Psychrophilic endophytic fungi with biological activity

Psychrophilic endophytic fungi with biological activity inhabit Cupressaceae plant family Mahdieh S. Hosseyni Moghaddam & Jalal Soltani

Symbiosis ISSN 0334-5114 Volume 63 Number 2 Symbiosis (2014) 63:79-86 DOI 10.1007/s13199-014-0290-2

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Author's personal copy Symbiosis (2014) 63:79–86 DOI 10.1007/s13199-014-0290-2

Psychrophilic endophytic fungi with biological activity inhabit Cupressaceae plant family Mahdieh S. Hosseyni Moghaddam & Jalal Soltani

Received: 19 March 2014 / Accepted: 4 August 2014 / Published online: 14 August 2014 # Springer Science+Business Media Dordrecht 2014

Abstract Psychrophilic microorganisms are cold-adapted organisms that have an optimum growth temperature below 15 °C, and often below 5 °C. Endophytic microorganisms live inside healthy plants and biosynthesize an array of secondary metabolites which confer major ecological benefits to their host. We provide information, for the first time, on an endophytic association between bioactive psychrophilic fungi and trees in Cupressaceae plant family living in temperate to cold, semi-arid habitats. We have recovered psychrophilic endophytic fungi (PEF) from healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens and Thuja orientalis (Cupressaceae, Coniferales). In total, 23 such fungi were found out of 110 endophytic fungal isolates. They were identified as ascomycetous fungi, more specifically Phoma herbarum, Phoma sp. and Dothideomycetes spp., all from Dothideomycetes. The optimal growth temperature for all these 23 fungal isolates was 4 °C, and the PEF isolates were able to biosynthesize secondary metabolite at this temperature. Extracted metabolites from PEF showed significant antiproliferative/cytotoxic, antifungal and antibacterial effects against phytopathogenic fungi and bacteria. Of special interest was their antibacterial activity against the ice-nucleation active bacterium Pseudomonas syringae. Accordingly, we suggest that evergreen Cupressaceae plants may benefit from their psychrophilic endophytic fungi during cold stress. Whether Mahdieh S. Hosseyni Moghaddam and Jalal Soltani contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13199-014-0290-2) contains supplementary material, which is available to authorized users. M. S. Hosseyni Moghaddam : J. Soltani Phytopathology Department, Bu-Ali Sina University, Hamedan, Iran J. Soltani (*) BioNanoTechnology Co. (BABiNT), Hamedan, Iran e-mail: [email protected]

such endosymbionts confer any ecological and evolutionary benefits to their host plants remains to be investigated in vivo. Keywords Psychrophilic endophytic fungi . Cold stress tolerance . Cupressaceae . Biological activity . Cupressus . Thuja

1 Introduction Many habitats on the earth are continuously or seasonally faced with low temperatures (Huston 2008; Margesin and Miteva 2011). Cold biosphere includes aquatic and terrestrial environments, but many temperate habitats often have cold temperatures during autumn and winter (Margesin and Miteva 2011). Psychrophilic microorganisms are defined as coldadapted organisms that best grow at temperatures below 5 °C. Psychrophilic fungi, including yeasts and filamentous fungi, are adapted to cold ecosystems like the Arctic and Antarctic zones (Robinson 2001; Frisvad 2008). However, except for the phytopathogenic snow molds (Hsiang et al. 1999), the endophytic association of psychrophilic fungi with plants in temperate biosphere has not been documented. Research in the last two decades has highlighted the fact that most healthy plants on the planet harbor one or more endophytic microorganism (Strobel et al. 2004). These endophytic microorganisms produce a vast array of secondary metabolites which can confer major ecological benefits to their host plants (Kusari et al. 2012; Kusari et al. 2013). Therefore endophytic microorganisms from many host plant species are currently being intensively investigated (Aly et al. 2010; Kusari et al. 2012, 2013; Strobel et al. 2004), especially for their use in pharmacology and plant stress management (Aly et al. 2010; Ryan et al. 2007; Ryan et al. 2008; Strobel et al. 2004).

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The healthy plants of the globally dispersed Cupressaceae (Coniferals) harbor a vast number of endophytic microorganisms (Fralish and Franklin 2002; Hoffman and Arnold 2008, 2010; Hosseyni Moghaddam 2013) with a broad range of bioactivities (Hosseyni Moghaddam et al. 2013; Hosseyni Moghaddam and Soltani 2014; Kumaran et al. 2008; Kour et al. 2008; Kusari et al. 2009; Soltani and Hosseyni Moghaddam 2014a, b). Because of their worldwide distribution, the evergreen Cupressaceae plants are frequently exposed to freezing temperatures. To remain evergreen and tolerate the low temperatures during the whole year, special adaptations are needed but the mechanisms are not known. Recent findings on other plants provide evidence that endophytic fungal symbionts may play a role in plant stress tolerance, especially to disease, drought, heat, high soil salinity, oxidative stress and heavy metal toxicity (Singh et al. 2011), but there is no information on symbiont-mediated cold tolerance in plants. In the present study the endophytic association of psychrophilic fungi with healthy plants of Cuppresaceae was explored and the contribution of such psychrophilic endophytic fungi to the ecology of these evergreen plants was investigated in vitro.

