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Short communication
A new endophytic species of Arthrinium (Apiosporaceae) from Jatropha podagrica Rohit Sharma a,*, Girish Kulkarni b, Mahesh S. Sonawane a, Yogesh S. Shouche a a
Microbial Culture Collection, National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune 411 007, Maharashtra, India b Molecular Biology Unit, National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune 411 007, Maharashtra, India
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abstract
Article history:
During study of endophytic fungal diversity in tropical region, a new species of Arthrinium
Received 15 August 2012
was isolated from healthy petiole of Jatropha podagrica. It is characterized by aseptate, dark
Received in revised form
brown, circular, lenticular conidia with hyaline margin, broad, hyaline conidiophores with
13 June 2013
numerous brown septa and ampulliformelageniform conidiophore mother cells. The new
Accepted 18 June 2013
species superficially resembles A. phaeospermum and A. saccharicola in having circular
Available online 2 August 2013
lenticular conidia, but differs in conidia and conidiophore size, and shape of conidiophore mother cells. Phylogenetic study using sequence analysis of ITS region (ITS1e5.8SeITS2)
Keywords: India
separates the new species (A. jatrophae) from other related species of Arthrinium. ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
ITS Lenticular conidia Phylogeny Taxonomy
The genus Arthrinium Kunze (teleomorph Apiospora: Sordariomycetes, Ascomycota) currently comprises 31 species (Kirk et al. 2008) having widespread distribution in various habitats. They are primarily plant pathogens but also isolated from erythematous nodules of human beings and endophytes from Juncus trifidus L. (Juncaceae) and Vignea kobomugi (Ohwi) Soja´k (syn. Carex kobomugi Ohwi) (Cyperaceae) (Rai 1989; Chlebicki 2009; Khan et al. 2009). Several species have been described by Ellis (1965, 1971, 1976), Pollack and Benjamin (1969), Calvo and Guarro (1980), Larrondo and Calvo (1990, 1992) and Bhat and Kendrick (1993). Although species of the genus Arthrinium have been reported to produce secondary metabolites with antimicrobial properties (Agut and Calvo 2004; Ramos
et al. 2010), there has been no novel species described since 1993. During present study, three isolates of an unknown species of Arthrinium were obtained from Jatropha podagrica Hook. (Euphorbiaceae). Although the plant is native to tropical America, it is also found in Africa, Asia and used as traditional medicine in Africa having several pharmaceutical and antimicrobial activities (Aiyelaagbe et al. 2000, 2007). Present study was undertaken to characterize Arthrinium jatrophae sp. nov. and ascertain its phylogenetic relationship with other known species of the genus. Healthy parts of J. podagrica shrub plant was collected from Pune (Pimpri), Maharashtra, India (18 370 07.0400 N, 73 480 13.4300 E). They were cleaned under running tap water
* Corresponding author. Tel.: þ91 020 25708237. E-mail addresses:
[email protected],
[email protected] (R. Sharma). 1340-3540/$ e see front matter ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2013.06.004
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(10 min), sterile distilled water (5 min, 2e3 times) and cut in to small pieces (1 cm). Surface sterilization done by modified Schulz et al. (1993) method. Plant parts inserted in 75% ethanol for 3 min (2e3 times) followed by 100% ethanol for 30 s. Dry, sterilized pieces were placed aseptically on potato dextrose agar (PDA; HieMedia, India) and Czapek Dox agar (CDA; HieMedia, India) plates. After 3e4 d, mycelium originating from periphery of plant pieces was transferred to fresh culture medium for further study. Three strains of Arthrinium were isolated from petiole, designated as MMI00050 (MCC 1037), MMI00051 (MCC 1038) and MMI00052 (MCC 1014). To test optimum temperature for growth, culture plates [PDA, CDA, malt extract agar (MEA; HieMedia, India), potato carrot agar (PCA; HieMedia, India), 2% oatmeal agar (OA)] were incubated at different temperatures (5e35 0.2 C). Measurements were made in lactophenol mounts (HieMedia, India) and microphotographs taken on Olympus BX53 (Olympus Corporation, Japan) fitted with ProgRes C5 camera (Jenoptik, USA). For molecular study, DNA was extracted from pure cultures grown in Petri dishes using QIAamp DNA Mini Kit (Qiagen, Inc., Valencia CA) as per manufacturer’s instructions and quantified by NanoDrop spectrophotometer (NDe1000, Thermo scientific, USA). Universal primer