Curr Microbiol DOI 10.1007/s00284-014-0594-8
Antiproliferative, Antifungal, and Antibacterial Activities of Endophytic Alternaria Species from Cupressaceae Jalal Soltani • Mahdieh S. Hosseyni Moghaddam
Received: 5 January 2014 / Accepted: 4 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Recent research has shown the bioprospecting of endophytic fungi from Cupressaceae. Here, we further uncover that the healthy cypress plants such as Cupressus arizonica, Cupressus sempervirens var. cereiformis, and Thuja orientalis host highly bioactive endophytic Alternaria fungal species. Indeed, endophytic Alternaria alternata, Alternaria pellucida, and Alternaria tangelonis were recovered from healthy Cupressaceous trees. Biodiversity and bioactivity of recovered endophytic Alternaria species were a matter of biogeography and host identity. We further extracted such Alternaria’s metabolites and highlighted their significant antiproliferative, growth inhibitory, and antibacterial activities against the model target fungus Pyricularia oryzae and the model pathogenic bacteria Bacillus sp., Erwinia amylovora, and Pseudomonas syringae. In vitro assays also indicated that endophytic Alternaria species significantly inhibited the growth of cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. In conclusion, since the recovered Alternaria species were originally reported as pathogenic and allergenic fungi, our findings suggest a possible ecological niche for them inside the foliar tissues of Cupressaceous trees. Moreover, in this study, the significant bioactivities of endophytic Alternaria species in association with Cupressaceae plant family are reported.
Jalal Soltani and Mahdieh S. Hosseyni Moghaddamhave contributed equally to this work J. Soltani (&) M. S. Hosseyni Moghaddam Phytopathology Department, Bu-Ali Sina University, Hamedan, Iran e-mail: [email protected] J. Soltani Bu-Ali BioNanoTechnology Co. (BABiNT), Hamedan, Iran
Introduction Currently, biodiversity and bioactivity of endophytic microorganisms from many host plant species are being intensively investigated [2, 22, 24, 40]. Recent studies have highlighted that the plant family Cupressaceae hosts a diverse number of endophytic microorganisms [13, 15]. Our group has further shown that such endophytic fungi from Cupressaceae plant family demonstrate a broad range of bioactivities, e.g., antiproliferative, antifungal, and antibacterial activities [15, 17, 18]. In recent years, it has been frequently shown that endophytic microorganisms of any host plant were potentially capable of producing the same bioactive molecules as the host plant [24]. Members of Cupressaceae plant family are used widely in ethnomedicine [12]. Therefore, the bioactivity of metabolites that are produced by endophytic microorganisms and recovered from Cupressaceous trees may resemble the bioactivity of metabolites from host plant itself. Indeed, drug discovery programs have indicated that some endophytic fungi from Cupressaceae were capable of biosynthesizing anticancer biomolecules [20, 21, 23]. Therefore, investigating endophytic fungi colonizing Cupressaceae plant family may reveal novel sources of bioactive molecules, besides fungal biodiversity. The fungus Alternaria (Dothideomycetes, Ascomycota) is a cosmopolitan genus widely distributed in plants, soil, food, and indoor air environment. Alternaria species have adapted a variety of life styles as phytopathogen, weak facultative parasite, saprophyte, and endophyte [42]. A number of Alternaria species have emerged as opportunistic human pathogens particularly in immunocompromised patients [29, 44]. They are also common allergens in humans [6]. Cases of asthma, phaeohyphomycosis, onychomycosis, sinusitis, ulcerated cutaneous infections, keratitis, visceral infection osteomyelitis
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Antibacterial Activities of Endophytic Alternaria Species
due to Alternaria species, among which Alternaria alternata was the most common one, have been reported [3, 11, 26, 33]. Alternaria fungi are also well known for their repertoire of biochemicals, like phytotoxins and mycotoxins [5, 7, 28, 38]. During the last few decades, at least 268 biomolecules have been reported from Alternaria fungi with phytotoxic, cytotoxic, and antimicrobial properties [5, 25, 43]. To our knowledge, despite the large number of endophytic fungi reported from Cupressaceae, only Alternaria mali and Alternaria sp. are recovered as endophyte from Thuja orientalis [13, 14], without any reference to their bioactivities. Cupressaceae plant family (Coniferals) inhabits earth in a near-global dispersion pattern [9]. The former studies have shown that the abundance, diversity, species composition, and bioactivity of endophytes in Cupressaceae can be significantly influenced by host plant and the locality in which the host plant grows [13, 17, 18]. Moreover, there is an ongoing need for novel sources of biomolecules which could be effective against cancerous and infectious diseases in humans, as well as against biotic stresses in plants. Our group has formerly reported biodiversity and bioactivity of endophytic Penicillium and Trichoderma fungal species from Cupressaceae [17, 18]. Here, we address the association with and bioactivity of Alternaria fungal species from the members of Cupressaceae plant family. We, particularly, show the significant antiproliferative, antifungal, and antibacterial capabilities of endophytic Alternaria spp. from Cupressaceae.
and fruit) were collected from Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and Thuja orientalis (Table 1). Recovery and Identification of Endophytic Fungi Asymptomatic fresh foliar tissues were collected from mature healthy Cupressaceous plants according to the references [13, 14], at least three individuals, in each locality during June to October, 2011. From each plant specimen, three randomly selected pieces were surface sterilized according to the references [17, 18] and in total 820 plant pieces (*0.5 9 0.5 9 1 cm from inner layers) were incubated on potato dextrose agar (PDA) and/or water agar (WA) culture media, at 26–28 °C, for 2–12 weeks. Hyphal tips, from emerging fungi, were isolated, purified, and maintained at 4 °C. Identification of the endophytic Alternaria isolates was achieved by studying their colony morphology and the mechanism of spore production on PCA (Potato Carrot Agar), after 7 days at 22–25 °C, under 16/8-h light intervals. Fungal specimen was stained and studied under microscope, according to the reference [36].
Bioassays Model Target Fungi and Bacteria
Materials and Methods Sampled Locations and Cupressaceae Host Species Several localities at four distinct provinces of Iran, representing the versatility of the country i.e., Fars (South of Iran), Guilan (North of Iran), Hamedan (West of Iran), and Markazi (Center of Iran) were explored for the plant material collection, based on their distinctive biogeographic histories. Plant specimens (twig, leaf, trunk bark,
Antiproliferative and antimicrobial bioactivities of Alternaria-extracted metabolites were examined on the model target fungus Pyricularia oryzae HS-1390 [16–19] and the target phytopathogenic bacteria Bacillus sp., Erwinia amylovora and Pseudomonas syringae (provided by A. Ghasemi, Plant Protection Institute, Tehran, Iran). Also, antifungal activity of endophytic isolates was initially tested, in vitro, against P. oryzae and subsequently against the cypress phytopathogens i.e., Diplodia seriata and
Table 1 Endophytic Alternaria species from each cypress host plant species, according to sampling sites, plant tissue, and fungal isolate Location (Iran)
Host plant species
Fars (Shiraz); South (29.61°N, 52.54°E; 1486 m)
Cupressus arizonica C. sempervirens var. cereiformis C. sempervirens var. cereiformis
Hamedan (Hamedan); West (34.79°N, 48.51°E; 1,900 m)
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Plant segment
Isolate
Endophyte species
Twig
CAE92
A. alternata
Twig
CSE165
A. alternata
Leaf
CSE177
A. alternata
Thuja orientalis
Leaf
POE43
A. alternata
T. orientalis
Leaf
POE72
A. alternata
T. orientalis
Twig
POE103
A. tangelonis
T. orientalis
Twig
POE182
A. pellucida
T. orientalis
Twig
POE104
A. alternata
Antibacterial Activities of Endophytic Alternaria Species
Spencermartinsia viticola [J. Abdollahzadeh; Kurdistan University, Iran; Unpublished] and Phaeobotryon cupressi [1], all from Botryosphaeriales, Ascomycota. All fungal and bacterial strains were maintained at 4 °C for long-term usage. Alternaria’s Metabolite Extraction Extraction of both intra- and extracellular metabolites was performed as described formerly [17]. Briefly, the fresh Alternaria isolates were inoculated into potato dextrose broth (PDB) and incubated for 15 days at 28 °C, 120 rpm, under dark condition. Then, each individual culture broth was extracted with methanol. For extracellular metabolites, the fermentation broth was filtered. Then, the organic solvent methanol (1:1) was added to each individual culture broth and subsequently transferred to 4 °C for 12 h to remove waxy materials. The organic phase was collected, and the solvent was removed by evaporation under 50 °C. The dried methanol extracts were redissolved in double distilled water or DMSO to obtain a final concentration of 250 lg mL-1. Finally, the concentrated extracts were passed through a filtration membrane (d = 0.22 lm) before their bioactivity was assayed. For intracellular metabolites, the mycelia biomasses were harvested, thoroughly washed, and macerated in methanol (1:5, 2 days). Then, the mycelia were homogenized thoroughly. The supernatants were further treated as explained above for the extracellular metabolites. Until being used for bioassays, the secondary metabolites were kept at -20 °C.