M.S. Hosseyni Moghaddam, J. Soltani

2.3 Identification of endophytic fungi Fungal growth was obtained by plating the isolates onto PDA culture media. The plates were continuously monitored for spore formation. Specimens were stained by Aniline blue and studied under Leica DMLB and Leitz bright-field light microscopes. The endophytic fungi were identified on the basis of their morphology in culture media using Barnett (1960) and Boerema et al. (2004), and analysis of the internal transcribed spacer sequences of nuclear ribosomal DNA (ITS1-5.8S-ITS2 rDNA sequence). Six representative isolates, from four identical groups, were selected for ITS rDNAs amplification and sequencing. For DNA extraction, the colonies of six representative fungal isolates were grown in 150 ml Erlenmeyer flasks containing 20 mL Potato-Dextrose-Broth (PDB) at 4 °C. After 15 days, genomic DNA of each isolate was extracted using SDS-CTAB method (Zhang et al. 1996) to amplify ITS15.8S-ITS2 rDNA sequence using the universal primers ITS1 and ITS4 (White et al. 1990). The PCR products were sequenced using ITS1 primer (Pouya-Gostar Gene, Tehran, Iran) and the sequences were analyzed online with BLAST in the GenBank database (available at: http://www.ncbi.nlm. nih.gov/BLAST). The megablast ITS rDNA queries identified GenBank accessions with the greatest identity (≥97 %) and Evalues of zero (Table 2).

2 Materials and methods 2.4 Metabolite extraction 2.1 Plant material Plant specimens (from stem, twig, and leaf) were collected from healthy foliar tissues of three cypress species i.e. Cupressus arizonica Greene, Cupressus sempervirens L. var. cereiformis Rehd and Thuja orientalis L. (Syn. Platycladus orientalis) (Cupressaceae) at four distinct locations in Iran (Table 1).

The fresh psychrophilic endophytic fungal isolates were cultivated, individually, in PDB in Erlenmeyer flasks and incubated for 15 days at 4 °C, under dark condition. After the fermentation process, each individual culture broth was extracted with methanol according to Hosseyni Moghaddam and Soltani (2014). We extracted separately the extracellular and intracellular secondary metabolites. Until used for bioassays, the extracted metabolites were kept at −20 °C.

2.2 Endophytic fungi recovery

2.5 Antiproliferative bioassay

Fresh asymptomatic foliar tissues from mature healthy Cupressaceous plants were sampled from branches at the outer canopy, during summer and autumn of 2011, using the method of Hoffman and Arnold (2010). Recovery of psychrophilic endophytic fungi was initially performed according to Soltani and Hosseyni Moghaddam (2014a) at 28 °C. However, subsequently we used low temperature (4 °C) to recover symbionts from apparently uninfected tissues. Accordingly, the Petri plates containing such tissue fragments were incubated at 4 °C and inspected for hyphae growth daily for 12 weeks. Hyphal tips, from emerged fungi, were then sub-cultured onto PotatoDextrose-Agar (PDA) medium, again at 4 °C, and brought into pure culture.

Antiproliferative bioactivity of the intra- and extra-cellular secondary metabolites from PEF isolates were examined on the conidial germination of Pyricularia oryzae (Tel. Magnaporthe grisea) (Kobayashi et al. 1996; Hosseyni Moghaddam and Soltani 2013). Accordingly, conidial suspension of P. oryzae (4×104 mL−1; 50 μL including 0.02 % yeast extract) was seeded into each well of a 96 well microtiter plate. The sample intra- or extra-cellular extract (50 μL) 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 μg mL−1. The assay plates were incubated at 28 °C for 16 h. The germination and the size of germ tubes originated from conidia were observed microscopically and compared with those of the control to determine the minimum inhibitory

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Table 1 Locations and characteristics of sampled sites and plant species as well as plant tissue and psychrophilic endophytic fungal isolate Location (Iran)

Elevation (m)

Annual average temperature (°C)

Annual precipitation (mm)

Isolate code

Host plant species

Plant segment

Fars (Shiraz) (29°61’N, 52°54’E) South of Iran

1486

Max. 25.62 Min. 9.19

305.6

Guilan (Manjil; Astara) (36°32’N, 49°11’E; 38°41’N, 48°87’E) North of Iran Hamedan (Hamedan) (34°79’N, 48°51’E) West of Iran

1050; −27

Max. 20.54 Min. 11.13

1,355.5

CSE8 CSE187 CSE192 CAE130 CAE205 CAE208 POE119 POE215 POE196 POE226

Cupressus sempervirens var. cereiformis C. sempervirens var. cereiformis C. sempervirens var. cereiformis Cupressus arizonica C. arizonica C. arizonica Thuja orientalis T. orientalis T. orientalis T. orientalis

