isolate from the order Diaporthales. Molecular identification was based on sequencing of the ITS1-5.8S-ITS2 region of rDNA (GenBank accession numbers JF766988 to JF767008).

The plant pathogenic fungi Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa were obtained from the Laboratório João Lúcio Azevedo, ESALQ, Universidade de São Paulo, Brazil.

For the experiments, all fungi were previously grown in Petri dishes containing potato dextrose agar (PDA) medium (Smith and Onions, 1983) at 28 °C under biochemical oxygen demand (BOD) for seven days.

In vitro antagonism and competitive interactions between endophytic and phytopathogenic fungi in dual culture

A modified version of the dual culture method of Campanile et al. (2007) was used. Briefly, 6-mm endophyte and phytopathogen plugs were combined in triplicate and inoculated onto PDA dishes, with a 4-cm distance between each plug. Filter paper plugs inoculated with 10 μL of fungicide Derosal plus® (with a 10−1 dilution of methyl benzimidazol-2-ylcabamato + tetramethylthiuram disulfide) or fungicide Tiofanil® (with a 200 mg/mL dilution of chlorothalonil + thiophanate-methyl) were used as positive controls, and autoclaved distilled water was used as a negative control.

The antagonism index (AI) was calculated as previously described (Campanile et al., 2007) using the following formula: AI = (RM - rm)/RM × 100, where rm represents the ray of the colony toward the antagonist, and RM represents the average of the three rays of the colony in the other directions. The competitive interaction (CI) between endophytes and phytopathogens was determined according to the Badalyan rating scale (Badalyan et al., 2002), which considers three main types of interactions (A, B and C) and four interaction sub-types (CA1, CB1, CA2 and CB2). Types A and B represented deadlock (mutual inhibition) at mycelial contact (A) or at a distance (B), whereas type C was replacement or overgrowth without initial deadlock. The intermediate interaction subtypes scored consisted of partial (CA1) or complete (CA2) replacement after initial deadlock with mycelial contact and partial (CB1) or complete (CB2) replacement after initial deadlock at a distance.

Conditions for protease production and cup plate assay

The endophytic fungi were grown as previously described (Sena et al., 2006) in liquid inducer medium (IM) containing powdered skim milk (Nestlé®) as the inducer substrate to stimulate protease secretion. The cultivation conditions were adapted from Sena et al. (2006), and the endophytes were also grown in IM containing two different substrates (carbon sources): rice or soy flour (5 g/L). Liquid medium incubated without fungal inoculation was used as a negative control. The cultures were incubated under stationary conditions (BOD at 28 °C for 10 days). Subsequently, the liquid medium was filtered using sterile gauze to separate the fungal mycelia.

For the cup plate assay, the filtered media were inoculated (50 μL) onto Petri dishes (9 cm) containing gelatin milk agar medium (Sena et al., 2006) with the surface perforated with cup plates (6-mm diameter). A commercial protease from Aspergillus oryzae (Sigma®) (≥ 500 U/g) was used as a positive control.

The experiment was performed in triplicate, and the dishes were incubated under BOD at 28 °C for 24 h. The enzymatic activity was evaluated as the presence of clear halos on an opalescent background and measured in millimeters (Dingle et al., 1953).

Statistical analyses

All experiments were performed using a completely randomized design (CRD) and analyzed by ANOVA (analysis of variance). The mean values were compared using the Scott-Knott test (p < 0.05) in the statistical program SISVAR 4.3 (Ferreira, 1999).

Results and Discussion

Evaluation of in vitro antagonism (AI) and competitive interaction (CI) between endophytic fungi and phytopathogens

The dual culture method has been broadly applied in antagonism studies because this analysis facilitates the in vitro screening of agents that can be used for biological control (Faria et al., 2002; Mariano, 1993). In the present study, ANOVA showed differences among the in vitro antagonistic actions, as varying degrees of phytopathogen mycelial growth inhibition were observed. The results obtained after screening all 98 P. hispidum endophytes are shown in Figure 1-A, and the types of CI observed between the endophytes and A. alternaria, Colletotrichum sp., P. citricarpa and M. perniciosa are shown in Figure 1-B. More details regarding AI and CI between the 21 molecularly identified endophytes tested and phytopathogens are shown in Table 1.

