AGATA GORYLUK-SALMONOWICZ¹*, MONIKA PIÓREK¹, HANNA REKOSZ-BURLAGA¹, MARCIN STUDNICKI² and MIECZYSŁAW BŁASZCZYK¹

¹Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland
²Department of Experimental Design and Bioinformatics, Faculty of Agriculture and Biology Warsaw University of Life Sciences, Poland

*Corresponding author: A. Goryluk-Salmonowicz Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland; e-mail: agatagoryluk@interia.pl.

Submitted 17 June 2014, revised 30 April 2015, accepted 11 February 2016
DOI: 10.5604/17331331.1215617

Abstract

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group  and Erwinia persicina.

Key words: Alternaria alternata, antifungal activity, endophytic bacteria, herbs

It is widely believed that all plants are colonized by an endophytic microflora composed of microscopic fungi and bacteria that live inside plant tissues without causing them any harm. Endophytes have been isolated from above-ground parts of plants (stems, flowers, leaves and fruits), from roots and from seeds (Reinhold-Hurek and Hurek, 1998; Tan and Zou, 2001). Numerous studies confirmed that endophytes have a great applicable potential. They have found uses in three main fields connected with crop yield enhancement and growth promotion (1), industrial and medical applications (2) and environmental pollution control (3).

Numerous studies reported that endophytes control plant pathogens through synthesis of different antimicrobial compounds (1). Miller et al. (1998) described endophytic bacteria Pseudomonas viridiflava isolated from grasses and producing ecomycins B and C. It was investigated that these lipopeptides inhibit the human pathogens Candida albicans. Similar studies were conducted by Guan et al. (2005), who reported antimicrobial agent producing strain Streptomyces griseus inhabiting Kandelia candel. Most research  has focused on the antifungal properties of isolated endophytes. Strobel et al. (2004) for example isolated oocydin A producing endophytes classified as Serratia marcescens. Antifungal compound producing endophytes (Paenibacillus polymyxa) were also reported by Beck et al. (2003). Endophytic-induced plant growth promotion is also achieved through  fixation of atmospheric nitrogen, production of iron-chelating siderophores, solubilisation of minerals and production of phytohormones.

Endophytes can act as mini-factories and often produce novel compounds (2). Researchers reported endophytes producing novel antibiotics, anticancer and antiviral compounds, volatile organic compounds, insecticidal agents, immunosuppressive compounds and antioxidants (Strobel and Daisy, 2003; Owen and Hundley, 2004). Castillo et al. (2002) for example isolated from Kennedia nigricans endophytes classified as Streptomyces strain NRRL 30562 that produces antibiotic and antimalarial agents – munumbicins. Antimalarial compounds were also reported by Ezra et al. (2004), who obtained coronamycin producing Streptomyces sp. isolated from Monstera sp. Few studies have been published describing anticancer compounds producing endophytes. The first one was by Stierle et al. (1993), who reported taxol producing endophytic fungus Taxomyces andreane, isolated from Taxus brevifolia.

Some reports confirmed that endophytes enhance phytoremediation (3). These endophytes inhabit plants grown in xenobiotic contaminated soil and express different mechanisms necessary to degradation of contaminants  (Germaine et al., 2004; 2006).

In the past few years the search for endophytes inhabiting medicinal plants intensified. It is now recognized that herbs are a very rich source of microorganisms with different biochemical properties. Numerous recent studies have been devoted to the identification of endophytes colonizing herbs from Asian countries. One study isolated 18 endophytic bacteria from herbal plants in Indonesia, such as citrus, turmeric, Andrographis paniculata and Piper crocatum (Soka et al., 2012). Another study obtained 19 bacterial endophytes and 113 fungal endophytes from plants grown in India: Digitalis lanata, Digitalis purpurea, Plantago ovata and Dioscorea bulbifera (Ahmed et al., 2012). Indian herbs were also investigated by Amirita and colleagues, who managed to isolate 334 fungal strains inhabiting the internal tissues of Adhatoda vasica, Costus igneus, Coleus aromaticus and Lawsonia (Amirita et al., 2012). Another study of medicinal plants grown in Taiwan isolated 156 fungal endophytes from 20 species from the Lauraceae and Rutaceae family (Ho et al., 2012). In 2014 our team presented endophytic microflora of Hypericum perforatum (Rekosz-Burlaga et al., 2014). From stems and leaves of the tested plants four bacterial strains were isolated.

The aim of the present study was to describe the endophytic microflora of selected medicinal plants inhabiting European countries. Isolated bacteria were tested for their antifungal properties against a plant pathogenic fungi and strains displaying the greatest antifungal activity have been classified according to their morphological, physiological and molecular characteristics.

