Mycologia, 102(5), 2010, pp. 989–995. DOI: 10.3852/09-261 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 25 August 2010
Gibberellin production and plant growth promotion from pure cultures of Cladosporium sp. MH-6 isolated from cucumber (Cucumis sativus L.) Muhammad Hamayun1, 2
5.18 ng/mL; GA4, 13.35 ng/mL and GA7, 2.4 ng/ mL) in conjunction with physiologically inactive (GA9 [0.69 ng/mL], GA12 [0.24 ng/mL], GA15 [0.68 ng/ mL, GA19 [1.94 ng/mL and GA20 [0.78 ng/mL]) gibberellins. The CF of MH-6 produced greater amounts of GA3, GA4, GA7 and GA19 than wild type Fusarium fujikuroi, a fungus known for high production of GA. The fungal isolate MH-6 was identified as a new strain of Cladosporium sp. on the basis of sequence homology (99%) and phylogenetic analysis of 18S rDNA sequence. Key words: Cladosporium, cucumber, endophytic fungi, gibberellins, plant growth promotion
Department of Botany, Abdul Wali Khan University, Mardan, Pakistan
Sumera Afzal Khan2 Center of Biotechnology and Microbiology, University of Peshawar, Pakistan
Abdul Latif Khan School of Applied Bioscience, Kyungpook National University, Korea, and Department of Chemistry, Kohat University of Science and Technology, Kohat, Pakistan
Gauhar Rehman Department of Genetic Engineering, Kyungpook National University, Korea
Youn-Ha Kim
INTRODUCTION
School of Applied Bioscience, Kyungpook National University, Korea
Soil is the most diverse of terrestrial microbial habitats, and soil fungal biota perform an important function in the soil ecosystem as they decompose plant residues, releasing nutrients that sustain and stimulate plant growth in the process (Wardle and Giller 1997). Soils with high biodiversity appear to be more resistant to stress (Griffiths et al. 2000), and low diversity might be associated with impaired ecosystem function (Tilman et al. 1997). Symbiotic relationships between plants and microorganisms have been known for more than a century (Peterson et al. 2008), however fungal symbiosis has not yet been studied widely and used for improvement of agriculture. Symbiotic fungal-crop associations of an endophytic nature have been shown to improve crop growth and yield without requiring extensive use of chemical fertilizers, while simultaneously improving the ability of these crops to tolerate a range of biotic and abiotic stresses (Rodriguez et al. 2008). The ability of fungal endophytes to improve plant tolerance to herbivory, salt, drought and heat might help farmers grow crops on marginal lands (Marquez et al. 2007, Waller et al. 2005). Fungal endophytes should be included among alternative modern technologies to support food production because they seem to offer solutions to several of the problems facing modern agriculture. The soil rhizosphere is considered to be a rich source of root endophytes because most of the root endophytes are reported from this region (Sessitsch et al. 2002). These endophytes may enter the plant by local cell wall degradation and/or fractures in the root system (Gough et al. 1997) and are involved in the promotion of plant growth and protection against
Ilyas Iqbal Department of Botany, University of Malakand, Chakdra, Pakistan
Javid Hussain Department of Chemistry, Kohat University of Science and Technology, Kohat, Pakistan
Eun-Young Sohn In-Jung Lee School of Applied Bioscience, Kyungpook National University, Korea
Abstract: Gibberellin (GA) production by soil fungi has received little attention, although substantial work has been carried out on other aspects of plant growth promoting fungi (PGPF). In our studies we investigated GA production and growth-promoting capacity of a novel fungal strain isolated from the roots of soilgrown cucumber. Pure cultures of 19 endophytic fungi were tested for shoot length promotion of Waito-C rice to identify the GA production capacity of these fungal isolates. Isolate MH-6 significantly increased shoot length (12.9 cm) of Waito-C, in comparison to control treatments. Bioassay with culture filtrate (CF) of MH-6 also significantly promoted growth attributes of cucumber plants. Analysis of MH-6 CF showed the presence of physiologically active (GA 1 , 1.97 ng/mL; GA 3 , Submitted 13 Oct 2009; accepted for publication 13 Jan 2010. 1 Corresponding author. E-mail:
[email protected] 2 Authors contributed equally to this study.