each Alternaria isolate and each target fungus was performed on PDA plates. A 5-mm mycelia disk of each Alternaria isolate was subcultured in one side of a Petri plate and incubated at 28 °C. When the Alternaria isolate covered the half of the plate, the target fungus was inoculated at the opposite side and the dual culture was incubated at 28 °C. Daily growth of the target fungus, in the presence of each Alternaria isolate, was compared to that of control until the control strain covered the whole Petri plate. The magnitude of antifungal effect was assessed by the diameter (in mm) of inhibition zones relative to that of control. Growth inhibition rate was calculated by the following formula: Growth inhibition rate (%) = (Dcontrol - Dtreated/Dcontrol) 9 100 %. Bacteriostatic (MIC) and Bactericidal (MBC) Assays The antibacterial activity of intra- and extracellular Alternaria’s metabolites was examined against Bacillus sp., Erwinia amylovora, and P. syringae. First, bacteria were grown to obtain 1 9 106 CFU mL-1. Then, microbroth dilution assays were performed as described for anti-P. oryzae assay, but in nutrient broth (NB) medium. The assay plates were incubated at 28 °C for 16 h. The growth of target bacteria was observed and compared with control to determine the MIC and the minimum bactericidal concentration (MBC). The experiments were performed in triplicate. Data were obtained and presented as IC80 value which represents the concentration of a metabolite that was required for 80 % inhibition in vitro.
Antiproliferative Assays
Statistical Analyses
Antiproliferative/cytotoxic bioactivity of both intra- and extracellular Alternaria’s metabolites was examined against the conidial germination of P. oryzae, as a model. P. oryzae conidial suspension (4 9 104 mL-1; 50 lL including 0.02 % yeast extract) was seeded into each well of a 96-well microtiter plate. The sample extract (50 lL) was added to each well in a serially dilution manner to yield the final concentrations of 250, 125, 62.5, 31.25, 15.62, and 7.81 lg mL-1. The assay plates were incubated at 28 °C for 16 h. For each sample extract, the germination and the size of germ tubes originated from 75 conidia were observed microscopically and compared with the control to determine the minimum inhibitory concentration (MIC). The experiments were performed in triplicate.
Data obtained from antifungal assays were subjected to analysis of variances (ANOVA) and means were compared by least significant differences (LSD) test, using SAS statistical software [37]. The differences among different treatments were determined at 5 % level (P \ 0.05).
Antifungal Assays The in vitro antifungal activity of Alternaria isolates was initially tested against P. oryzae and then against D. seriata, P. cupressi and S. viticola. For this, a dual culture of
Results Host Identity, Biogeography, and Biodiversity of Alternaria Species A total of 820 plant specimens were collected from Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and Thuja orientalis (Cupressaceae) growing at four provinces of Iran. Out of 110 fungal isolates recovered from Cupressaceous host plants (13.4 % colonization rate), 35 fungal species from 19 fungal genera were obtained, among which
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Antibacterial Activities of Endophytic Alternaria Species
Alternaria represented 7.7 % of the isolates. In total, eight Alternaria isolates were recovered which included Alternaria alternata (75 %), Alternaria pellucida, and Alternaria tangelonis. The species Alternaria alternata, with a ubiquitous dispersion pattern, was recovered from three hosts and at two different localities. Furthermore, endophytic Alternaria isolates were recovered only from Fars and Hamedan provinces and only from Cupressus arizonica, Cupressus sempervirens var. cereiformis, and T. orientalis (Table 1). As seen, the majority of Alternaria isolates were recovered at Fars province which has an annual average temperature of 16.58 °C and from twigs and leaves of T. orientalis (Syn. Platycladus orientalis) (Table 1). Antiproliferative/Cytotoxic Bioactivity of Intraand Extra-Cellular Metabolites from Alternaria isolates Pyricularia oryzae has often served as a predictive target model for primary screening of anticancer and antifungal activity of microbial metabolites [17–19]. Our former findings indicate that the extracellular metabolites of cypress fungal endophytes were more bioactive than the intracellular metabolites [17, 18]. Hence, conidial germination and development of germ tube from P. oryzae were adapted for evaluating antiproliferative and growth inhibition activities of both intra- and extracellular metabolites extracted from endophytic Alternaria cultures according to reference [19]. The data are represented in Tables 2 and 3. It was found that Alternaria’s extracellular metabolites significantly inhibited conidial germination of P. oryzae (Table 2). Notably, Alternaria alternata CSE177 and A. alternata CSE165 (both isolated from C. sempervirens var. cereiformis) showed antiproliferative activities at 62.5 lg mL-1 and inhibition of germ tube elongation at a concentration of 7.8 lg mL-1. The isolates A. tangelonis POE103 and A. pellucida POE182 (both from T. orientalis) also showed strong capacities in this regard. Also, as shown in Table 3, Alternaria’s intracellular metabolites were significantly effective against the conidia of P. oryzae. Surprisingly, the intracellular metabolites of A. alternata CAE92 (extracellular metabolite of which showed the least antiproliferative activity among all) here showed the highest cytotoxicity. Indeed, its intracellular metabolite was cytotoxic at 125 lg mL-1 and showed strong inhibition of germ tube elongation at 31.2 lg mL-1. In the next order, were extracellular metabolites from Alternaria alternata CSE177 and A. alternata CSE165. Antifungal Bioactivity of Alternaria Isolates Data on P. oryzae conidia led us to evaluate, using a Petri plate dual culture assay, the antifungal activity of endophytic Alternaria against the model fungus P. oryzae and
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Table 2 Antiproliferative activity (cytotoxicity) of extracellular metabolites from endophytic Alternaria isolates against the conidia of Pyricularia oryzae Isolate
The final concentrations of extracellular metabolites (lg mL-1) 250.0
125.0
62.5
31.2
15.6
7.8
A. alternata CSE177
*
*
*
???
??
??
A. alternata CSE165
*
*
*
???
??
??
A. tangelonis POE103
*
*
*
???
–
–
A. pellucida POE182
*
*
???