Figure 1 Antagonism index (AI) and competitive interaction (CI) between 98 P. hispidum endophytic fungi and phytopathogenic fungi in dual culture. A) AI indicates the reduction (%) in phytopathogen mycelial growth. *Means of triplicates. Different letters indicate that the AI intervals are significantly different according to the Scott-Knott test (p < 0.05). B) *Badalyan rating scale (Badalyan et al., 2002): A = deadlock with mycelial contact; B = deadlock at a distance; CA1 = partial replacement after initial deadlock with contact; CB1 = partial replacement after initial deadlock at a distance. **N = no competitive interaction was observed (absence of endophyte antagonism). 

Table 1 Antagonism index (AI) and competitive interaction (CI) between the 21 molecularly identified endophytic fungi tested and five phytopathogenic fungi in dual culture. 

Endophytic fungi/controls Phytopathogenic fungi

A. alternata Colletotrichum sp. P. citricarpa M. perniciosa




AI* CI** AI CI AI CI AI CI
L. theobromae JF766989 64.79a CA1 54.16a CA1 56.07a CA1 60.09a CB1
Diaporthales isolate JF767007 38.69b A 42.10b A 51.18a A 37.22a A
Diaporthe sp. JF766998 37.64b A 31.00b A 36.89b A 27.03a A
Diaporthe sp. JF767000 35.33b A 26.90c A 30.44c A 34.11a A
P. herbarum JF766995 33.03c A 21.46c A 28.93c B 31.56a A
Bipolaris sp. JF767007 32.33c A 00.00e N*** 25.58c A 22.29a B
Phlebia sp. JF766997 31.40c A 27.67c A 27.65c A 31.36a A
Bipolaris sp. JF767001 30.49c A 25.25c A 24.84c CA1 27.01a A
Colletotrichum sp. JF766996 30.00c A 11.00e A 23.41c A 31.91a A
C. gloesporioides JF767002 26.65c A 21.78c A 29.08c B 16.12b A
Bipolaris sp. JF766993 25.28c B 18.49d A 22.25c A 24.54a A
M. cladophyllus JF767003 22.30d A 25.00c A 37.00b CA1 08.98b A
Bipolaris sp. JF766992 21.49d A 32.36b A 28.91c A 32.23a A
Alternaria sp. JF766991 20.26d A 25.87c A 24.05c A 28.14a A
Alternaria sp. JF766990 19.63d A 25.70c A 19.58d A 29.43a B
Colletotrichum sp. JF767006 17.50d A 13.60d A 17.42d A 15.85b A
Bipolaris sp. JF767005 13.68e A 16.40d A 22.01c A 25.16a A
S. commune JF766994 13.19e A 17.74d CA1 11.43d A 08.88b CA1
Colletotrichum sp. JF767004 12.70e A 28.18c A 36.69b A 17.13b A
Colletotrichum sp. JF766999 09.00e A 14.28d A 10.14d A 21.13b A
P. capitalensis JF766988 04.07e B 11.56e A 10.90d B 18.18b B
Fungicide Derosal Plus® (C+1) 12.16e - 23.00c - 05.18e - 23.01a -
Fungicide Tiofanil® (C+2) 12.16e - 04.00e - 03.63e - 12.72b -
Distilled water (C−) 00.00e - 00.00e - 00.00e - 00.00e -

*Means of triplicates. The mean values followed by different letters indicate that the AI intervals are significantly different according to the Scott-Knott test (p < 0.05).

**Badalyan rating scale (Badalyan et al., 2002): A = deadlock with mycelial contact; B = deadlock at a distance; CA1 = partial replacement after initial deadlock with contact; CB1 = partial replacement after initial deadlock at a distance.

***N = no competitive interaction (absence of endophyte antagonism).

(C+1)Positive control (10−1 in distilled water); (C+2)positive control (200 mg/mL in distilled water); (C−)negative control.