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pathogens (Waller et al. 2005). The plant-growth promoting capacity of fungal endophytes is partly due to the production of phytohormones, such as indole3-acetic acid (IAA), cytokines, and other plant growthpromoting substances (Zou and Tan 1999) and/or partly owing to the fact that endophytes enhance the host uptake of nutrients such as nitrogen (Reis et al. 2000) and phosphorus (Malinowski and Belesky 1999). Gibberellins play a vital role in plant growth and development. However only 12 fungi, associated with plants and/or soil have been reported as GA producers (Kawaide 2006, MacMillan 2002, Vandenbussche et al. 2007). Endophytic strains of the Cladosporium sphaerospermum and Penicillium citrinum have been reported as GA producers (Hamayun et al. 2009a, Khan et al. 2008). Genus Cladosporium includes several plant-associated saprophytic and some plant pathogenic species. Some species are widely distributed dematiaceous molds, and some are predominant in tropical and subtropical regions (de Hoog et al. 2000). Some Cladosporium species such as C. fulvum are pathogenic to plants, causing scab diseases as well as leaf spots and blights. Information on fungal endophyte GA production capacity is limited, although endophytes have been reported as plant-growth promoters (Hamayun et al. 2009b, Khan et al. 2009a). GA-producing fungal endophytes might have potential to increase crop yields due to increasing concern about the excessive use of fertilizers in agricultural and the subsequent negative effect on the environment. The main objective of this research was to identify potential fungal inoculums for plant growth promotion to reduce the use of fertilizer in agricultural fields. MATERIALS AND METHODS
Isolation of fungal endophytes from cucumber roots.—Endophytic fungi were isolated from the roots of cucumber plants (Cucumis sativus L.) grown in soil under greenhouse conditions. To isolate endophytic fungi we randomly selected cucumber plants and the fine roots near root tips were washed with tap water to remove soil. Root samples were suspended in Tween 80 solution (2–3 drops in 50 mL distilled water) and placed in a shaking incubator set at 120 rpm for 5 min at room temperature. Roots were rinsed in distilled water to remove any residual Tween 80. The cleaned samples were surface sterilized by suspending them in 50 mL 1% perchloric acid and agitating on a shaking incubator (120 rpm, 5 min). The roots were washed with autoclaved distilled water, dried between a layer of sterile filter paper, cut into pieces (0.5 cm each), cultured on Hagem media plates (Hamayun et al. 2009c) and incubated at 25 C until the emergence of fungal cells (Khan et al. 2008). Hagem media plates were supplemented with 80 mg/mL
streptomycin (Yamada et al. 2001) to avoid bacterial contamination. The effectiveness of surface sterilization was tested by the imprinting technique (Schulz et al. 1999) and sterilized root pieces placed on Hagem media plates. Absence of any microbial growth on imprinted media plates after 4–7 d incubation was considered confirmation of the effectiveness of the procedure (Khan et al. 2008). Pure fungal cultures were isolated, grown on potato dextrose agar (PDA) media plates and slants (Khan et al. 2009b). PDA slants were used for storage. Czapek broth medium, containing 1% glucose and peptone was used for GA production (Hassan 2002) by incubating the fungal isolates at 30 C, 120 rpm, 7 d. The wild type strain of Fusarium fujikuroi was used as a positive control for GA production during this experiment (provided by the Korean Culture Center of Microorganisms [KCCM]). Bioassay on Waito-C and cucumber.