Plant samples were collected from two areas in central Poland, near Kozienice town (51.575°N, 21.750°E) and in Warsaw city (52.259°N, 21.020°E), during the vegetative seasons of 2007 and 2008. Bacterial endophytes were isolated from stems of 13 native growing herbal plant species: Chelidonium majus L., Elymes repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentla arvensis L., Papaver rhoeasL., Rosmarinus officinalis L. Solidago gigantea L. and Vinca minor L. Bacterial isolation from plant material was performed according to the procedure of Hung and Annapurna (2004). Five plants of each species were tested. Samples were first washed in distilled water for 5 minutes and the surfaces were sterilized by bathing in a mixture of 0,1% mercuric chloride (HgCl₂) and 70% ethanol. The samples were then rinsed four times in sterile distilled water and dried on sterile filter paper. To verify the efficiency of sterilization, samples were  placed on nutrient agar. The water from the fourth rinse was also plated on nutrient agar to confirm sterilization. To isolate endophytes, the samples were cut longitudinally and placed cut side down on nonselective media, including water agar, nutrient agar, potato dextrose agar and tryptic soya agar. All plates were then incubated at 28°C for 2-7 days. The produced colonies were sub-cultured several times to obtain pure cultures. To confirm that the colonies originated from a single cell and were not mixed, their morphology was recorded and the bacteria were Gram-stained and observed under light microscope. Pure isolates were maintained at –20°C.

The antifungal properties of the bacterial isolates were assayed using an in vitro test with the following fungal strains: Alternaria alternata ES11, Paecilomyces variotti ES23, Chaetomium sp. E13, Byssochlamys sp. E9, Aureobasidium sp. E4 and Fusarium sp. E23. Fungal strains were obtained from Department of Microbial Biology, Warsaw University of Life Sciences in Poland. Fungal spores were spread onto the surface of potato-dextrose agar (PDA) and then three 20 µl drops of each bacterial culture (grown in nutrient broth at 30°C for 24 h with shaking at 180 rpm) were spotted onto the surface of the inoculated plate. All plates were then incubated for 7 days at room temperature (Goryluk et al., 2009). If bacteria inhibited fungal growth, a zone of inhibition appeared around the colonies. For the isolates with the strongest antifungal properties, the diameters of the hyaline inhibition zones were measured (mm). This test was repeated five times for each bacterial isolate. Data were analyzed using one-way analysis of variance. Homogenous groups of means were determined with the Tukey’s procedure of multiple comparisons at the significance level 0.05. The analyses were performed using Statgraphics 4.1 statistical package. The isolates with the strongest antifungal activity were selected for further identification.

The identification of selected isolates was made based on morphological observations (1) and the biochemical properties (2) of the bacteria. The results were analysed according to Bergey’s Manual of Systematic Bacteriology (Brenner et al., 2005; Vos et al., 2009). To confirm the species  identification, molecular analysis of 16S rRNA gene sequences (3 and 4) was performed.

1. For colony characterization of the selected strains, the nature of the colonies, their pigmentation and shape were recorded. To describe the properties of the bacterial cells, microscopic observations were made to determine their shape and size, their ability to move and form spores as well as their Gram-staining characteristics.
2. The biochemical properties of the bacterial isolates were tested following standard protocols. Seven different carbon compounds (lactose, saccharose, glucose, arabinose, mannitol, rahmnose, citrate) were used to check the ability to substrate utilization, provided as the sole carbon source. The ability to gelatin, starch, urea and arginine hydrolysis was monitored. Mixed-acid fermentation test (Methyl-Red test) was performed. The activities of catalase, sulfhydrolase and lysine decarboxylase were determined. Production of fluorescent pigments, indole and acetoine was evaluated. The ability to grow at extreme temperatures (4°C and 55°C) was tested.
3. Molecular characterization
   Genomic DNA was isolated from the selected strains as described by Hung and Annapurna (2004). Using these DNAs as templates and primers 8f (5’-AGAGTTTGATCCTGGCTCAG-3’) and pH (5’-AAGGAGGTGATCCAGCCGCA-3’) (Johri and Johri, 2004), fragments of the 16S rRNA genes were amplified by PCR. The amplicons were analyzed by electrophoresis on a 1% agarose gel, purified using a commercial kit (Clean up; A&A Biotechnology) and then sequenced using an automated DNA sequencer (454 GS FLX Titanium, Roche). The gene sequences were submitted to NCBI GenBank (accession numbers KJ130483-KJ130486). Bacterial strains were deposited in collection of Department of Microbial Biology, Warsaw University of Life Sciences in Poland.
4. Bioinformatics analysis
   The 16S rRNA gene sequences from the bacterial isolates were compared with 16S ribosomal RNA sequences (Bacteria and Archaea) in the NCBI database using Standard Nucleotide BLAST with the default settings, to identify the most similar sequences. For each strain, 11 nucleotide sequences (unknown sequence and the 10 most similar) were aligned using CLUSTAL W2 (Larkin et al., 2007). The multiple sequence alignments were then used to create phylogenetic trees by the Neighbor Joining method with complete deletion of gaps, implemented in MEGA5 software (Saitou and Nei, 1987; Tamura et al., 2004; 2011).