The AI values obtained for the best antagonist (L. theobromae JF766989) varied between 54.16 and 64.79%, and these results were higher than those obtained in a previous study (Campanile et al., 2007), where the best result for antagonism was 28.5%. Badalyan et al. (2002) observed that most of xylotrophic mushrooms and cereal phytopathogens present subtypes of the type C interaction. According to the scale proposed by the same authors, interaction types A and B indicate a deadlock or mutual inhibition in which neither organism overgrows in the presence of the other; in contrast, type C and associated subtype interactions indicate a replacement involving the inhibition of one organism. L. theobromae JF766989 partially overgrew (interactions CA1 and CB1) in the presence of all phytopathogens; however, most of the 98 P. hispidum endophytes presented deadlock interactions with mycelial contact (A).

Although these results suggest L. theobromae JF766989 as an antagonist of phytopathogenic fungi, most of the endophytes tested were more effective than fungicides for reducing the growth of the phytopathogens.

Evaluation of the proteolytic activity of endophytic fungi

Screening for new producers of novel and industrially useful enzymes is of great interest for biotechnology research (Kumar and Takagi, 1999). Proteases are physiologically necessary and have been isolated from a wide diversity of sources, such as plants, animals, and microorganisms (Rao et al., 1998). Microbial proteases have several characteristics necessary for biotechnological application and represent a large portion of the total worldwide sale of enzymes, with low production costs compared with animal or plant proteases. Moreover, microorganisms are preferred as a source of proteases due to their rapid growth, limited space requirements for cultivation and ease of genetic manipulation to generate new enzymes with desirable properties (Najafi et al., 2005; Said and Pietro, 2002).

The cup plate assay in the present study showed that 28 of the 98 endophytes (28.57%) presented proteolytic activity when grown in inducer medium. An ANOVA showed differences in the observed enzymatic halos, with means ranging from 1.33 to 16.40 mm in diameter; the highest value was observed for S. commune JF766994 (Fig. 2 and Table 2).

Figure 2 Cup plate assay: A) Schizophyllum commune enzymatic halos (16.40 mm) produced after growth in inducer medium (IM); B) S. commune halos (15.40 mm) produced after growth in IM + rice flour; C) Phoma herbarum halos (17.67 mm) produced after growth in IM + soy flour. 

Table 2 Proteolytic activity of endophytic fungi grown in liquid inducer medium (IM) with and without additional substrates compared with a commercial fungal protease. 

Fungi Halos (in mm) obtained for each culture condition

IM IM + rice flour IM + soy flour
S. commune JF766994 16.40Ba* 15.40Ba 15.00Ba
Endophyte G62-75 15.40B - -
Endophyte G23-54 15.00C - -
Colletotrichum sp. JF767004 14.47Ca 14.53Ba 15.00Ba
Endophyte G38-111 14.00Ca 14.27Ba 13.40Ca
Endophyte G42-108 10.20Da 14.46Ba -
Endophyte G13-13 10.00D - -
L. theobromae JF766989 09.53D - -
Endophyte G04-24 09.47Da 09.87Ca 15.00Ba
Endophyte G17-101 08.27D - -
Endophyte G57-82 06.67D - -
Alternaria sp. JF766991 06.00D - -
Endophyte G04-04 06.00Da 15.00Ba 11.33Da
P. herbarum JF766995 05.67Eb 10.27Cb 17.67Ba
Endophyte G36-112 05.47Eb 14.20Ba -
Endophyte G39-39 05.47Ea 10.33Ca -
Bipolaris sp. JF767001 05.40E - -
Endophyte G07-23 05.20E - -
Bipolaris sp. JF767005 04.87Ea 10.00Ca 10.26Da
Endophyte G07-138 04.47E - -
Endophyte G01-01 03.93F - -
Endophyte G54-69 03.93F - -
Endophyte G53-83 03.67Fb 13.87Ba 05.00Eb
Colletotrichum sp. JF766999 03.60F - -
Endophyte G62-127 03.53Fa 07.86Da -
Endophyte G11-11 03.47F - -
Endophyte G05-05 02.27Gb 07.27Db 15.00Ba
Endophyte G35-35 01.33Ga 10.00Ca 09.73Da
Positive control (C+) 23.63A 23.63A 23.63A

*Means of triplicates. The mean values followed by upper-case letters in columns or lower-case letters in rows are not significantly different according to the Scott-Knott test (p < 0.05).