pairs ITS1 and ITS4 (White et al. 1990) were used to amplify ITS1e5.8SeITS2 region using PE 9700 thermocycler (PE Applied Biosystems, Singapore). The amplification products was purified by PCR Cleanup Kit (Qiagen Inc., Valencia, CA) and sequenced on ABI 3730xl DNA sequencer using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA). A BLASTn sequence homology search was done to compare it with available sequences of Arthrinium in GenBank database (Zhang et al. 2000). Alignment (CLUSTAL W; Thompson et al. 1994) and phylogenetic analysis based on the neighbor joining (NJ), the maximum parsimony (MP) and maximum likelihood (ML) method was done with retrieved GenBank sequences of Arthrinium in MEGA 5.0 (Tamura et al. 2011). Nigrospora sphaerica (Sacc.) E.W.E Mason CY 256 (HQ608063) was taken as outgroup. The alignment is submitted to TreeBASE (Submission Id: 13584). Arthrinium jatrophae Rohit Sharma, sp. nov. Figs. 1, 2. MycoBank no.: MB 564302. Diagnosis: The species is distinguished by production of anomalous conidia along with regular lenticular conidia and short, thick conidiophore mother cells which are simple and narrow but broad at the point of septation forming groove and furrow appearance. These cells are colorless except for transverse septa which are brown or dark brown. Type: India, Pune (Pimpri), Maharashtra, from healthy petiole of Jatropha podagrica Hook., 3 March 2011, leg. Rohit Sharma [Holotype, AMHe9557 (exeMCC 1014; ¼ NFCCI 2599 ¼ CBS 134262); paratype, AMHe9556 (exeMCC 1037)]. rDNA sequence ex holotype MCC 1014 (JQ246355), ex paratype MCC 1037 (HE981191), MCC 1038 (HE981192). Etymology: jatrophae, refers to the host plant Jatropha podagrica from which this endophyte species was recovered. Teleomorph: Unknown. Colonies on 2% MEA white or creamyewhite, effused, circular, 70 mm diam. in 7 d at 25 0.2 C, reverse cream, black
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Fig. 1 e Arthrinium jatrophae (holotype). a: Conidia. b: Conidiophores. c: Conidiophore mother cells. Bars aec 10 mm.
where conidia are produced after 90 d of incubation. Fructifications are superficial and pulvinate with powdery appearance. Mycelium partly immersed and partly superficial formed by extensively branched mycelia, colorless, smooth, soluble pigment not produced in media. Hyphae diameter is 1.6e2 mm. Conidiophores basauxic, macronematous, mononematous, arising from short but thick ampulliformelageniform conidiophore mother cells, simple often narrow but broad at the point of septation forming groove and furrow appearance, usually colorless except for transverse septa which are brown or dark brown, 49.1e88.6 2.7e6.5 (mean 68.5e5.3) mm. Conidiophore mother cells polyblastic, integrated and terminal. Conidia borne in grape like bunch, lateral and terminal, brown with hyaline rim, smooth and aseptate, with scar of attachment, initially spherical later changing to lenticular in shape, 6.5e9.7 (mean 8.4) mm in face view and 3.2e6.5 (mean 4.3) mm in side view, some are irregular in shape, young conidia are fragile and forms a depression in lactophenol and SEM given an impression of cup shape conidia. Some anomalous conidia are also produced. Anomalous conidia are elongated, smooth, brown, tapering at one end, 8.2e12.2 4.1e5.4 (mean 10.6e4.5) mm (Fig. 2geh). All three isolates of A. jatrophae were obtained from petiole. The fungus has an optimum temperature of 25 0.2 C growing best on PDA and MEA (Fig. 3), sporulating after 80e90 d incubation at 25 0.2 C. Arthrinium jatrophae MCC 1014 produce two types of conidia viz. round lenticular conidia with hyaline germ pore and few anomalous conidia (Fig. 1aeb) both borne on short, thick and multieseptate conidiophores. The conidiophore mother cells are swollen and bottle shaped (Fig. 1c). The new species is morphologically similar to the group of species with circular or nearly circular lenticular conidia i.e. A. euphorbiae M.B. Ellis, A. saccharicola F. Stevens,
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Fig. 2 e Microscopy of Arthrinium jatrophae a: Conidial bunches (MCC 1014). bef: Conidial bunch (brown) attached to hyaline conidiophore with brown septation (MCC 1014). g: Broad septation of conidiophore (SEM; MCC 1014). hek: Anomalous conidia (MCC 1037). Bars a, d, hek 20 mm; bec, eef 10 mm; g 2 mm.
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Fig. 3 e Temperatureegrowth relationship of three stains of Arthrinium jatrophae on Czapek Dox Agar (CDA), Malt Extract Agar (MEA), Oatmeal Agar (OA), Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) (mm diam. of 7 d old colonies).