To evaluate the antifungal properties of the selected bacterial isolates, their influence on the growth and development of A. alternata ES11 was assayed in vitro. A fungal spore suspension was prepared and added to 300 ml of PDB containing a suspension of bacterial cells. Control cultures were also prepared: PDB with fungal spores or PDB with bacteria. All cultures were incubated for 10 days at 25°C with shaking (1500 rpm). The numbers of bacteria and fungi were then evaluated using the plate count method and by  microscopic analysis of the cultures. To prepare the fungal spore suspension A. alternata was cultured on PDA for one week at 25°C and the spores were harvested aseptically and suspended in sterile distilled water. To prepare the suspension of bacteria, the strains were cultured in nutrient broth for 12 h at 30°C with shaking (1500 rpm) and then 0.5 ml of these cultures were used to inoculate PDB. The plate count method was used to determine the concentration of fungal spores and bacterial cells.

Our results presented in this paper revealed that 12 tested herbs – Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. are inhabited by bacterial endophytes. This is the first examination of endophytic microflora of the tested herbs, except C. majus L. which was tested for the first time by our team in 2007 (Goryluk et al., 2009). From the tested plants 181 bacterial isolates were obtained. The highest number of bacterial isolates came from C. majus (48 isolates) and from G. pratense (30 isolates) and the smallest number (6 isolates) from: F. vulgare, S. gigantea and Matricaria chamomilla. No isolates were obtained from Papaver rhoeas. This is not the first time, that a studied plant has apparently lacked endophytes. Soka et al. (2012) failed to isolate any endophytes from a specimen of the herb Nothopanax scuttelarium. It is not known whether the inability to isolate endophytic microorganisms from some species of plants is because they are naturally sterile, or if there is some methodological problem. All of the plants examined in our work share some common properties: they are very expansive, they grow in poor environments and have medicinal properties like anti-inflammatory, antibacterial, antifungal or anticancer activities. Plant extracts have frequently been used to treat medical conditions, and of the plants studied here, C. majus L. has been the source of numerous medicines: Ukrain (anti-cancer activity), Chelifungin (anti-fungal properties) and Di-Sancor (anti-HIV properties). In addition, infusions of C. majus have been shown to have a positive influence on the nervous and digestive systems (Ożarowski, 1976). This herb contains a number of active substances such as alkaloids. One outstanding issue is the contribution made by endophytic microorganisms to the medicinal properties of such plants. There are some examples where endophytes produce biologically active substances. One report presented in 1993 showed that Taxomyces andreanae, an endophytic fungus isolated from the yew tree Taxus brevifolia, produced paclitaxel (Stierle et al., 1993). This compound is used as an anticancer medicine (Taxol^®) and previously had to be isolated from yew trees by a process that was long and costly due to low yield. The discovery that the endophyte is responsible for synthesizing paclitaxel has made the production of this medicine much easier. Another example is the endophytic fungus Chaetomium globosum, which produces hypericin isolated from the herb Hypericum perforatum (Kusari et al., 2008).