—Culture filtrates (CF) of all fungal isolates were bioassayed on Waito-C (Oryza sativa L. cv. Waito-C) seedlings to identify the plant growthpromoting capacity of these isolates on rice. Seeds of WaitoC were surface sterilized and treated with 20 mg/mL uniconazol 24 h. Seeds were washed thoroughly and soaked in autoclaved distilled H2O until germination. Two Waito-C seedlings were transplanted in each glass tube (50 mL), which contained 20 mL 0.8% water agar medium. The plants were grown in a controlled environmental chamber with a 16 h, 30 C day (light intensity of 1000 mmol m22s21) and 8 h, 20 C night regimes (Jang et al. 2008). Two young seedlings were transplanted in glass tubes containing a 0.8% water agar medium. Forty milliliters of culture solution was centrifuged at 5000 g, 4 C, 15 min, and the resulting pellet and supernatant were stored immediately at 270 C (Khan et al. 2008) until lyophilized (ISE Bondiro freeze dryer). The lyophilized supernatant was mixed with 1 mL autoclaved distilled water and 10 mL of this solution was applied to the apical meristem of rice seedlings, at the two-leaf stage (Hamayun et al. 2009d). Shoot length was measured after 7 d and compared to Waito-C rice seedlings, which had been treated either with distilled water (negative control) or Czapek medium (positive control). Each sample was assayed in triplicate. In a separate bioassay experiment cucumber seeds were surface sterilized with 5% NaClO for 15 min and then washed with distilled water. Seeds were sown in autoclaved soil under greenhouse conditions (30 6 2 C). Fungal isolate MH-6 induced maximum stem elongation during screening and was tested for plant growth promotion on cucumber. Ten milliliters MH-6 CF were taken directly from filtered culture solution and applied to 3 wk old cucumber seedlings, and the growth attributes (i.e. plant length, shoot length, plant fresh weight, plant dry weight and leaf area) were recorded 2 wk after CF treatment. The results were directly compared with plants applied with H2O and Czapek media. Each treatment comprised three replicates with15 plants per replicate. Extraction and quantification of gibberellins.— Gibberellins were extracted from CF of MH-6 by following the protocol of Lee et al. (1998). GA were chromatographed on a 3.9 3 300 m Bondapak C18 column (Waters Corp., Milford,
HAMAYUN ET AL.: GIBBERELLIN PRODUCTION Massachusetts) and eluted at 1.5 mL/min with this gradient: 0–5 min, isocratic 28% MeOH in 1% aqueous acetic acid; 5–35 min, linear gradient 28–86% MeOH; 35– 36 min, 86–100% MeOH; 36–40 min, isocratic 100% MeOH. Forty-eight 1.5 mL fractions were collected. The fractions were prepared for gas chromatograph/mass spectrometer (GC/MS) with selected ion monitoring (SIM) (6890N network GC system and 5973 network mass selective detector; Agilent Technologies, Palo Alto, California). For each GA 1 mL sample was injected in 30 m 3 0.25 mm i.d., 0.25 mm thick film DB-1 capillary column ( J&W Scientific Co., Folsom, California). The GC oven temperature was programmed for a 1 min hold at 60 C, then to rise at 15 C min21 to 200 C followed by 5 C min21 to 285 C. Helium carrier gas was maintained at a head pressure of 30 kPa. The GC was interfaced directly with a mass selective detector with an interface and source temperature of 280 C, an ionizing voltage of 70 eV and a dwell time of 100 ms. Full scan mode (the first trial) and three major ions of the supplemented (2H2) GA internal standards (obtained from Prof Lewis N. Mander, Australian National University, Canberra, Australia) and fungal GA were monitored simultaneously. The retention time was determined with hydrocarbon standards to calculate the KRI (Kovats retention index) value, while the GA quantification was based on the peak area ratios of non-deuterated (extracted) GA to deuterated GA. Genomic DNA extraction and fungal identification.