Stem Leaf Leaf Leaf Twig Twig Twig Twig Stem Stem

1900

Max. 19.26 Min. 2.13

323.7

POE115 POE167 POE191 POE198 POE199 CAE2 CAE3 CAE161

T. orientalis T. orientalis T. orientalis T. orientalis T. orientalis C. arizonica C. arizonica C. arizonica

Stem Leaf Stem Twig Stem Stem Stem Leaf

1775

Max. 20.78 Min. 6.94

345.7

CAE4 CAE5 CAE6 CAE7 CAE143

C. arizonica C. arizonica C. arizonica C. arizonica C. arizonica

Leaf Stem Leaf Leaf Stem

Markazi (Mahalat) (33°91’N, 50°45’E) Center of Iran

concentration (MIC). The experiments were performed in triplicate. 2.6 Antibacterial bioassay To test the antibacterial activity of PEF metabolites, the target plant-associated bacteria Bacillus sp., Erwinia amylovora and Pseudomonas syringae (provided by A. Ghasemi, Plant Protection Institute, Tehran, Iran) were grown overnight to yield 1×106 colony forming unit (CFU) mL−1. Microbroth dilution assays were performed as described for antiproliferative assay in a final volume of 100 μL. The assay plates were incubated at 28 °C for 16 h. The growth of target bacteria were observed and compared with that of control to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The experiments were performed in triplicate. Data is reported as IC80 value which represents the concentration of a metabolite that was required for 80 % inhibition in vitro.

Phaeobotryon cupressi, Spencermartinsia viticola (Abdollahzadeh et al. 2009) and also Pyricularia oryzae. To this end, a dual culture of each PEF isolate and the target fungus was performed on PDA Petri plates. Because of the different optimal temperatures that PEF isolates and the target fungus needed, firstly a five mm mycelial disk of each PEF isolate was subcultured in one side of a Petri plate and incubated at 4 °C, until it covered the half of the plate. This allows the PEF isolates to secrete their extrolites to the culture medium. Then the target fungus was inoculated at the opposite side and the dual culture was incubated at optimal temperature for the growth of target fungus, i.e. 28 °C. Daily growth of the target fungus, in the presence of each PEF isolate was compared to that of target control, until the control strain covered the whole Petri plate. The diameter (D) of the colony of target fungus was measured (mm) and growth inhibition rate was calculated by the following formula: Growth inhibition rate (%)=(Dcontrol – Dtreated/Dcontrol)×100 %. 2.8 Statistical analyses

2.7 Antifungal bioassays Antifungal activity of endophytic isolates were tested against cypress phytopathogenic fungi Diplodia seriata,

Statistical analyses were applied using ANOVA and SAS procedures and programs. In cases where the F-test showed significant differences among means, the differences among



Table 2 Psychrophilic endophytic fungal isolates (PEF) from Cupressaceae plant family identified based on sequence data from the internal transcribed spacer regions of nuclear ribosomal DNA (ITS rDNA) Group of identical PEF isolates

Host cypress species

Representative PEF isolate

PEF identification (GenBank accession with greatest identity)a

GenBank accessions of fungi from this study

POE115 POE119 POE167 POE191 POE196 POE198 POE199 POE215 CAE130 CAE161 CAE205 CAE208 CSE8 CSE187 CSE192

T. orientalis T. orientalis T. orientalis T. orientalis T. orientalis T. orientalis T. orientalis T. orientalis C. arizonica C. arizonica C. arizonica C. arizonica C. s. var. cereiformis C. s. var. cereiformis C. s. var. cereiformis C. arizonica C. arizonica C. arizonica C. arizonica C. arizonica C. arizonica C. arizonica T. orientalis

CAE208

Phoma herbarum (AY293803 99 %)

KF309191

CSE8

Dothideomycetes sp. (GQ152998 72 %, E-value 5e−62)

KM220773

CAE3 CAE4 CAE7

Dothideomycetes sp. (GQ152998 99 %) Dothideomycetes sp. (GQ152998 98 %) Dothideomycetes sp. (GQ152998 96 %)

KM220774 KF309190 KM220775

POE226

Phoma sp. (JX164068 99 %)

CAE2 CAE3 CAE4 CAE5 CAE6 CAE7 CAE143 POE226

GenBank accessions having the greatest identity (≥97 %) to the PEF based on megablast of ITS rDNA sequences. All megablast ITS rDNA queries, except for CAE8, identified GenBank accessions with E-values of zero

treatments were compared using least significant differences (LSD) test at 5 % significance level (Steel et al. 1997).

from these plants and these psychrophilic endophytic fungi were present in all sampled locations and on all three cypress host species in Iran (Table 1). 3.2 Biodiversity of psychrophilic endophytic fungi (PEF)