A. phaeospermum (Corda) M.B. Ellis, A. sacchari (Speg.) M.B. Ellis and A. arundinis (Corda) Dyko & B. Sutton (syn. Apiospora montagnei Sacc.) but differ in conidia and conidiophore size, and shape of conidiophore mother cells (Table 1). An NCBIe BLASTn search using ITS sequence showed maximum similarity 89% with A. phaeospermum (Po17). Additional homologous ITS sequences of 13 Arthrinium species (24 strains) were retrieved from GenBank and aligned with A. jatrophae. The aligned dataset (Submission Id: 13584) contained 559 characters including gaps and showed less sequence similarity in ITS1 region (72%evariable) as compared to ITS2 (39%e variable). Phylogenetic analyses of ITS region using MP, ML and NJ (Fig. 4) placed A. jatrophae in a monophyletic clade within the genus Arthrinium. NJetree distributes 14 species of Arthrinium into seven clades and three separate branches consisting single species, A. aureum, A. jatrophae and A. urticae M.B. Ellis. Although A. jatrophae is an endophyte isolated from petiole of J. podagrica, there are several reports of Arthrinium genus as endophyte isolated from various plant parts (Petrini et al. 1990; Mene´ndez et al. 1995; Girlanda et al. 1997; Bettucci et al. 1999; Chlebicki 2009; Jones et al. 2009; Khan et al. 2009). Results of study confirm that A. jatrophae is distinct from Arthrinium species described so far. Although A. jatrophae resembles A. saccharicola and A. phaeospermum morphologically, phylogenetic analysis of ITS region clustered it separately. NJetree shows clustering of different Arthrinium species and A. jatrophae making a separate branch supporting novel status of A. jatrophae. In NCBI database, many isolates (at least 10) are registered as A. phaeospermum (not included in present study). However, phylogenetic analysis shows these isolates are different from A. phaeospermum and need to be assigned proper species status. A thorough study of these strains is required to authenticate their position within Arthrinium. Record of other species from different parts of world, especially as endophyte and their role in plant growth could help in
Table 1 e Comparison of Arthrinium jatrophae with other related species of Arthrinium. Characters
Conidia shape
Conidia size in mm; face side view (mean) Conidiophore dimensions in mm (mean) Conidiophore (morphology; color)
A. jatrophae MCC 1037
A. jatrophae MCC 1014
Lenticular, dark brown, equatorial germ slit, some deformed 5.92e9.57 (7.56)
Lenticular, dark brown, equatorial germ slit, some deformed 6.5e9.7 3.2e6.5 (8.4 4.3)
32.3e58.4 2.3e5.5 (39.2 4) Thick, numerous septa
49.1e88.6 2.7e6.5 (68.5 5.3) Thick, numerous septa
A. phaeospermuma A. saccharicolaa
Lenticular, dark brown, equatorial germ slit
Arthrinium state of Ap. montagneia
A. saccharia
A. euphorbiaea
Lenticular, pale brown, hyaline band at junction of two sides
Lenticular, dark brown, hyaline/ pale band at junction of two sides
Lenticular, brown/ olivaceous brown, smooth walled
8e12 5e7 (9.9 5.9)
Lenticular, brownedark brown, hyaline band at junction of two sides 7e10 4e6 (9 5.1)
5.5e8 3e4.5 (6.5 3.8)
6e8 3e4 (7 3.8)
4e5.5 3e4 (4.7 3.2)
65 1e1.5
25e130 2e4
4e50 0.5
40e130 1e1.5
15e110 0.5e1
Thread like, flexuose
Simple, straight/ flexuous, cylindrical
Simple flexuous, threadelike, few septa
Simple, flexuous, cylindrical, many septa
Simple, flexuous, cylindrical, numerous septa
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Table 1 e (continued ) Characters
Conidiophore mother cell (shape; size in mm) Hypha (color; thickness in mm)
a
A. jatrophae MCC 1037
A. jatrophae MCC 1014
A. phaeospermuma A. saccharicolaa
Ampulliforme Ampulliforme lageniform lageniform
Lageniform; 5e10 3e5
Subsphericale flask shaped; 5e7 4.5e6
Colorlesse brown or dark brown; 1.3e2.5
Colorless to pale brown
Subhyalinee brown; 2e5
Colorlesse brown or dark brown; 1.6e2
Arthrinium state of Ap. montagneia Subsphericale oval or barrel form; 5e7 3e5 Colorlessepale brown; 1e4
A. saccharia
A. euphorbiaea
Subsphericale clavate; 3e6 4e5.5
Clavate, subspherical/ barrel shaped; 4.5e5 3.5e5 Subhyalinee pale olivaceous brown; 2.5e5 (aerial) 1e3 (immersed)
Subhyalinee pale olivaceous brown; 2e5
Data from Ellis (1965).
Fig. 4 e Neighbor joining (NJ) tree of the Arthrinium strains studied. Bootstrap support of branches indicated on the node was obtained using 1000 replicates. Only statistically significant bootstrap values (‡50%) are indicated. Branch lengths are indicated as 0.01 substitutions per positions according to the scale bar underneath the tree. Number on right side of species name denotes the strain number/culture collection accession number. Number in parentheses denotes accession number of the sequence deposited to online database.
better understanding of fungal ecology and their role as endophyte.
Acknowledgments The authors thank DBT, India for funding and DirectoreNCCS, India for laboratory facilities, D. Dhotre, MCCeNCCS, India for his discussion on phylogenetic analysis and R. Sharma, ARI, India for going through manuscript. Authors also thank Montserrat Agut,
Universitat Ramon Llull, Spain for valuable suggestions. Anonymous reviewers are also acknowledged for their suggestions.
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