???
??
–
A. alternata POE43
*
*
???
??
–
–
A. alternata POE104
*
*
???
??
–
–
A. alternata POE72
*
*
???
??
–
–
A. alternata CAE92
*
*
???
?
–
–
The observations were averages of 4–6 assays * The P. oryzae conidial germination was completely inhibited, ??? strong growth inhibition of germ tube (B1/3 of control); ?? moderate growth inhibition of germ tube (1/3–2/3 of control), ? low growth inhibition of germ tube (C2/3 but less than control); – not inhibited (as control)
Table 3 Antiproliferative activity (cytotoxicity) of intracellular metabolites from endophytic Alternaria isolates against the conidia of Pyricularia oryzae Isolate
The final concentrations of intracellular metabolites (lg mL-1) 250.0
125.0
62.5
31.2
15.6
7.8
A. alternata CAE92
*
*
???
???
?
–
A. alternata CSE177
*
*
???
??
?
–
A. alternata CSE165
*
*
???
??
?
–
A. tangelonis POE103
*
*
???
?
–
–
A. pellucida POE182
*
???
??
??
–
–
A. alternata POE43
*
???
???
??
–
–
A. alternata POE104
*
???
???
?
–
–
A. alternata POE72
*
???
???
?
–
–
The observations were averages of 4–6 assays * The P. oryzae conidial germination was completely inhibited; ??? strong growth inhibition of germ tube (B1/3 of control); ?? moderate growth inhibition of germ tube (1/3–2/3 of control); ? low growth inhibition of germ tube (C2/3 but less than control); – not inhibited (as control)
the cypress fungal phytopathogens D. seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. Indeed, all endophytic Alternaria isolates showed *65–92 % in vitro antifungal activity against all target fungi, especially against P. oryzae (Table 4). In total, A. alternata CAE92 and A. pellucida POE182 were the most effective isolates against all target fungi. Moreover, A. alternata CSE177 and A. alternata CSE165 were also ranked as highly effective.
Antibacterial Activities of Endophytic Alternaria Species Table 4 Antifungal activity of cypress endophytic Alternaria against the cypress phytopathogenic fungi and the model fungus P. oryzae Endophytic isolate
A. pellucida POE182 A. alternata CAE92
Radius zone of hyphae growth (mm) and growth inhibition (%) of the target fungi Diplodia seriata
Phaeobotryon cupressi
5.3 ± 0.57c (88)
6.3 ± 0.57
b
7.3 ± 1.15 (83)
(86)
5.6 ± 0.57 (87)
Pyricularia oryzae
5.3 ± 1.15c (88)
3.6 ± 0.57d (91)
c
3.3 ± 0.57e,d (92)
b
5.3 ± 1.15 (88)
A. alternata CSE177 A. alternata CSE165
8.0 ± 1.73 (81) 8.6 ± 0.57b (80)
7.3 ± 1.52 (83) 7.0 ± 1.0 cd (84)
6.6 ± 0.57 (85) 6.6 ± 0.57b (85)
7.3 ± 1.15c (83) 6.6 ± 0.57c (85)
A. alternata POE43
14.6 ± 0.57a (67)
16.3 ± 0.57a (63)
14.0 ± 1.00a (68)
12.3 ± 0.57b (70)
A. alternata POE104
14.6 ± 0.57a (67)
15.3 ± 0.57a (66)
14.6 ± 1.15a (67)
15.6 ± 0.57a (65)
A. alternata POE72
b
c,d d
Spencermartinsia viticola
a
14.3 ± 1.15 (67) a
c
a
16.6 ± 0.57 (63)
a
14.6 ± 1.15a (67)
a
14.3 ± 1.15 (68)
b
A.tangelonis POE103
15.3 ± 1.15 (65)
13.3 ± 0.57 (70)
15.0 ± 1.00 (66)
12.3 ± 0.57b (72)
Control
44.3 ± 0.57 (0)
45.0 ± 0.00 (0)
45.0 ± 0.00 (0)
44.6 ± 0.57 (0)
Data (significant at P B 0.05) were averages (±standard deviation) of three replicates. Similar letters indicate no significant difference. The rows are ordered according to Diplodia seriata statistical data
Table 5 The antibacterial activities of extra- and intra-cellular metabolites from endophytic Alternaria species Fungal isolate
Target bacteria
Extract concentration (lg mL1-) MICa
MBCb
Extracellular A. alternata CAE92
A. tangelonis POE103
Extracellular
Intracellular
Pseudomonas syringae
7.8
31.2
15.6
Erwinia amylovora
7.8
31.2
15.6
62.5
15.6 7.8
62.5 31.2
31.2 15.6
125 62.5
7.8
62.5
15.6
125
Bacillus sp. Pseudomonas syringae Erwinia amylovora
A. pellucida POE182
Intracellular
62.5
Bacillus sp.
15.6
62.5
31.2
125
Pseudomonas syringae
31.2
62.5
62.5
125
Erwinia amylovora
31.2
62.5
62.5
125
7.8
15.6
15.6
Bacillus sp.
31.2
Data (significant at P B 0.05) were obtained from three replicates. Data are reported as IC80 values a
Minimum inhibitory concentration
b
Minimum bactericidal concentration
A. tangelonis POE103, extracellular metabolite of which was highly effective, showed the least antifungal capability, using Petri plate assay (Table 4). Bacteriostatic and Bactericidal Bioactivity of Intraand Extra-Cellular Metabolites from Alternaria Isolates Considering the data represented in Tables 2, 3, and 4, both intra- and extra-cellular metabolites from endophytic isolates A. alternata CAE92, A. tangelonis POE103, and A. pellucida POE182 were further evaluated for their antibacterial activities against the model bacteria Bacillus sp., E. amylovora, and P. syringae. As shown, all endophytic Alternaria’s metabolites showed bacteriostatic and bactericidal activities against all the three gram-negative and gram-positive bacterial targets (Table 5).
The extracellular metabolites were bacteriostatic at a range of 7.8–31.2 lg mL-1 and bactericidal at a range of 7.8–31.2 lg mL-1. The intracellular metabolites were bacteriostatic at a range of 15.6–62.5 lg mL-1 and bactericidal at a range of 31.2–125 lg mL-1. Furthermore, A. alternata CAE92 and A. tangelonis POE103 were more effective against gram-negative bacteria, while A. pellucida POE182 was more effective against gram-positive bacteria.