Endophytic bacteria isolated from tested plants were screened for antifungal properties against plant pathogenic fungi – A. alternata, Paecilomyces variotti, Chaetomium sp., Byssochlamys sp., Aureobasidium sp. and Fusarium sp. These facultative fungal pathogens causes diseases of different plants all over the world, including various vegetables, fruits and cereal crops. Some of them, for example A. alternata, produces mycotoxins, which can be transferred from the infected plant to the tissues used as food. Fusarium species also produce mycotoxins, like zearalenone or fumonisins. Fusarium spp. they are important pathogens of many agricultural plants, like corn, wheat or soybean (Muthomi and Mutitu, 2003). Antifungal activity of bacteria has been detected in many genera and one of the significance of this kind of researches is the possibility to use of these bacteria as plant protection agents. Of the 181 endophytic isolates tested in our study, 53 displayed antifungal properties (29%). The highest number of bacteria with such activity was obtained from C. majus (22 isolates, 45%). 10 out of 18 isolates from Erigeron annuus L. (55%)  and almost half of the isolates from Mentha arvensis L. (6 out of 13, 46%) had antifungal properties. Only one isolate with antifungal properties was obtained from G. pratense L. (3%), Humulus lupulus L. (12%), S. gigantea L. (16%) and R. officinalis L. (12%). Among the fungal species tested, A. alternata was the most sensitive to bacterial influence. The growth of this fungus was inhibited by 39 of the bacterial isolates. On the other hand, the least sensitive was Fusarium sp., which was inhibited by only one isolate: 30B. The growth of Byssochlamys species was inhibited by 7 isolates. The other fungi tested, P. variotti, Chaetomium sp. and Aureobasidium sp., were inhibited by 25, 26 and 27 of the bacterial isolates, respectively. The obtained results are in accordance with those achieved by our team earlier (Goryluk et al., 2009) when isolates obtained from C. majus exhibited antifungal activity against A. alternata, Chaetomium sp. and P. variotti. In another study, Sgroy et al. (2009) used A. alternata to show that two endophytic bacteria (Brevibacterium halotolerans, Bacillus pumilus) isolated from Prosopis strombulifera, can inhibit fungal growth. Hui et al. (2012) isolated one endophytic species (Bacillus subtilis) from Prunus mume with antifungal properties against A. alternata and Fusarium sp.

Endophytic bacteria with the strongest antifungal properties were selected for further researches – 2-5b and 30B isolate from C. majus, P2 and P3 from E. repens and N2-1a from S. gigantea. First, taxonomic classification was conducted based on morphological, physiological and molecular characteristics. Afterwards, the diameter of fungal growth inhibition zones produced by these isolates and their influence on mycelium development of the fungus A. alternata was examined.

Only one of the isolates produced spores and was Gram-positive (30B). This isolate, in contrast to the remaining isolates, produced bigger cells (2.5 ´ 1.2 µm), formed chains and was not able to move. Gram-negative isolates formed smaller single cells (1.6 ´ 1.1 µm) and were able to move but very weakly. Some of the four Gram-negative isolates could be differentiated by the nature of their colonies. Isolate 2-5b produced round pink colonies with a regular shape, and a glossy and smooth appearance. Colonies of isolates P2 and P3 were similar in shape and appearance to those of 2-5b, but they produced fluorescent pigments. Isolate N2-1a also produced similar colonies, but without any pigmentation. In contrast, colonies of isolate 30B had an irregular crater-like shape and matt appearance.

All of the isolates could utilize lactose, sucrose, glucose, arabinose and mannitol, they were indole negative and catalase positive (Table I).

Table I
Biochemical characteristics of selected endophytic isolates.

	Isolate
	2-5b	30B	P2	P3	N2-1a
Substrate utilization
Lactose/saccharose	+/+	+/+	+/+	+/+	+/+
Glucose/arabinose	+/+	+/+	+/+	+/+	+/+
Mannitol/rhamnose	+/+	+/–	+/–	+/–	+/+
Citrate/gelatin	+/–	–/+	+/+	+/+	+/–
Starch/urea	–/–	+/–	–/–	–/–	–/–
Arginine/lysine	–/–	+/–	+/+	+/+	–/+
Methyl-Red test	–	–	–	–	–
Metabolite production
Fluoresceine/Pyocyanine	–/–	–/–	+/–	+/–	–/–
Hydrogen sulfide	–	+	–	–	+
Indole/ catalase	–/+	–/+	–/+	–/+	–/+
Acetoine	+	+	–	–	+
Growth temperature
4°C / 55°C	–/–	–/–	+/–	+/–	–/–

Based on the morphological and biochemical properties of the isolates, they were identified as Bacillus sp. (30B), Erwinia sp. (2-5b) and Pseudomonas spp. (P2, P3, N2-1a). The PCR amplification, sequencing and bioinformatic analysis of 16S rRNA gene sequences from each of the isolates enabled the identification of four of them to species level (Table II). Their phylogenetic affiliation was estimated based on the phylogenetic tree constructed to visualize the relationship between the sequences of isolates and related organisms from the GenBank database. Isolate 2-5b was determined as Erwinia persicina (GenBank accession number KJ130483), isolate P2 and P3 as Pseudomonas azotoformans (GenBank accession numbers KJ130484 and KJ130485, respectively) and isolate N2-1a as Pseudomonas cedrina (GenBank accession number KJ130486). Isolate 30B was placed within the Bacillus subtilis group, but further investigations are required to verify this species identification.