—Genomic DNA isolation and PCR were performed according to the protocol of Khan et al. (2008). Fungal isolate MH-6 was identified by sequencing the internal transcribed region (ITS) of 18S rDNA, with universal primers ITS-1 (59-TCC GTA GGT GAA CCT GCG G-39) and ITS-4 (59-TCC TCC GCT TAT TGA TAT GC-39). BLASTn (http://www.ncbi. nlm.nih.gov/BLAST/) was used to look for nucleotide sequence homology. The sequences obtained were aligned by Clustal W with MEGA 4 software (Tamura et al. 2007), and the neighbor joining tree was generated with the same software. The bootstrap replications (100 000) were used as statistical support for the nodes in the phylogenetic tree. Growth parameters.—Plant height, shoots length, plant fresh and dry biomass and leaf area were measured 14 d after fungal CF application. Three replicates of 10 plants each were selected randomly for measuring these growth attributes. The dry weights were measured after drying samples at 70 C for 48 h in an oven (Bohm 1979). The leaf area was calculated with CI-203 portable laser area meter (CID Inc., USA). Statistical analysis.—These data were analyzed statistically for standard deviation with MS-EXCEL software. The mean values were compared with Duncan’s multiple range test (DMRT) at P , 0.05 (ANOVA SAS release 9.1; SAS, Cary, North Carolina). RESULTS
Screening of endophytic fungi for plant growth promotion.—CF from pure cultures of 19 endophytic fungi
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FIG. 1. Influence of fungal CF (10 mL) on shoot length of Waito-C rice seedlings after 7 d. Error bars show standard deviations. Control-1 stands for distilled water; Control-2 stands for Czapek medium. MH-1–MH-19 represent the 19 fungal endophytes isolated from cucumber.
were screened for plant-growth promotion. It was found that 16 fungal isolates promoted shoot length of Waito-C rice while three isolates (i.e. MH-4, MH-9 and MH-18) inhibited plant growth. Isolate MH-6 significantly promoted shoot length (12.9 cm compared to Czapek (8.4 cm) and distilled water (7.9 cm) treated plants (FIG. 1). Bioassay of MH-6 on cucumber plants.—The CF of isolate MH-6 was bioassayed on cucumber plants. We observed that all growth attributes were enhanced significantly by MH-6 CF application as compared to control treatments. The mean plant length (23.6 cm), shoot length (13.78 cm), fresh weight (6.74 g), dry weight (1.25 g) and leaf area (209.8 cm2) all were significantly greater than distilled H2O and Czapektreated plants. The effect of MH-6 on growth attributes of cucumber was not significantly different from F. fujikuroi-treated plants (TABLE I). Analyses of MH-6 CF for gibberellins.—GA analysis of the CF of isolate MH-6 showed the presence of physiologically active gibberellins (GA1, 1.97 ng/mL; GA3, 5.18 ng/mL; GA4, 13.35 ng/mL and GA7, 2.4 ng/mL) along with physiologically inactive (GA9 [0.69 ng/mL], GA12 (0.24 ng/mL), GA15 (0.68 ng/ mL), GA19 (1.94 ng/mL) and GA20 (0.78 ng/mL]) GA. CF of MH-6 produced higher amounts of GA3, GA4, GA7 and GA19 than wild type Fusarium fujikuroi, which was used as a positive control (FIG. 2). GC-MS SIM spectra for bioactive GA (SUPPLEMENTARY FIGS. 1, 2, 3 and 4). Identification of fungal isolate MH-6.— BLASTn showed that fungal isolate MH-6 had a 99% sequence homology and 98% query coverage with Cladosporium species. In the consensus tree isolate MH-6 formed a subclade (98% bootstrap support) with isolate Cladosporium sp. SPRY16 (FIG. 3). On the basis of