Discussion It is becoming evident that all higher plants host one or more untapped endophytic microorganisms [39]. These microorganisms primarily reside in tissues beneath the epidermal cell layers, without causing any symptom of
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Antibacterial Activities of Endophytic Alternaria Species
disease [4]. The secondary metabolites produced by endophytic microorganisms have unique insecticidal, nematicidal, antimicrobial, plant growth-promoting, and plant-strengthening properties which render their hosts resistant to environmental stresses, or function as signaling molecules which trigger such phenomena in their host plants [24, 31, 32, 40]. Furthermore, the bioactive metabolites produced by endophytic microorganisms are most likely sources of novel biomolecules and seem to have unique genetic and biological systems that may have applications in forestry, agriculture, medicine, and bioindustry [2]. The members of Cupressaceae plant family (Coniferales) have a near-global distribution [9] and are in use in forestry, agriculture, and ethnomedicine [12]. This plant family includes, among others, the genera Cupressus, Juniperus, and Thuja. Of these, Cupressus sempervirens is native to the eastern Mediterranean and has a disjunct population in Fars province of Iran. It is recently shown that healthy Cupressaceous trees, i.e., Juniperus virginiana, Cupressus arizonica, and T. orientalis, host a vast number of endophytic fungi [13, 14]. Also, research in our group has shown that almost the same number of fungal species, although with a different subset of species, associates Cupressaceous trees in Iran, i.e., Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and T. orientalis [15]. It has further been indicated that the host plant and the locality of sampling affect the diversity of endophytic mycobiota recovered from the plant family Cupressaceae [13, 17, 18]. Moreover, our research on bioactivity of endophytic fungi from the cypress family has indicated the significant antiproliferative, antifungal, and antibacterial capabilities of its endophytic mycobiota [15, 17, 18]. Others also have demonstrated that endophytic mycobiota from cypress trees could serve as a novel source for producing anticancerous drugs like Taxol and Podophyllotoxin [20, 21, 23]. Here, we explored the endophytic Alternaria mycobiota of aboveground tissues of the healthy members of Cupressaceae plant family and bioactivities thereof. As shown, eight endophytic Alternaria isolates were recovered, representing Alternaria alternata, A. pellucida, and A. tangelonis. This further confirms the dominance of ascomycetous fungi recovered as endophytes from Cupressaceae [13, 14, 17, 18]. The majority of isolates (75 %) belonged to Alternaria alternata, which showed a ubiquitous dispersion at two localities, and on three cypress hosts i.e., Cupressus arizonica, C. sempervirens var. cereiformis, and T. orientalis. The fungi A. pellucida and A. tangelonis were recovered only from T. orientalis plant and at Fars province. Interestingly, the only report of Alternaria mali at US has also been from the host plant T. orientalis [13,
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14]. It seems from our findings that Fars province harbors a more diverse endophytic Alternaria than the other locations. This is in agreement with our former reports [17, 18] and confirms the results obtained at US on the effect of biogeography on endophytic populations [13]. Moreover, taking into account our previous and current results, Cupressus sempervirens var. cereiformis (called Sarve Naz), which is endemic to Fars, Iran, demonstrates a great potential of hosting highly significant bioactive endophytic fungi. This may indicate an evolutionary relationship between the Iranian endemic cypress hosts and the highly effective endophytic fungi. This relationship may play a protective role in cypress hosts against e.g., biotic stresses. Moreover, our findings indicate that endophytic mycobiota of Cupressaceae is a novel source of biodiversity. Indeed, only A. alternata has been previously reported from Cupressaceous trees in Iran [8] and just as a phytopathogen. So, reporting the endophytic association of A. alternata, A. pellucida, and A. tangelonis is novel to the mycoflora of Iran. This further highlights the possibility of finding more fungal taxa by exploring endophytic niches. These findings also indicate that pathogenic fungi can reside inside Cupressaceous plants without causing any disease symptom, as it is shown previously [17, 18]. Notably, it is recently highlighted that endophytic fungi (like Alternaria mali from T. orientalis) may themselves harbor endohyphal bacteria [14], which indicates a sophisticated interrelationship among these microorganisms and the host plant. Alternaria alternata, commonly being a serious plant pathogen and a human opportunistic pathogen, has already been introduced as an endophytic fungus of different host plants with a capability for biosynthesizing promising biomolecules, like anticancer alkaloid Camptothecine [10, 27, 30, 34, 35]. Alternaria pellucida and A. tangelonis, primarily known as plant pathogens, are also recovered as endophytic fungi from the plant Suaeda microphylla, Amaranthaceae [41]. Here, we introduced the endophytic association of such Alternaria species with healthy members of the plant family Cupressaceae. Furthermore, we have shown for the first time the antiproliferative, antifungal, and antibacterial capacities of such endophytic fungi from Cupressaceae. Our data indicated that metabolites from endophytic Alternaria were significantly cytotoxic, with a special reference to extracellular metabolites. For A. alternata CAE92, extracellular metabolite showed the least cytotoxicity, but the intracellular metabolites showed the highest cytotoxicity among all. This isolate, recovered from Cupressus arizonica, was also the most effective fungus, in Petri plate dual culture assays, against the hyphae growth of the target fungi P. oryzae, Phaeobotryon cupressi, and Spencermartinsia viticola with 92, 87, and 88 percent growth inhibition, respectively. So,
Antibacterial Activities of Endophytic Alternaria Species
complicated arrays of factors were probably involved in bioactivity of this isolate. Moreover, both intra- and extracellular metabolites from Alternaria alternata CSE177, and A. alternata CSE165 were among the most cytotoxic metabolites. These two isolates could also inhibit the hyphae growth of all target fungi, in Petri plate dual culture assays, about 80–85 % of that of control. It is interesting that these isolates were recovered from the Iranian indigenous cypress Cupressus sempervirens var. cereiformis (Sarve Naz). Our antifungal assays indicated that all Alternaria isolates could inhibit the hyphae growth of P. oryzae and of the cypress phytopathogens D. seriata, P. cupressi, and S. viticola. In this respect, A. pellucida POE182 and A. alternata CAE92, CSE177 and CSE165 were the most effective isolates. However, A. pellucida POE182 metabolites had shown moderate cytotoxicity. So, close contact of A. pellucida POE182 with other fungi probably involves more complicated factors. The isolate A. tangelonis POE103, compared to other isolates, showed the least antifungal abilities. We have further shown, here, that metabolites from A. alternata CAE92, A.tangelonis POE103, and A. pellucida POE182 exerted bacteriostatic and bactericidal effects against bacteria. In general, the extracellular metabolites were more effective than the intracellular ones. Moreover, the metabolites from A. alternata CAE92 and A. tangelonis POE103 were more effective against gram-negative bacteria than the gram-positive ones. Also, in general, antibacterial properties of metabolites from these two isolates were higher than their antifungal effects on target fungi. This may indicate the significance of those metabolites for surviving in niches occupied mainly by dominant gramnegative bacteria. The metabolites from A. pellucida POE182 were more effective against gram-positive bacteria than the gram-negative ones. The antibacterial potential of metabolites from this isolate was almost identical to its antifungal effects. Considering the strong antifungal effects of this isolate in Petri plate assays, this may suggest a broader range of antibiosis for this isolate, especially toward competing fungi. In conclusion, we have recovered, for the first time, highly bioactive endophytic Alternaria species from the members of cypress family. Our current and former researches indicate that the most bioactive endophytic isolates have been recovered from Iranian indigenous cypress Cupressus sempervirens var. cereiformis (Sarve Naz, at Shiraz, Fars province) which has a disjunct population of Mediterranean cypress. Besides introducing an ecological niche for such pathogenic and allergenic fungi, this raises the possibility of an evolutionarily relationship between this host plant and its endophytic fungi. The antiproliferative, antifungal, and antibacterial capabilities
of the recovered Alternaria isolates potentially could find application in biocontrol and in agrochemical/drug discovery programs. Especially, since endophytic fungi from Cupressaceae and endophytic Alternaria alternata from other host plants have shown ability for production of anticancer compounds [20, 21, 23, 35], endophytic Alternaria from Cupressaceae may emerge as a novel source for anticancer biomolecule discovery. Acknowledgments Dr. Jafar Abdollahzadeh (PhD), Dr. Salar Jamali (PhD), and Abolghasem Ghasemi (MSc) are appreciated for their generous gifting of target fungi and bacteria. We are thankful to our colleagues Dr. Soheila Mirzaei (PhD), Freydoun Babalhavaeji (MSc), and Dr. Javad Hamzei (PhD) at Bu-Ali Sina University of Hamedan, Iran, for their helpful technical discussions. J. S. dedicates this work to the memory of Adrina Ezmiri.