3 Results 3.1 Discovery and host identity of psychrophilic endophytic fungi (PEF) During the attempt to recover and purify colonies of endophytic fungi from aboveground tissues of three closely related cypress species in the Cupressaceae, we noticed that there were 23 extremely slow growing colonies that emerged, out of the 110 isolates, and these grew best at 4 °C (Supplementary material, Table S1). The growth rate of some isolates was up to ten-fold higher at 4 °C vs. 15 °C (Table S1). Actually, increasing the temperature from 4°C to 15°C, resulted in five isolates i.e. CAE3, POE215, CAE2, CAE4, CAE6 failing to grow. Furthermore raising the temperature to 20°C resulted in all of the other endophytic fungal isolates, except one, failing to develop over the study time. Thus the coldadapted endophytic fungi recovered from the Cupressaceae were clearly psychrophilic endophytic fungi (PEF). Our survey indicated a 20.5 % recovery frequency for PEF strains

All PEF isolates represented Ascomyceteous fungi based on their morphology, and could be grouped into four colony types. BLAST sequence analyses of ITS rDNA sequences of six representative identical isolates revealed that all six fungal isolates belonged to Pleosporales, Pleosporomycetidae, Dothideomycetes (Ascomycota) (Table 2). All megablast ITS rDNA queries, except for CAE8, identified GenBank accessions with E-values of zero. The representative PEF isolates were identified as Phoma herbarum, Phoma sp., and four Dothideomycetes spp. The relevant ITS rDNA sequences were deposited in GenBank under the accession numbers KF309190, KF309191, KF309192, KM220773, KM220774 and KM220775 (Table 2). 3.3 Antiproliferative and growth inhibition activities of PEF metabolites The results, shown in Table 3, indicated significant antiproliferative and growth inhibition bioactivity for all cypress PEF

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Table 3 The antiproliferative/cytotoxic activities of extra- and intracellular metabolites from psychrophilic endophytic fungi on the conidial germination and germ tube elongation of Pyricularia oryzae The final concentrations of metabolites (μg mL1−) Isolate code POE226 CAE7 CAE4 CAE208 CAE161 CAE130 POE191 CSE187 POE198 POE119 CSE192 POE115 POE215 POE167 POE196 CAE205 CAE143 CAE5 CAE2 CSE8 CAE3 CAE6 POE199

Table 3 (continued) The final concentrations of metabolites (μg mL1−) Intra-

Metabolite ExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtra-

250.0 * * * * * * * * * * * * *

125.0 * * * * * * * * * * * * *

62.5 * * * * * +++ * * * * * +++ *

31.2 +++ +++ +++ * +++ +++ +++ +++ +++ +++ +++ + +++

15.6 +++ ++ ++ +++ ++ + + ++ + ++ + − +

7.8 ++ − + + + + − − − − − − −

IntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtra-

* * * * * * * * * * * * * * * * * *

* * * * * * * * * * * * * * * * * *

+++ * +++ * +++ * +++ * +++ * ++ * ++ * ++ +++ ++ +++

+ +++ + +++ + +++ + +++ + ++ + ++ + ++ + + + +

− + − + − + − + − − − − − − − − − −

− − − − − − − − − − − − − − − − − −

IntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtraIntraExtra-

* * * * * * * * * +++ +++ +++ * +++

+++ +++ * +++ * +++ +++ ++ * ++ +++ ++ +++ ++

++ ++ +++ ++ +++ ++ + + +++ + ++ − + −

+ − +++ − +++ − − − + − + − + −

− − ++ − ++ − − − − − − − − −

− − − − − − − − − − − − − −

+++

++

−

−

−

−

Symbols: (*) The P. oryzae conidial germination was inhibited; (+++) strong growth inhibition of germ tube (≤1/3 of control); (++) moderate growth inhibition of germ tube (1/3–2/3 of control); (+) low growth inhibition of germ tube (≥2/3 but less than control); (−) no inhibition (same as control). The observations were averages of 4–6 assays

isolates. As seen, both intra- and extra-cellular metabolites from all PEF showed antiproliferative effects on P. oryzae conidia and on its germ tube elongation in a range of concentrations, in vitro (Table 3). At the concentration of 62.5 μg mL−1, the extracellular metabolites of all PEF showed higher cytotoxic and growth inhibitory effects than the intracellular metabolites. However, intracellular metabolites from CAE7 isolate showed cytotoxicity at a concentration of 31.2 μg mL−1 and growth inhibition at 15.6 μg mL−1. Metabolites from PEF isolates POE226 (Phoma sp.), CAE4 (Dothideomycetes sp.), CAE 7 (Dothideomycetes sp.), CAE208 (Phoma herbarum) and CAE161 showed the highest cytotoxic and growth inhibitory bioactivities (Table 3). 3.4 Antifungal bioactivity of PEF isolates Data represented in Table 4 show that all PEF isolates, pregrown at 4 °C, inhibited the hyphae growth of P. oryzae, in Petri plate dual culture assays. Meanwhile, PEF isolates POE191, CAE161 and CAE208 exhibited the highest antifungal bioactivities against P. oryzae, in vitro (77–90 % growth inhibition). Surprisingly, POE226 and CAE4, metabolites of which had shown high cytotoxicity and growth inhibitory on conidia of P. oryzae, here in Petri plate dual culture assays showed the least to moderate antifungal bioactivities on this target fungus (58 and 66.5 % growth inhibition) compared to the other isolates. Moreover, in vitro assays indicated hyphae growth inhibition of cypress pathogenic fungi, D. seriata, P. cupressi and S. viticola by all PEF isolates. Of special interest, were the isolates CAE208, CAE161, POE191 and CAE130 and POE119 which inhibited the growth of target fungi by up to 90 %. The rest of the PEF isolates were also capable of target growth inhibition in a range of 55 to 75 % of the control (Table 4). 3.5 Antibacterial activity of PEF metabolites Extra- and intra-cellular metabolites from four superior PEF isolates in former experiments, i.e. CAE161, CAE208, POE226 and POE191 were further tested for antibacterial activity against Bacillus sp., E. amylovora and P. syringae. As MIC and MBC bioassays indicated, metabolites exerted promising