(C+)Positive control: commercial protease from Aspergillus oryzae Sigma® (≥ 500 U/g) used directly in the cup plate assay.

Approximately 28.57% of the P. hispidum endophytes evaluated presented proteolytic activity under the conditions assayed. In some cases, the enzyme production was significantly higher after the medium was changed. Species of the genus Mucor are protease producers of commercial value (Alves et al., 2002), with 82% of 56 isolates belonging to 11 different species presenting proteolytic activity. Djamel et al. (2009) reported that only 10 (3.9%) of 253 Penicillium strains examined presented significant proteolytic activity, as based on the hydrolysis of milk casein (clear zones around the colony) and the mycelium colony diameter, with clear halos greater than 9 mm.

The 28 P. hispidum endophytic isolates that initially presented enzymatic activity were grown in the presence of rice or soy flour (Table 2). When rice flour was used as the substrate, 14 endophytes produced enzymatic halos, ranging from 7.27 to 15.40 mm, with the best values observed for S. commune JF766994. In addition, two unidentified isolates (G53-83 and G36-112) presented statistically superior enzymatic activity when grown on this substrate. In the presence of soy flour, positive results were obtained for 10 endophytes, with enzymatic halos ranging from 5.0 to 17.67 mm in diameter. The best result was obtained for P. herbarum JF766995, which presented statistically superior enzymatic activity when grown on this substrate; similar results were obtained for isolate G05-05.

Agro-industrial and other wastes can be used as substrates for fermentation, suggesting a cost-effective approach to enhance enzymatic production, as these substrates are cheap and abundant natural carbon sources (Blesson, 2009; Singh et al., 2012). Singh et al. (2012) showed that sugarcane bagasse, wheat bran, corncob, wheat straw and, in particular, rice bran are suitable substrates for the production of amylases and xylanases from thermophilic actinobacteria. In addition, substrates such as soy, wheat and rice bran, mango and banana peel, gelatin and fish flour have been used for the production of microbial proteases (Murthy and Naidu, 2010; Paranthaman et al., 2009; Souza et al., 2008).

Consistent with the results of the present study, Souza et al. (2008) used the cup plate assay to investigate the production of enzymes from Amazonian basidiomycetes cultivated on different substrates, obtaining enzymatic halos of up to 23.80 mm in diameter. Smaller halos (up to 18.07 mm in diameter) were obtained using a medium supplemented with protein sources, and halos of up to 19.11 and 18.64 mm in diameter were obtained on soy bran and fish flour, respectively. Paranthaman et al. (2009) verified protease production under the solid-state fermentation of Aspergillus niger using different varieties of broken rice as substrates, and the results varied between 44.7 and 67.7 U/g.

Conclusions

Endophytes constitute a novel and important new source of active substances that can be employed in different biotechnological industries. Diverse strains, even members of the same endophytic fungal species, can exhibit characteristic metabolite production with enzymatic or antifungal potential. Some positive antifungal phenotypes of endophytes might reflect competition for space or nutrients, as demonstrated through dual culture experiments. The results of the present study suggest that in the assemblage of P. hispidum endophytes, certain strains are important sources of antifungal properties, particularly L. theobromae JF766989, which reduced the growth of A. alternaria, Colletotrichum sp., P. citricarpa and M. perniciosa by approximately 54 to 65%.

Investigators in Brazil should further explore the potential to generate new enzymes from microbial sources, as this country has a continental area that includes hundreds of plant species with diverse endophytes. The results of the present study highlight the proteolytic activity of the endophytes P. herbarum JF766995 and S. commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. As some isolates showed increased activity in the presence of rice or soy flour as a substrate, these endophytes have the potential to produce enzymes from agriculture wastes.

Acknowledgments

The authors would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the Master's scholarship awarded to Ravely Casarotti Orlandelli. The authors would also like to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (process n. 480726/2011-6) for financial support.

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Received: October 22, 2013; Accepted: September 05, 2014

Send correspondence to J.A. Pamphile. Laboratório de Biotecnologia Microbiana, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, 87020-900 Maringá, PR, Brazil. E-mail: prof.pamphile@gmail.com; japamphile@uem.br.

Associate Editor: Raquel Silva Peixoto

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