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Antiproliferative, Antifungal, and Antibacterial Activities of Endophytic Alternaria Species from Cupressaceae Jalal Soltani • Mahdieh S. Hosseyni Moghaddam
Received: 5 January 2014 / Accepted: 4 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Recent research has shown the bioprospecting of endophytic fungi from Cupressaceae. Here, we further uncover that the healthy cypress plants such as Cupressus arizonica, Cupressus sempervirens var. cereiformis, and Thuja orientalis host highly bioactive endophytic Alternaria fungal species. Indeed, endophytic Alternaria alternata, Alternaria pellucida, and Alternaria tangelonis were recovered from healthy Cupressaceous trees. Biodiversity and bioactivity of recovered endophytic Alternaria species were a matter of biogeography and host identity. We further extracted such Alternaria’s metabolites and highlighted their significant antiproliferative, growth inhibitory, and antibacterial activities against the model target fungus Pyricularia oryzae and the model pathogenic bacteria Bacillus sp., Erwinia amylovora, and Pseudomonas syringae. In vitro assays also indicated that endophytic Alternaria species significantly inhibited the growth of cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. In conclusion, since the recovered Alternaria species were originally reported as pathogenic and allergenic fungi, our findings suggest a possible ecological niche for them inside the foliar tissues of Cupressaceous trees. Moreover, in this study, the significant bioactivities of endophytic Alternaria species in association with Cupressaceae plant family are reported.
Jalal Soltani and Mahdieh S. Hosseyni Moghaddamhave contributed equally to this work J. Soltani (&) M. S. Hosseyni Moghaddam Phytopathology Department, Bu-Ali Sina University, Hamedan, Iran e-mail: [email protected] J. Soltani Bu-Ali BioNanoTechnology Co. (BABiNT), Hamedan, Iran
Introduction Currently, biodiversity and bioactivity of endophytic microorganisms from many host plant species are being intensively investigated [2, 22, 24, 40]. Recent studies have highlighted that the plant family Cupressaceae hosts a diverse number of endophytic microorganisms [13, 15]. Our group has further shown that such endophytic fungi from Cupressaceae plant family demonstrate a broad range of bioactivities, e.g., antiproliferative, antifungal, and antibacterial activities [15, 17, 18]. In recent years, it has been frequently shown that endophytic microorganisms of any host plant were potentially capable of producing the same bioactive molecules as the host plant [24]. Members of Cupressaceae plant family are used widely in ethnomedicine [12]. Therefore, the bioactivity of metabolites that are produced by endophytic microorganisms and recovered from Cupressaceous trees may resemble the bioactivity of metabolites from host plant itself. Indeed, drug discovery programs have indicated that some endophytic fungi from Cupressaceae were capable of biosynthesizing anticancer biomolecules [20, 21, 23]. Therefore, investigating endophytic fungi colonizing Cupressaceae plant family may reveal novel sources of bioactive molecules, besides fungal biodiversity. The fungus Alternaria (Dothideomycetes, Ascomycota) is a cosmopolitan genus widely distributed in plants, soil, food, and indoor air environment. Alternaria species have adapted a variety of life styles as phytopathogen, weak facultative parasite, saprophyte, and endophyte [42]. A number of Alternaria species have emerged as opportunistic human pathogens particularly in immunocompromised patients [29, 44]. They are also common allergens in humans [6]. Cases of asthma, phaeohyphomycosis, onychomycosis, sinusitis, ulcerated cutaneous infections, keratitis, visceral infection osteomyelitis
123
Antibacterial Activities of Endophytic Alternaria Species
due to Alternaria species, among which Alternaria alternata was the most common one, have been reported [3, 11, 26, 33]. Alternaria fungi are also well known for their repertoire of biochemicals, like phytotoxins and mycotoxins [5, 7, 28, 38]. During the last few decades, at least 268 biomolecules have been reported from Alternaria fungi with phytotoxic, cytotoxic, and antimicrobial properties [5, 25, 43]. To our knowledge, despite the large number of endophytic fungi reported from Cupressaceae, only Alternaria mali and Alternaria sp. are recovered as endophyte from Thuja orientalis [13, 14], without any reference to their bioactivities. Cupressaceae plant family (Coniferals) inhabits earth in a near-global dispersion pattern [9]. The former studies have shown that the abundance, diversity, species composition, and bioactivity of endophytes in Cupressaceae can be significantly influenced by host plant and the locality in which the host plant grows [13, 17, 18]. Moreover, there is an ongoing need for novel sources of biomolecules which could be effective against cancerous and infectious diseases in humans, as well as against biotic stresses in plants. Our group has formerly reported biodiversity and bioactivity of endophytic Penicillium and Trichoderma fungal species from Cupressaceae [17, 18]. Here, we address the association with and bioactivity of Alternaria fungal species from the members of Cupressaceae plant family. We, particularly, show the significant antiproliferative, antifungal, and antibacterial capabilities of endophytic Alternaria spp. from Cupressaceae.
and fruit) were collected from Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and Thuja orientalis (Table 1). Recovery and Identification of Endophytic Fungi Asymptomatic fresh foliar tissues were collected from mature healthy Cupressaceous plants according to the references [13, 14], at least three individuals, in each locality during June to October, 2011. From each plant specimen, three randomly selected pieces were surface sterilized according to the references [17, 18] and in total 820 plant pieces (*0.5 9 0.5 9 1 cm from inner layers) were incubated on potato dextrose agar (PDA) and/or water agar (WA) culture media, at 26–28 °C, for 2–12 weeks. Hyphal tips, from emerging fungi, were isolated, purified, and maintained at 4 °C. Identification of the endophytic Alternaria isolates was achieved by studying their colony morphology and the mechanism of spore production on PCA (Potato Carrot Agar), after 7 days at 22–25 °C, under 16/8-h light intervals. Fungal specimen was stained and studied under microscope, according to the reference [36].
Bioassays Model Target Fungi and Bacteria
Materials and Methods Sampled Locations and Cupressaceae Host Species Several localities at four distinct provinces of Iran, representing the versatility of the country i.e., Fars (South of Iran), Guilan (North of Iran), Hamedan (West of Iran), and Markazi (Center of Iran) were explored for the plant material collection, based on their distinctive biogeographic histories. Plant specimens (twig, leaf, trunk bark,
Antiproliferative and antimicrobial bioactivities of Alternaria-extracted metabolites were examined on the model target fungus Pyricularia oryzae HS-1390 [16–19] and the target phytopathogenic bacteria Bacillus sp., Erwinia amylovora and Pseudomonas syringae (provided by A. Ghasemi, Plant Protection Institute, Tehran, Iran). Also, antifungal activity of endophytic isolates was initially tested, in vitro, against P. oryzae and subsequently against the cypress phytopathogens i.e., Diplodia seriata and
Table 1 Endophytic Alternaria species from each cypress host plant species, according to sampling sites, plant tissue, and fungal isolate Location (Iran)
Host plant species
Fars (Shiraz); South (29.61°N, 52.54°E; 1486 m)
Cupressus arizonica C. sempervirens var. cereiformis C. sempervirens var. cereiformis
Hamedan (Hamedan); West (34.79°N, 48.51°E; 1,900 m)
123
Plant segment
Isolate
Endophyte species
Twig
CAE92
A. alternata
Twig
CSE165
A. alternata
Leaf
CSE177
A. alternata
Thuja orientalis
Leaf
POE43
A. alternata
T. orientalis
Leaf
POE72
A. alternata
T. orientalis
Twig
POE103
A. tangelonis
T. orientalis
Twig
POE182
A. pellucida
T. orientalis
Twig
POE104
A. alternata
Antibacterial Activities of Endophytic Alternaria Species
Spencermartinsia viticola [J. Abdollahzadeh; Kurdistan University, Iran; Unpublished] and Phaeobotryon cupressi [1], all from Botryosphaeriales, Ascomycota. All fungal and bacterial strains were maintained at 4 °C for long-term usage. Alternaria’s Metabolite Extraction Extraction of both intra- and extracellular metabolites was performed as described formerly [17]. Briefly, the fresh Alternaria isolates were inoculated into potato dextrose broth (PDB) and incubated for 15 days at 28 °C, 120 rpm, under dark condition. Then, each individual culture broth was extracted with methanol. For extracellular metabolites, the fermentation broth was filtered. Then, the organic solvent methanol (1:1) was added to each individual culture broth and subsequently transferred to 4 °C for 12 h to remove waxy materials. The organic phase was collected, and the solvent was removed by evaporation under 50 °C. The dried methanol extracts were redissolved in double distilled water or DMSO to obtain a final concentration of 250 lg mL-1. Finally, the concentrated extracts were passed through a filtration membrane (d = 0.22 lm) before their bioactivity was assayed. For intracellular metabolites, the mycelia biomasses were harvested, thoroughly washed, and macerated in methanol (1:5, 2 days). Then, the mycelia were homogenized thoroughly. The supernatants were further treated as explained above for the extracellular metabolites. Until being used for bioassays, the secondary metabolites were kept at -20 °C.