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TABLE I. Effect of fungal culture filtrates (CF) on growth attributes of soil grown cucumber measured 3 wk after CF application Plant length (cm/plant)
Treatment Cladosporium sp. MH-6 (10 mL)b F. fujikuroi (10 mL) Control (distilled H2O) Czapek medium (10 mL)
23.6 23.9 16.3 17.2
6 6 6 6
1.41 1.83 0.61 1.05
Ac A B B
Shoot length (cm/plant) 13.78 14.15 10.2 10.8
6 6 6 6
0.34 0.62 0.25 0.56
A A B B
Plant FWa (g/plant) 6.74 6.93 4.42 4.91
6 6 6 6
0.22 0.67 0.18 0.25
Plant DWa (g/plant) A A B B
1.25 1.16 0.79 0.86
6 6 6 6
0.04 0.06 0.03 0.02
Leaf area (cm2/plant) A A B B
209.8 187.3 165.04 172.2
6 6 6 6
11.3 A 7.9 A 7.4 B 9.1 B
a
FW stands for fresh weight; DW stands for dry weight. CF of MH-6 and F. fujikuroi (10 mL each) were taken directly from their respective culture solutions. The effect of Czapek medium on plant growth was measured by applying 10 mL of this medium as a positive control. c Treatment means having common letters are not significantly different at the 5% level by Duncan’s multiple range test. b
sequence homology and neighbor joining phylogenetic analysis, isolate MH-6 appears to be a novel strain of Cladosporium sp., closely related to isolate SPRY 16, which was identified as a new strain of Cladosporium species. The 18S rDNA sequence of isolate MH-6 was submitted to NCBI GenBank under accession number FJ950740.
to promote plant growth and tolerance to abiotic and biotic stresses. As such the practical applications of endophytes as potential sources of bioorganic nutrients and as biocontrol agents can significantly improve yields in an environmentally sound way (Diene and Narisawa 2009). Endophytic fungi are well known plant symbionts, although information on GA production and plant
DISCUSSION
Agriculture in the 21st century faces the daunting task of satisfying the increasing demand for food in a context of continuous depletion of natural resources and the need to respect international environmental standards. Among the alternatives to conventional agriculture developed in this context, symbiotic fungal associations with crops show considerable promise because of their effectiveness, habit-specific mode of action and ability to provide multiple benefits (Diene and Narisawa 2009). Endophytism represents a new area of research based on the benefits of mutualistic interactions between host crops and nonpathogenic fungi. The advantages conferred by endophytic fungi include their ability
FIG. 2. Mean amounts of various gibberellins secreted by fungal isolate MH-6 and F. fujikuroi. The quantification of GA was accomplished with GC/MS SIM. Values given are means of three replicates, while error bars show standard deviation.
FIG. 3. Phylogenetic tree constructed by the neighbor joining method with the 18S rDNA sequence (ITS region) of Cladosporium sp. MH-6 and related fungi (21 references and 1 clone). Fungal isolate MH-6 formed a subclade with 98% bootstrap consensus with Cladosporium sp. SPRY16, which identify MH-6 as a new strain of Cladosporium species. A. niger was taken as outgroup.