Table 4 The antifungal activity of psychrophilic endophytic fungi, pre-grown at 4°C, on cypress phytopathogenic fungi and the model fungus P. oryzae, at 28°C, using Petri plate dual culture assays Radius zone of mycelia growth (mm) and growth inhibition (%) of target fungi PEF Isolate CAE208 CAE161 CAE130

Diplodia seriata 7.30±1.52 j (83.8) 7.60±0.57 ij (81.1) 9.30±0.57 ij (79.3)

Phaeobotryon cupressi 6.00±1.73 I (86.2) 5.00±1.73 I (88.5) 10.60±1.15 fgh (75.7)

Spencermartinsia viticola 4.30±1.52 l (90.4) 5.30±0.57 l (88.1) 11.30±0.57 jk (74.7)

Pyricularia oryzae 5.30±1.15 l (87.8) 4.60±1.15 l (89.4) 9.30±0.57 k (78.7)

POE119 POE191 POE196 CAE205 CSE192 CSE187 CAE7 POE215 CAE4 CSE8 POE167 CAE2 CAE6 POE115 CAE5 CAE143 POE198 POE226

9.60±1.15 ij (78.7) 10.30±0.57 hi (77.1) 10.30±0.57 hi (77.1) 11.00±1.00 hi (75.6) 12.60±1.15 gh (72.0) 12.60±1.52 gh (72.0) 13.60±0.57 hi (69.8) 13.60±1.15 gh (69.8) 14.30±0.57 gef (68.2) 14.30±1.15 gef (68.2) 14.60±1.15 gef (67.6) 14.60±1.15 efg (67.6) 15.30±1.52 defg (66.0) 15.60±1.52 def (65.3) 16.30±0.57 cdef (63.8) 16.60±1.15 bcde (64.0) 17.60±0.57 abcd (60.9) 18.60±0.57 abc (58.7)

12.60±0.57 ef (71.1) 5.00±1.00 I (88.5) 12.60±1.15 ef (71.1) 10.30±1.73 gf (76.4) 13.60±1.15 ed (68.8) 16.30±1.15 bc (62.2) 11.30±1.15 gf (74.1) 11.30±0.57 gf (74.1) 15.00±1.73 cd (65.6) 15.60±1.52 b (64.2) 13.60±0.57 ed (68.8) 19.30±0.57 a (55.7) 13.60±0.57 ed (68.8) 11.60±1.15 gf (73.4) 9.30±0.57 h (78.7) 19.30±0.57 a (55.7) 14.60±1.15 cd (66.5) 18.00±1.73 ab (58.7)

11.00±1.00 k (75.3) 4.60±2.08 l (89.7) 12.60±0.57 ghijk (71.8) 11.30±1.52 jk (74.7) 14.00±1.73 efgh (68.6) 14.30±1.52 gfe (67.9) 13.00±1.00 fghij (70.9) 14.60±1.15 def (67.3) 12.30±1.15 hijk (72.4) 15.30±0.57 cde (65.7) 16.00±1.00 bc (61.1) 16.30±0.57 bcd (63.5) 14.00±1.15 efgh (68.6) 13.30±1.52 fghi (70.2) 16.60±1.15 bc (62.8) 16.30±1.15 bcd (63.5) 17.30±1.15 ab (61.2) 18.60±1.52 a (58.3)

9.30±1.15 k (78.7) 4.30±1.15 l (90.1) 11.00±1.73 hi (74.8) 11.30±0.57 ijk (74.1) 13.30±1.15 gh (69.5) 14.00±1.00 fg (67.9) 11.6±1.15 hi (73.4) 13.30±0.57 gh (69.5) 14.60±1.15 efg (66.5) 14.60±1.15 efg (66.5) 14.60±0.57 efg (66.5) 16.00±1.73 cde (63.3) 13.30±0.57 gh (69.5) 13.30±1.15 gh (69.5) 9.60±1.15 jk (78.0) 17.6±0.57 abc (59.6) 16.30±0.57 bcd (62.6) 18.30±1.52ab (58.0)