Table II
Phylogenetic affiliation of isolates based on the analysis of 16S rDNA fragments.

Isolatea (GenBank acc. No.)	Best match with database b (GenBank acc. No.)	Microbial affiliation
2-5b (KJ130483) P2 (KJ130484) P3 (KJ130485) N2-1a (KJ130486)	E. persicina (NR026049) P. azotoformans (NR037092) P. cedrina (NR042147) P. cedrina (NR024912)	E. persicina P. azotoformans P. cedrina

^aThe sequence of isolate
^bThe sequences of isolates were compared with nucleotide sequences from database (similitude in 99%) and the phylogenetic tree was constructed; the best match was selected as the closest sequence from the phylogenetic tree

Endophytes classified as Bacillus and Pseudomonas species are very often isolated by researchers (Goryluk et al., 2009; Lodewyckx et al., 2001; Narayan et al., 2013; Rekosz-Burlaga et al., 2014). Liu et al. (2014) for example isolated Bacillus sp. strain and P. azotoformans strain as an endophytes from xerophilous moss Grimmia montana. P. cedrina was isolated by Behrendt et al. (2003) as plant associated bacteria inhabiting phyllosphere of Solanum tuberosum L. Bacillus and Pseudomonas genera are well-known for its production of diverse secondary metabolic products. Our results are in accordance with this statement because the strongest antifungal activity against A. alternata was reported for Bacillus sp. strain 30B (Table III).

Table III
Antifungal activities of bacterial endophytes (named 2-5b, 30B, P2, P3, N2-1a) against six fungal strains.

Isolates	Fungal growth inhibition zone (mm)
	A. alternata	Chaetomium sp.	P. variotti	Byssochlamys sp.	Aureobasidium sp.	Fusarium sp.
2-5b	18.6(b)	10.6(a)	n	n	n	n
30B	24.0(c)	11.2(a)	19.0	6.2	7.0	14.4
P2	8.4(a)	16.4(bc)	n	n	n	n
P3	9.4(a)	18.0(c)	n	n	n	n
N2-1a	15.2(b)	12.4(ab)	n	n	n	n

n – no inhibition zone
Letters in parenthesis indicate homogenous groups of means, which do not differ significantly at α  =  0.05. Analysis were prepared separately for each fungal strain.

Fungal growth inhibition zone had 24 mm. Smaller inhibition zones were observed for E. persicina strain 2-5b and P. cedrina strain N2-1a, while the narrowest inhibition zones were produced by P. azotoformans strains P2 and P3. In contrast, these strains had the strongest activity against Chaetomium sp. while other strains produced smaller inhibition zones. Only one strain, Bacillus sp. 30B, inhibit the growth of all tested fungi, even Fusarium sp. strain. Similar results were obtained by Narayan et al. (2013) who isolated B. tequilensis endophytic strains with strong and broad spectrum of antifungal activity against all tested pathogenic fungi, like Alternaria panax and Fusarium oxysporum. This strain produced fungal inhibition zones wider than 8 mm. Other researchers, Tschen and Tseng (1989) isolated Bacillus sp. strains active against Fusarium sp. and Paecilomyces sp. which produced bacereutin. Axelrood et al. (1996) in turn, obtained B. amyloliquefaciens strains with antifungal activity against Fusarium oxysporum. B. amyloliquefaciens strains with strong activity against Fusarium sp. were also isolated by Cuijuan et al. (2014). To confirm the antifungal activity against A. alternata, dual-culture experiment was performed. After 10 days of incubation of fungal spores with the bacteria, no fungal colonies were detected in any of the cultures, while the bacterial number were slightly reduced (E. persicina strain 2-5b and Bacillus sp. strain 30B) or unchanged (Pseudomonas spp. strains P2, P3 and N2-1a). Microscopic observations showed that fungal spores incubated in presence of bacteria were deformed and unable to grow and form mycelium.

Presented researches revealed that tested herbs: C. majus L., E. repens L., E. annuus L., Euphrasia rostkoviana Hayne, F. vulgare L., G. pratense L., H. lupulus L., Matricaria chamomilla L., Mentha arvensis L., , Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. are inhabited by endophytic bacteria. 29% of the isolates displayed antifungal properties against plant pathogens. The highest number of endophytes with antifungal activity was  obtained from C. majus L., E. annuus L. and Mentha arvensis L. (45%, 55% and 46%, respectively). The highest antifungal activity was recorded for Bacillus sp. strain 30B which was isolated from C. majus L.

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