each Alternaria isolate and each target fungus was performed on PDA plates. A 5-mm mycelia disk of each Alternaria isolate was subcultured in one side of a Petri plate and incubated at 28 °C. When the Alternaria isolate covered the half of the plate, the target fungus was inoculated at the opposite side and the dual culture was incubated at 28 °C. Daily growth of the target fungus, in the presence of each Alternaria isolate, was compared to that of control until the control strain covered the whole Petri plate. The magnitude of antifungal effect was assessed by the diameter (in mm) of inhibition zones relative to that of control. Growth inhibition rate was calculated by the following formula: Growth inhibition rate (%) = (Dcontrol - Dtreated/Dcontrol) 9 100 %. Bacteriostatic (MIC) and Bactericidal (MBC) Assays The antibacterial activity of intra- and extracellular Alternaria’s metabolites was examined against Bacillus sp., Erwinia amylovora, and P. syringae. First, bacteria were grown to obtain 1 9 106 CFU mL-1. Then, microbroth dilution assays were performed as described for anti-P. oryzae assay, but in nutrient broth (NB) medium. The assay plates were incubated at 28 °C for 16 h. The growth of target bacteria was observed and compared with control to determine the MIC and the minimum bactericidal concentration (MBC). The experiments were performed in triplicate. Data were obtained and presented as IC80 value which represents the concentration of a metabolite that was required for 80 % inhibition in vitro.
Antiproliferative Assays
Statistical Analyses
Antiproliferative/cytotoxic bioactivity of both intra- and extracellular Alternaria’s metabolites was examined against the conidial germination of P. oryzae, as a model. P. oryzae conidial suspension (4 9 104 mL-1; 50 lL including 0.02 % yeast extract) was seeded into each well of a 96-well microtiter plate. The sample extract (50 lL) was added to each well in a serially dilution manner to yield the final concentrations of 250, 125, 62.5, 31.25, 15.62, and 7.81 lg mL-1. The assay plates were incubated at 28 °C for 16 h. For each sample extract, the germination and the size of germ tubes originated from 75 conidia were observed microscopically and compared with the control to determine the minimum inhibitory concentration (MIC). The experiments were performed in triplicate.
Data obtained from antifungal assays were subjected to analysis of variances (ANOVA) and means were compared by least significant differences (LSD) test, using SAS statistical software [37]. The differences among different treatments were determined at 5 % level (P \ 0.05).
Antifungal Assays The in vitro antifungal activity of Alternaria isolates was initially tested against P. oryzae and then against D. seriata, P. cupressi and S. viticola. For this, a dual culture of
Results Host Identity, Biogeography, and Biodiversity of Alternaria Species A total of 820 plant specimens were collected from Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and Thuja orientalis (Cupressaceae) growing at four provinces of Iran. Out of 110 fungal isolates recovered from Cupressaceous host plants (13.4 % colonization rate), 35 fungal species from 19 fungal genera were obtained, among which
123
Antibacterial Activities of Endophytic Alternaria Species
Alternaria represented 7.7 % of the isolates. In total, eight Alternaria isolates were recovered which included Alternaria alternata (75 %), Alternaria pellucida, and Alternaria tangelonis. The species Alternaria alternata, with a ubiquitous dispersion pattern, was recovered from three hosts and at two different localities. Furthermore, endophytic Alternaria isolates were recovered only from Fars and Hamedan provinces and only from Cupressus arizonica, Cupressus sempervirens var. cereiformis, and T. orientalis (Table 1). As seen, the majority of Alternaria isolates were recovered at Fars province which has an annual average temperature of 16.58 °C and from twigs and leaves of T. orientalis (Syn. Platycladus orientalis) (Table 1). Antiproliferative/Cytotoxic Bioactivity of Intraand Extra-Cellular Metabolites from Alternaria isolates Pyricularia oryzae has often served as a predictive target model for primary screening of anticancer and antifungal activity of microbial metabolites [17–19]. Our former findings indicate that the extracellular metabolites of cypress fungal endophytes were more bioactive than the intracellular metabolites [17, 18]. Hence, conidial germination and development of germ tube from P. oryzae were adapted for evaluating antiproliferative and growth inhibition activities of both intra- and extracellular metabolites extracted from endophytic Alternaria cultures according to reference [19]. The data are represented in Tables 2 and 3. It was found that Alternaria’s extracellular metabolites significantly inhibited conidial germination of P. oryzae (Table 2). Notably, Alternaria alternata CSE177 and A. alternata CSE165 (both isolated from C. sempervirens var. cereiformis) showed antiproliferative activities at 62.5 lg mL-1 and inhibition of germ tube elongation at a concentration of 7.8 lg mL-1. The isolates A. tangelonis POE103 and A. pellucida POE182 (both from T. orientalis) also showed strong capacities in this regard. Also, as shown in Table 3, Alternaria’s intracellular metabolites were significantly effective against the conidia of P. oryzae. Surprisingly, the intracellular metabolites of A. alternata CAE92 (extracellular metabolite of which showed the least antiproliferative activity among all) here showed the highest cytotoxicity. Indeed, its intracellular metabolite was cytotoxic at 125 lg mL-1 and showed strong inhibition of germ tube elongation at 31.2 lg mL-1. In the next order, were extracellular metabolites from Alternaria alternata CSE177 and A. alternata CSE165. Antifungal Bioactivity of Alternaria Isolates Data on P. oryzae conidia led us to evaluate, using a Petri plate dual culture assay, the antifungal activity of endophytic Alternaria against the model fungus P. oryzae and
123
Table 2 Antiproliferative activity (cytotoxicity) of extracellular metabolites from endophytic Alternaria isolates against the conidia of Pyricularia oryzae Isolate
The final concentrations of extracellular metabolites (lg mL-1) 250.0
125.0
62.5
31.2
15.6
7.8
A. alternata CSE177
*
*
*
???
??
??
A. alternata CSE165
*
*
*
???
??
??
A. tangelonis POE103
*
*
*
???
–
–
A. pellucida POE182
*
*
???
???
??
–
A. alternata POE43
*
*
???