POE199 CAE3 Control

19.30±1.52 ab (57.1) 19.60±2.30 a (56.5) 45.00±0.00 (−)

18.00±1.73 ab (58.7) 19.60±1.52 a (55.1) 43.60±1.15 (−)

18.00±1.00 ab (59.7) 11.60±1.15 ijk (74.0) 44.60±0.75 (−)

18.30±0.57 ab (58.0) 19.00±1.00 a (56.4) 43.60±1.15 (−)

Data (significant at P≤0.05) were averages (±standard deviation) of three replicates Similar letters indicate no significant difference. The rows are ordered according to the statistical significance of Diplodia seriata data

bacteriostatic and bactericidal bioactivities, in a range of 7.8– 125 μg mL1− (Table 5). The antibacterial activity of extracellular metabolites was more effective than interacellular ones.

4 Discussion Psychrophilic fungi are adapted for growing at temperatures below 10 °C, and these fungi are particularly common in alpine, Arctic and Antarctic environments (Vincent 1988; Del Frate and Caretta 1990; Vishniac 1996; Deming 2002; Gocheva et al. 2005). Psychrophilic fungi are also found in man-made refrigerated environments (Gounot 1986; Pitt and Hocking 1997). Some species such as Phoma herbarum which is a mesophilic filamentous fungus, found mostly in soils of the temperate and subtropical zones (Domsch et al. 1980), has also been isolated from tundra soil (Flanagan and Scarborough 1974) and Antarctica (Selbmann et al. 2005; Singh et al. 2006). However, endophytic association of these psychrophilic fungi with plants has not been documented.

The Cupressaceae plant family hosts a vast number of ascomycetous endophytic fungi which grow best at temperatures between ca. 21–28 °C (Hoffman and Arnold 2008; Hosseyni Moghaddam 2013; Hosseyni Moghaddam et al. 2013; Hosseyni Moghaddam and Soltani 2014; Soltani and Hosseyni Moghaddam 2014a, b). However, when we attempted to recover the 23 extremely slow growing endophytic isolates (out of 110) that were associating with the Cupressaceae, we found that all of them grew best at 4 °C. Thus 20.5 % of the endophytic fungal population which has adapted to an endophytic life style in cypress plants are pychrophilic endophytic fungi (PEF). There are reports of endophytic fungi that show limited or no growth at room temperatures (Aly et al. 2010; Strobel et al. 2004). Thus, as shown by our findings, the initial incubation temperature can bias the biodiversity of recovered endophytic fungi. Our findings also revealed the presence of psychrophilic endophytic fungi at all sampling locations and in both indigenous and non-indigenous cypress host species in Iran. Although the annual average temperature of all locations

Author's personal copy Psychrophilic endophytic fungi with biological activity inhabit Table 5 The antibacterial activity of extra- and intra-cellular metabolites from the most potent psychrophilic endophytic fungi

Extract concentration (μg mL1−) MICa

Minimum inhibitory concentration

Target bacteria

Extracellular

Intracellular

Extracellular

Intracellular

CAE161

P. syringae E. amylovora Bacillus sp. P. syringae E. amylovora Bacillus sp. P. syringae E. amylovora Bacillus sp. P. syringae E. amylovora Bacillus sp.

15.6 15.6 7.8 15.6 15.6 7.8 15.6 15.6 7.8 7.8 7.8 15.6

31.2 62.5 15.6 31.2 62.5 15.6 31.2 62.5 15.6 31.2 31.2 62.5

31.2 31.2 15.6 31.2 31.2 15.6 31.2 31.2 15.6 15.6 15.6 31.2

31.2 125.0 31.2 31.2 125.0 31.2 125.0 125.0 31.2 62.5 62.5 125.0

POE191

b

Minimum bactericidal ?concentration Data (significant at P≤0.05) were obtained from three replicates Data are reported as IC80 values

MBCb

PEF isolate

CAE208

a

85

POE226

was sub-optimal for the recovered PEF (Table 1), the PEF isolates may have evolved to be active during the cold season of the year when freezing temperatures pertain inside evergreen Cupressaceous foliage. This may have significant implications for ecology of cypress plant family. The representative PEF isolates were identified as the members of Dothideomycetes, Ascomycota. Previously, Phoma glomerata and Phoma sp. have been reported as endophytic fungi associated with Thuja orientalis and Cupressus arizonica (Hoffman and Arnold 2008, 2010). These Phoma species are recovered regularly at room temperatures (ca. 21.5°C), and nothing is reported on their bioactivities. Phoma fungal species are universal colonizers of plants and soils, and are also reported from various host plants in Iran (Abbasi and Aliabadi 2009; Ershad 2009). Phoma herbarum has been previously reported as a plant pathogen, as well as, a universal soil resident and a psychrophilic fungus in cold climates (Domsch et al. 1980; Flanagan and Scarborough 1974; Selbmann et al. 2005; Singh et al. 2006). However, the present study is the first to report a symbiotic association between psychrophilic endophytic ascomycetous fungi like Phoma species and the Cupressaceae in temperate zones. The metabolites from psychrophilic endophytic fungi showed significant bioactivities against a panel of phytopathogenic fungi and bacteria. In general, the extracellular metabolites from PEF exerted their bioactivity at lower concentrations than the intracellular ones. Of special interest was their antibacterial activity against the ice-nucleation active bacterium Pseudomonas syringae. The strains of P. syringae are abundant in rain, snow, alpine streams, and lakes and in wild plants (Morris et al. 2008). Most strains of Pseudomonas syringae are ice-nucleation active agents and are commonly known to produce proteins which cause water to freeze faster