HAMAYUN ET AL.: GIBBERELLIN PRODUCTION growth promotion capacity of this group is limited. In our current study we isolated 19 fungal endophytes from randomly selected cucumber plants grown under greenhouse conditions. The soil used for this experiment was collected from barren paddy fields in the outskirts of Daegu City, Korea, and the fungi most probably were present in the soil before entering the roots of cucumber. The majority of these isolates promoted growth of Waito-C, which suggests that they might secrete secondary metabolites that influence plant growth and development (Khan et al. 2008, Hamayun et al. 2009a). Fungal isolate MH-6 induced maximum shoot elongation of Waito-C during screening and so was studied in greater detail for production of GA. Because GA are known to affect shoot elongation the CF from the fungal isolate MH-6 thus was analyzed for presence of GA. Screening of fungal CF for secondary metabolites is an established procedure for the identification of biologically active molecules (Higgs et al. 2001). The use of nutrientfree water-agar growth media for rice plants helped in the determination of the sole effect of CF on rice seedling growth. We used Waito-C rice for the screening experiment because Waito-C is a dwarf and bioactive GA-deficient rice cultivar, with blocked C13 hydroxylation pathway for GA biosynthesis (Mitsunaga and Yamaguchi 1993). The effect of CF from MH-6 was similar to that of F. fujikuroi, a known GA producer. Similar observations on GA production capacity of endophytic fungi also have been reported by Khan et al. (2008) and Hamayun et al. (2009d). The CF of MH-6 also was bioassayed on cucumber because we were interested in investigating the effects of MH-6 on its host. We observed that growth attributes of cucumber were significantly promoted by such an application. Our current study had a result similar to a report on growth promotion of host soybean by applying CF of an endophytic fungus (Hamayun et al. 2009d). Plant growth promotion also has been observed by endophytic yeast due to production of auxins, as in the case of Williopsis saturnus in maize (Nasser 2005). Plant growth-promoting fungi (PGPF) associated with roots secrete a large number of secondary metabolites in the rhizosphere (Strobel 2003). These secretions have been and will continue to be an efficient source of novel secondary metabolites (Cragg et al. 1997). Secretion of GA by PGPF has been reported by Kawaide (2006) and Vandenbussche et al. (2007), which indicated the potential beneficial role of PGPF in plant growth and development, especially under nutrient-deficient conditions. Cladosporium sp. MH-6 produced nine different gibberellins that included bioactive GA1, GA3, GA4 and GA7. Isolate MH-6 CF contained greater amounts
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of bioactive GA3, GA4 and GA7 than wild type F. fujikuroi, which demonstrates the novelty of this fungal strain. The GA production potential of C. sphaerospermum was reported by Hamayun et al. (2009d) although GA1 was not detected in an earlier study, and the amounts of bioactive GA produced by C. sphaerospermum were far less than those reported for MH-6. GA were analyzed by GC/MS-SIM, which is a reliable tool. Compared to non-MS detection based chromatographic techniques (HPLC-DAD, GC-FID), where only compounds targeted by a special analytical protocol are found. GC-MS as a tool provides a unique opportunity to learn new interesting and unexpected knowledge regarding extract (Franck et al. 2005). Molecular and phylogenetic approaches now are used widely for the identification of fungi. Genomic DNA sequencing is an objective, reproducible and rapid technique for fungal identification. The internal transcribed spacer region (ITS) is the most commonly used sequence for molecular identification of fungi (Kim and Lee 2000, Lee et al. 2001, Sugita and Nishikawa 2003). In our current study we used the 5.8S and flanking ITS1/4 gene regions for fungal identification. The 5.8S gene is highly conserved and is suitable for higher taxonomic analysis, while the highly variable ITS region is useful for analysis at a lower taxonomic level. The phylogenetic tree construction is also crucial in molecular identification because BLASTn alone cannot overcome the possibility of statistical errors. Phylogenetic analysis shows that MH-6 is closely related to Cladosporium sp. SPRY16 but not identical because the bootstrap support and sequence homology of the two strains are less than 100%. The fungal isolate MH-6 is therefore a new strain of Cladosporium species. Our current study presents valuable information on the relationship of GA production capacity of Cladosporium sp. MH-6 and the potential role and application of fungal endophytes in plant growth and development. Our studies indicate that Cladosporium sp. MH-6 is a novel GA-producing fungus that secretes comparable amounts of GA to F. fujikuroi. Therefore this isolate might have potential for use as a biofertilizer with minimal environmental risks. Additional studies on the identification and characterization of GA-encoding gene cluster and optimization of GA-producing media for this fungus will need to be developed. ACKNOWLEDGMENTS
This work was financially supported by the Korea Research Foundation Grant (KRF-521-F00001) and Republic of Korea’s Brain Korea 21 Project.
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