??
–
–
A. alternata POE104
*
*
???
??
–
–
A. alternata POE72
*
*
???
??
–
–
A. alternata CAE92
*
*
???
?
–
–
The observations were averages of 4–6 assays * The P. oryzae conidial germination was completely inhibited, ??? strong growth inhibition of germ tube (B1/3 of control); ?? moderate growth inhibition of germ tube (1/3–2/3 of control), ? low growth inhibition of germ tube (C2/3 but less than control); – not inhibited (as control)
Table 3 Antiproliferative activity (cytotoxicity) of intracellular metabolites from endophytic Alternaria isolates against the conidia of Pyricularia oryzae Isolate
The final concentrations of intracellular metabolites (lg mL-1) 250.0
125.0
62.5
31.2
15.6
7.8
A. alternata CAE92
*
*
???
???
?
–
A. alternata CSE177
*
*
???
??
?
–
A. alternata CSE165
*
*
???
??
?
–
A. tangelonis POE103
*
*
???
?
–
–
A. pellucida POE182
*
???
??
??
–
–
A. alternata POE43
*
???
???
??
–
–
A. alternata POE104
*
???
???
?
–
–
A. alternata POE72
*
???
???
?
–
–
The observations were averages of 4–6 assays * The P. oryzae conidial germination was completely inhibited; ??? strong growth inhibition of germ tube (B1/3 of control); ?? moderate growth inhibition of germ tube (1/3–2/3 of control); ? low growth inhibition of germ tube (C2/3 but less than control); – not inhibited (as control)
the cypress fungal phytopathogens D. seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. Indeed, all endophytic Alternaria isolates showed *65–92 % in vitro antifungal activity against all target fungi, especially against P. oryzae (Table 4). In total, A. alternata CAE92 and A. pellucida POE182 were the most effective isolates against all target fungi. Moreover, A. alternata CSE177 and A. alternata CSE165 were also ranked as highly effective.
Antibacterial Activities of Endophytic Alternaria Species Table 4 Antifungal activity of cypress endophytic Alternaria against the cypress phytopathogenic fungi and the model fungus P. oryzae Endophytic isolate
A. pellucida POE182 A. alternata CAE92
Radius zone of hyphae growth (mm) and growth inhibition (%) of the target fungi Diplodia seriata
Phaeobotryon cupressi
5.3 ± 0.57c (88)
6.3 ± 0.57
b
7.3 ± 1.15 (83)
(86)
5.6 ± 0.57 (87)
Pyricularia oryzae
5.3 ± 1.15c (88)
3.6 ± 0.57d (91)
c
3.3 ± 0.57e,d (92)
b
5.3 ± 1.15 (88)
A. alternata CSE177 A. alternata CSE165
8.0 ± 1.73 (81) 8.6 ± 0.57b (80)
7.3 ± 1.52 (83) 7.0 ± 1.0 cd (84)
6.6 ± 0.57 (85) 6.6 ± 0.57b (85)
7.3 ± 1.15c (83) 6.6 ± 0.57c (85)
A. alternata POE43
14.6 ± 0.57a (67)
16.3 ± 0.57a (63)
14.0 ± 1.00a (68)
12.3 ± 0.57b (70)
A. alternata POE104
14.6 ± 0.57a (67)
15.3 ± 0.57a (66)
14.6 ± 1.15a (67)
15.6 ± 0.57a (65)
A. alternata POE72
b
c,d d
Spencermartinsia viticola
a
14.3 ± 1.15 (67) a
c
a
16.6 ± 0.57 (63)
a
14.6 ± 1.15a (67)
a
14.3 ± 1.15 (68)
b
A.tangelonis POE103
15.3 ± 1.15 (65)
13.3 ± 0.57 (70)
15.0 ± 1.00 (66)
12.3 ± 0.57b (72)
Control
44.3 ± 0.57 (0)
45.0 ± 0.00 (0)
45.0 ± 0.00 (0)
44.6 ± 0.57 (0)
Data (significant at P B 0.05) were averages (±standard deviation) of three replicates. Similar letters indicate no significant difference. The rows are ordered according to Diplodia seriata statistical data
Table 5 The antibacterial activities of extra- and intra-cellular metabolites from endophytic Alternaria species Fungal isolate
Target bacteria
Extract concentration (lg mL1-) MICa
MBCb
Extracellular A. alternata CAE92
A. tangelonis POE103
Extracellular
Intracellular
Pseudomonas syringae
7.8
31.2
15.6
Erwinia amylovora
7.8
31.2
15.6
62.5
15.6 7.8
62.5 31.2
31.2 15.6
125 62.5
7.8
62.5
15.6
125
Bacillus sp. Pseudomonas syringae Erwinia amylovora
A. pellucida POE182
Intracellular
62.5
Bacillus sp.
15.6
62.5
31.2
125
Pseudomonas syringae
31.2
62.5
62.5
125
Erwinia amylovora
31.2
62.5
62.5
125
7.8
15.6
15.6
Bacillus sp.
31.2
Data (significant at P B 0.05) were obtained from three replicates. Data are reported as IC80 values a
Minimum inhibitory concentration
b
Minimum bactericidal concentration
A. tangelonis POE103, extracellular metabolite of which was highly effective, showed the least antifungal capability, using Petri plate assay (Table 4). Bacteriostatic and Bactericidal Bioactivity of Intraand Extra-Cellular Metabolites from Alternaria Isolates Considering the data represented in Tables 2, 3, and 4, both intra- and extra-cellular metabolites from endophytic isolates A. alternata CAE92, A. tangelonis POE103, and A. pellucida POE182 were further evaluated for their antibacterial activities against the model bacteria Bacillus sp., E. amylovora, and P. syringae. As shown, all endophytic Alternaria’s metabolites showed bacteriostatic and bactericidal activities against all the three gram-negative and gram-positive bacterial targets (Table 5).
The extracellular metabolites were bacteriostatic at a range of 7.8–31.2 lg mL-1 and bactericidal at a range of 7.8–31.2 lg mL-1. The intracellular metabolites were bacteriostatic at a range of 15.6–62.5 lg mL-1 and bactericidal at a range of 31.2–125 lg mL-1. Furthermore, A. alternata CAE92 and A. tangelonis POE103 were more effective against gram-negative bacteria, while A. pellucida POE182 was more effective against gram-positive bacteria.