under conditions which would not normally cause water to freeze. Such bacteria catalyze the formation of ice crystals on and in the plant tissues which spread intercellularly and intracellularly, causing mechanical disruption of cell membranes and subsequent cell death and plant damage (Lindow 1983). Most plants have no significant frost-tolerance mechanism and during freezing season suffer from frost injury due to ice formation (Lindow 1983). Since Cupressaceae have a global distribution, they are often exposed to low temperatures. As they are evergreen plants, special adaptations would help them to stay green during the whole year. Based on our research, we suggest that symbiosis with psychrophilic microorganisms may provide a low temperature adaptation in Cupressaceae. We highlight for the first time the idea that psychrophilic endophytic fungi are a possible mean for cold stress tolerance in plants, as well as a source of fungal biodiversity in temperate zones, and a source for antiproliferative, antifungal and antibacterial activity. This may have significant implications for ecological and evolutionary studies, as well as for plant protection. The fungal metabolites produced as a result of endosymbiosis with plants, at temperatures at or below 5 °C, may contribute to the ecology and survival of the host plants and this aspect needs further study, in vivo.

Acknowledgments We appreciate generous gift of target fungi and bacteria by Jafar Abdollahzadeh (Kurdistan University, Sanandaj, Iran), Salar Jamali (Guilan University, Rasht, Iran) and Abolghasem Ghasemi (Plant Protection Institute, Tehran, Iran). We also thank Javad Hamzei, Freydoun Babalhavaeji, Soheila Mirzaei, Sonbol Nazeri, Dustmorad Zafari and Mohammad-Javad Soleimani Pari at Bu-Ali Sina University of Hamedan, Iran, for their helpful discussions and for sharing laboratory facilities. This work was financially supported by BABiNT, and a grant from BASU to J.S. Jalal Soltani dedicates this work to Mohammad Reza Soltani and Javad Soltani.


References Abbasi M, Aliabadi F (2009) The list of fungi recorded in proceedings of 12th to 18th Iranian plant protection congresses (1995–2008). Elm va Honar Press, Iran, 276 Abdollahzadeh J, Mohammadi Goltapeh E, Javadi A, Shams-Bakhsh M, Zare R, Phillips AJL (2009) Barriopsis iraniana and Phaeobotryon cupressi: two new species of the Botryosphaeriaceae from trees in Iran. Persoonia 23:1–8 Aly AH, Debbab A, Kjer J, Proksch P (2010) Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers 41:1–16 Barnett HL (1960) Illustrated genera of imperfect fungi. Burgess Publishing company, Minneapolis, pp 225 Boerema GH, Gruyter J, de Noordeloos ME, Hamers MEC (2004) Phoma identification manual. Differentiation of specific and infraspecific taxa in culture. CABI Publishing, 470 Del Frate G, Caretta G (1990) Fungi isolated from Antarctic material. Polar Biol 11:1–7 Deming JW (2002) Psychrophiles and polar regions. Curr Opin Microbiol 5:301–309 Domsch KH, Gams W, Anderson TH (1980) Compendium of soil fungi. Academic, London Ershad, J (2009) Fungi of Iran. Iranian Research Institute of Plant Protection Press. 3rd Eds, 531 Flanagan PW, Scarborough AM (1974) Physiological groups of decomposer fungi on tundra plant remains. In: Holding AJ, Heal OW, McLean SF Jr, Flannagan PE (Eds) Soil organisms and decomposition in tundra. Tundra Biome Steering Committee, Stockholm, pp 159–181 Fralish JS, Franklin SB (2002) Taxonomy and ecology of woody plants in North American forests. Wiley, New York Frisvad JC (2008) Fungi in cold ecosystems. In: Margesin R, Schinner F, Marx JC, Gerday C (Eds) Psychrophiles: from biodiversity to biotechnology, vol 9, Chapter., pp 137–156 Gocheva YG, Krumova E, Slokoska L, Gesheva V, Angelova M (2005) Isolation of filamentous fungi from Antarctica. C R Acad Bulg Sci 58:403–408 Gounot AM (1986) Psychrophilic and psychrotrophic microorganisms. Experientia 42:1192–1197 Hoffman MT, Arnold AE (2008) Geographic locality and host identity shape fungal endophyte communities in Cupressaceous trees. Mycol Res 112:331–334 Hoffman MT, Arnold AE (2010) Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal endophytes. Appl Environ Microbiol 76:4063–4075 Hosseyni Moghaddam MS (2013) Study on some biological effects of natural products from endophytes of cypress. MSc thesis, Bu-Ali Sina University of Hamedan, Iran, 180 Hosseyni Moghaddam MS, Soltani J (2013) An Investigation on the effects of photoperiod, aging and culture media on vegetative growth and sporulation of rice blast pathogen Pyricularia oryzae. Prog Biol Sci 3:135–143 Hosseyni Moghaddam MS, Soltani J (2014) Bioactivity of endophytic Trichoderma fungal species from the plant family Cupressaceae. Ann Microbiol 64:753–761 Hosseyni Moghaddam MS, Soltani J, Babalhavaeji F, Hamzei J, Nazeri S, Mirzaei S (2013) Bioactivities of endophytic Penicillia from Cupressaceae. J Crop Prot 2:421–433 Hsiang T, Matsumoto N, Millett SM (1999) Biology and management of Typhula snow molds of turfgrass. Plant Dis 83:783–798 Huston AL (2008) Biotechnological aspects of cold-adapted enzymes. In: Margesin R, Schinner F, Marx JC, Gerday C (Eds)Psychrophiles: from biodiversity to biotechnology, pp 347–363 Kobayashi H, Namikoshi M, Yoshimoto T, Yokochi T (1996) A screening method for antimitotic and antifungal substances using conidia