Discussion It is becoming evident that all higher plants host one or more untapped endophytic microorganisms [39]. These microorganisms primarily reside in tissues beneath the epidermal cell layers, without causing any symptom of
123
Antibacterial Activities of Endophytic Alternaria Species
disease [4]. The secondary metabolites produced by endophytic microorganisms have unique insecticidal, nematicidal, antimicrobial, plant growth-promoting, and plant-strengthening properties which render their hosts resistant to environmental stresses, or function as signaling molecules which trigger such phenomena in their host plants [24, 31, 32, 40]. Furthermore, the bioactive metabolites produced by endophytic microorganisms are most likely sources of novel biomolecules and seem to have unique genetic and biological systems that may have applications in forestry, agriculture, medicine, and bioindustry [2]. The members of Cupressaceae plant family (Coniferales) have a near-global distribution [9] and are in use in forestry, agriculture, and ethnomedicine [12]. This plant family includes, among others, the genera Cupressus, Juniperus, and Thuja. Of these, Cupressus sempervirens is native to the eastern Mediterranean and has a disjunct population in Fars province of Iran. It is recently shown that healthy Cupressaceous trees, i.e., Juniperus virginiana, Cupressus arizonica, and T. orientalis, host a vast number of endophytic fungi [13, 14]. Also, research in our group has shown that almost the same number of fungal species, although with a different subset of species, associates Cupressaceous trees in Iran, i.e., Cupressus arizonica, Cupressus sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp., and T. orientalis [15]. It has further been indicated that the host plant and the locality of sampling affect the diversity of endophytic mycobiota recovered from the plant family Cupressaceae [13, 17, 18]. Moreover, our research on bioactivity of endophytic fungi from the cypress family has indicated the significant antiproliferative, antifungal, and antibacterial capabilities of its endophytic mycobiota [15, 17, 18]. Others also have demonstrated that endophytic mycobiota from cypress trees could serve as a novel source for producing anticancerous drugs like Taxol and Podophyllotoxin [20, 21, 23]. Here, we explored the endophytic Alternaria mycobiota of aboveground tissues of the healthy members of Cupressaceae plant family and bioactivities thereof. As shown, eight endophytic Alternaria isolates were recovered, representing Alternaria alternata, A. pellucida, and A. tangelonis. This further confirms the dominance of ascomycetous fungi recovered as endophytes from Cupressaceae [13, 14, 17, 18]. The majority of isolates (75 %) belonged to Alternaria alternata, which showed a ubiquitous dispersion at two localities, and on three cypress hosts i.e., Cupressus arizonica, C. sempervirens var. cereiformis, and T. orientalis. The fungi A. pellucida and A. tangelonis were recovered only from T. orientalis plant and at Fars province. Interestingly, the only report of Alternaria mali at US has also been from the host plant T. orientalis [13,
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14]. It seems from our findings that Fars province harbors a more diverse endophytic Alternaria than the other locations. This is in agreement with our former reports [17, 18] and confirms the results obtained at US on the effect of biogeography on endophytic populations [13]. Moreover, taking into account our previous and current results, Cupressus sempervirens var. cereiformis (called Sarve Naz), which is endemic to Fars, Iran, demonstrates a great potential of hosting highly significant bioactive endophytic fungi. This may indicate an evolutionary relationship between the Iranian endemic cypress hosts and the highly effective endophytic fungi. This relationship may play a protective role in cypress hosts against e.g., biotic stresses. Moreover, our findings indicate that endophytic mycobiota of Cupressaceae is a novel source of biodiversity. Indeed, only A. alternata has been previously reported from Cupressaceous trees in Iran [8] and just as a phytopathogen. So, reporting the endophytic association of A. alternata, A. pellucida, and A. tangelonis is novel to the mycoflora of Iran. This further highlights the possibility of finding more fungal taxa by exploring endophytic niches. These findings also indicate that pathogenic fungi can reside inside Cupressaceous plants without causing any disease symptom, as it is shown previously [17, 18]. Notably, it is recently highlighted that endophytic fungi (like Alternaria mali from T. orientalis) may themselves harbor endohyphal bacteria [14], which indicates a sophisticated interrelationship among these microorganisms and the host plant. Alternaria alternata, commonly being a serious plant pathogen and a human opportunistic pathogen, has already been introduced as an endophytic fungus of different host plants with a capability for biosynthesizing promising biomolecules, like anticancer alkaloid Camptothecine [10, 27, 30, 34, 35]. Alternaria pellucida and A. tangelonis, primarily known as plant pathogens, are also recovered as endophytic fungi from the plant Suaeda microphylla, Amaranthaceae [41]. Here, we introduced the endophytic association of such Alternaria species with healthy members of the plant family Cupressaceae. Furthermore, we have shown for the first time the antiproliferative, antifungal, and antibacterial capacities of such endophytic fungi from Cupressaceae. Our data indicated that metabolites from endophytic Alternaria were significantly cytotoxic, with a special reference to extracellular metabolites. For A. alternata CAE92, extracellular metabolite showed the least cytotoxicity, but the intracellular metabolites showed the highest cytotoxicity among all. This isolate, recovered from Cupressus arizonica, was also the most effective fungus, in Petri plate dual culture assays, against the hyphae growth of the target fungi P. oryzae, Phaeobotryon cupressi, and Spencermartinsia viticola with 92, 87, and 88 percent growth inhibition, respectively. So,
Antibacterial Activities of Endophytic Alternaria Species
complicated arrays of factors were probably involved in bioactivity of this isolate. Moreover, both intra- and extracellular metabolites from Alternaria alternata CSE177, and A. alternata CSE165 were among the most cytotoxic metabolites. These two isolates could also inhibit the hyphae growth of all target fungi, in Petri plate dual culture assays, about 80–85 % of that of control. It is interesting that these isolates were recovered from the Iranian indigenous cypress Cupressus sempervirens var. cereiformis (Sarve Naz). Our antifungal assays indicated that all Alternaria isolates could inhibit the hyphae growth of P. oryzae and of the cypress phytopathogens D. seriata, P. cupressi, and S. viticola. In this respect, A. pellucida POE182 and A. alternata CAE92, CSE177 and CSE165 were the most effective isolates. However, A. pellucida POE182 metabolites had shown moderate cytotoxicity. So, close contact of A. pellucida POE182 with other fungi probably involves more complicated factors. The isolate A. tangelonis POE103, compared to other isolates, showed the least antifungal abilities. We have further shown, here, that metabolites from A. alternata CAE92, A.tangelonis POE103, and A. pellucida POE182 exerted bacteriostatic and bactericidal effects against bacteria. In general, the extracellular metabolites were more effective than the intracellular ones. Moreover, the metabolites from A. alternata CAE92 and A. tangelonis POE103 were more effective against gram-negative bacteria than the gram-positive ones. Also, in general, antibacterial properties of metabolites from these two isolates were higher than their antifungal effects on target fungi. This may indicate the significance of those metabolites for surviving in niches occupied mainly by dominant gramnegative bacteria. The metabolites from A. pellucida POE182 were more effective against gram-positive bacteria than the gram-negative ones. The antibacterial potential of metabolites from this isolate was almost identical to its antifungal effects. Considering the strong antifungal effects of this isolate in Petri plate assays, this may suggest a broader range of antibiosis for this isolate, especially toward competing fungi. In conclusion, we have recovered, for the first time, highly bioactive endophytic Alternaria species from the members of cypress family. Our current and former researches indicate that the most bioactive endophytic isolates have been recovered from Iranian indigenous cypress Cupressus sempervirens var. cereiformis (Sarve Naz, at Shiraz, Fars province) which has a disjunct population of Mediterranean cypress. Besides introducing an ecological niche for such pathogenic and allergenic fungi, this raises the possibility of an evolutionarily relationship between this host plant and its endophytic fungi. The antiproliferative, antifungal, and antibacterial capabilities
of the recovered Alternaria isolates potentially could find application in biocontrol and in agrochemical/drug discovery programs. Especially, since endophytic fungi from Cupressaceae and endophytic Alternaria alternata from other host plants have shown ability for production of anticancer compounds [20, 21, 23, 35], endophytic Alternaria from Cupressaceae may emerge as a novel source for anticancer biomolecule discovery. Acknowledgments Dr. Jafar Abdollahzadeh (PhD), Dr. Salar Jamali (PhD), and Abolghasem Ghasemi (MSc) are appreciated for their generous gifting of target fungi and bacteria. We are thankful to our colleagues Dr. Soheila Mirzaei (PhD), Freydoun Babalhavaeji (MSc), and Dr. Javad Hamzei (PhD) at Bu-Ali Sina University of Hamedan, Iran, for their helpful technical discussions. J. S. dedicates this work to the memory of Adrina Ezmiri.
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