M.S. Hosseyni Moghaddam, J. Soltani of P. oryzae, modification and application to tropical marine fungi. J Antibio 49:873–879 Kour A, Shawl AS, Rehman S, Sultan PH, Qazi PH, Suden P, Khajuria RK, Verma V (2008) Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva. World J Microbiol Biotechnol 24:1115–1121 Kumaran RS, Muthumary J, Hur BK (2008) Production of taxol from Phyllosticta spinarum, an endophytic fungus of Cupressus sp. Eng Life Sci 8:438–446 Kusari S, Lamshoft M, Spiteller M (2009) Aspergillus fumigatus fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin. Appl Microbiol 107:1364–5072 Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798 Kusari S, Pandey SP, Spiteller M (2013) Untapped mutualistic paradigms linking host plant and endophytic fungal production of similar bioactive secondary metabolites. Phytochemistry 91:81–87 Lindow SE (1983) The role of bacterial ice nucleation in frost injury to plants. Annu Rev Phytopathol 21:363–384 Margesin R, Miteva V (2011) Diversity and ecology of psychrophilic microorganisms. Res Microbiol 162:346–361 Morris CE, Sands DC, Vinatzer BA, Glaux C, Guilbaud C, Buffière A, Yan S, Dominguez H, Thompson BM (2008) The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. ISME J 2:321–334 Pitt JI, Hocking AD (1997) Fungi and food spoilage, 2nd edn. Blackie, London Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151:341–353 Ryan R, Ryan D, Dowling DN (2007) An acquired efflux system is responsible for copper resistance in Xanthomonas strain IG8. FEMS Microbiol Lett 268:40–46 Ryan R, Germaine K, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9 Selbmann L, de Hoog GS, Mazzaglia A, Friedman EI, Onofri S (2005) Fungi at the edge of life: cryptoendolithic black fungi from Antarctic desert. Stud Mycol 51:1–32 Singh SM, Puja G, Bhat DJ (2006) Psychrophilic fungi from Schirmacher Oasis, East Antarctica. Curr Sci 90:1388–1392 Singh LP, Gill SS, Tuteja N (2011) Unraveling the role of fungal symbionts in plant abiotic stress tolerance. Plant Signal Behav 6:175–191 Soltani J, Hosseyni Moghaddam MS (2014a) Antiproliferative, antifungal and antibacterial activities of endophytic Alternaria species from Cupressaceae. Curr Microbiol 69:349–356 Soltani J, Hosseyni Moghaddam MS (2014b) Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family. Arch Microbiol. doi:10.1007/s00203-014-0997-8 Steel RGD, Torrie JH, Dicky DA (1997) Principles and procedures of statistics- a biometrical approach, 3rd edn. McGraw Hill Book International Co, Singapore, pp 204–227 Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67:257–268 Vincent WF (1988) Microbial ecosystems of Antarctica. Cambrige University Press, Cambridge Vishniac HS (1996) Biodiversity of yeast and filamentous microfungi in terrestrial Antarctic ecosystems. Biodivers Conserv 5:1365–1378 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) “PCR protocols: A guide to methods and applications”. Academic, San Diego, pp 315–322 Zhang D, Yang Y, Castlebury LA, Cerniglia CE (1996) A method for the large scale isolation of high transformation efficiency fungal genomic DNA. FEMS Microbiol Lett 145:261–265