Biosynthetic pathway and optimal conditions for the production of indole-3-acetic acid by an endophytic fungus, Colletotrichum fructicola CMU-A109

Endophytic fungi are known to produce indole-3-acetic acid (IAA), which can stimulate plant growth. Twenty-seven isolates of endophytic fungi were isolated from Coffea arabica in northern Thailand. Only one isolate (CMU-A109) produced IAA in vitro. This isolate was identified as Colletotrichum fructicola based on morphological characteristics and molecular phylogenetic analysis of a combined five loci (internal transcribed spacer of ribosomal DNA, actin, β-tubulin 2, chitin synthase and glyceraldehyde-3-phosphate dehydrogenase genes). Identification of a fungal IAA production obtained from indole 3-acetamide (IAM) and tryptophan 2-monooxygenase activity is suggestive of IAM routed IAA biosynthesis. The highest IAA yield (1205.58±151.89 μg/mL) was obtained after 26 days of cultivation in liquid medium supplemented with 8 mg/mL L-tryptophan at 30°C. Moreover, the crude fungal IAA could stimulate coleoptile elongation of maize, rice and rye. This is the first report of IAA production by C. fructicola and its ability to produce IAA was highest when compared with previous reports on IAA produced by fungi.


Introduction
Endophytic fungi are defined as the fungi that colonize plant tissues without causing any disease-related symptoms [1][2][3]. Colonization by endophytic fungi can improve the ecological adaptation of the host plant by enhancing its growth and tolerance to biotic and abiotic stresses [4,5]. Several studies have reported the potential of endophytic fungi to produce bioactive compounds that protect the host plant against microbial influences and promote plant growth in both volatile and non-volatile substance productions [6][7][8]. Many studies have shown that plant growth promotion may be attributed to the secretion of plant-growth promoting secondary metabolites (phytohormones e.g. auxins, cytokinins, ethylene and gibberellins, and siderophore) and the ability to mobilize insoluble phosphate and provide nitrogen to their host plants by endophytic fungi [9][10][11][12][13]. PLOS

Fungal isolation
Endophytic fungi were isolated from healthy mature leaves and stems of coffee plants (Coffea arabica L.) collected from Khun Changkean of Highland Research and Training Center, Faculty of Agricultural, Chiang Mai University, Thailand (18˚50'43.0"N, 98˚54'26.0"E) in November 2012. The coffee leaves and stems were washed in running tap water for 15 min and cut into small pieces (5 mm 2 leaf and 1 mm length stem). The cut samples were triple surface-sterilized method (70% ethanol for 1 min, 2% sodium hypochlorite for 3 min and 95% ethanol for 30 sec) [34] under a laminar flow hood. Twenty-five leaf and stem pieces were placed on potato dextrose agar (PDA) supplemented with 50 ppm of chloramphenicol and 35 ppm of Rose Bengal. The plates were incubated at 25˚C. The fungi growing out from the tissue samples were transferred to fresh PDA plates. The pure cultures were preserved on PDA slants for the short-term and 20% glycerol at -20˚C for the long-term at The Culture Collection of the Sustainable Development of Biological Resources (SDBR) Laboratory, Faculty of Science, Chiang Mai University, Chiang Mai and Thailand Bioresource Research Center, Pathum Thani Province, Thailand.
mycelium, density and pigment production were recorded. Micromorphological characteristics were examined using a light microscope (Olympus CX51, Japan). Size data of the anatomical features are based on at least 50 measurements of each structure. Molecular studies. Molecular techniques were used to confirm the identification of IAA producing endophytic fungi. Genomic DNA was extracted from one week-old fungal mycelia on PDA (1-5 mg) using a DNA Extraction Mini Kit (FAVORGEN, Taiwan) following the manufacturer's protocol. The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), actin (ACT), β-tubulin 2 (TUB2), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with primers and annealing temperatures following the previous studies [35−38] as presented in Table 1. Polymerase chain reaction (PCR) was performed in 20 μL reaction containing 1.0 μL DNA template, 1.0 μL of each forward and reverse primers, 10.0 μL 2X Quick Taq HS DyeMix (TOYOBO, Japan) and 7 μL deionized water, and the following thermal conditions: 94˚C for 2 min, followed by 35 cycles of 94˚C for 2 min, the temperatures dependent on the amplified gene (Table 1) for 1 min and 72˚C for 1 min, and a final 72˚C for 10 min on a peqSTAR thermal cycler (PEQLAB Ltd., UK). PCR products were checked on 1% agarose gels stained with ethidium bromide under UV light. PCR products were purified using a PCR clean up Gel Extraction NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Germany). The purified PCR products were directly sequenced. Sanger sequencing was carried out by 1 ST Base Company (Kembangan, Malaysia) using the PCR primers mentioned above. Sequences were used to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html).
For phylogenetic analysis, the sequences obtained from this study and from previous studies along with sequences from GenBank database were used. The multiple sequence alignment was carried out using MUSCLE [39]. A maximum likelihood (ML) phylogenetic tree from a combined ITS, ACT, TUB2, GAPDH and CHS-1 sequences was constructed using RAxML v7.0.3 [40], applying the rapid bootstrapping algorithm for 1000 replications using the GTRGAMMA model. The ML trees were viewed with TreeView32 [41]. Bayesian phylogenetic analyses were carried out using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method in MrBayes version 3.2 [42].

Determination of fungal indole compound production
One fungal disc (5 mm in diameter) was inoculated into 5 mL of potato dextrose broth (PDB) supplemented with 2 mg/mL L-Trp (Sigma-Aldrich, Germany), pH 6.0, in 18 (internal diameter) × 180 mm test tubes and incubated in the dark on a shaker for one week at room temperature (25±2ºC). The fungal culture was collected by centrifugation at 1250 × g for 5 min. The Table 1. Details of primers, annealing temperature and the obtained product size of the amplification of gene targets in this study.

Region
Primer colorimetric assay [43] was performed to determine fungal indole compound production. The fungal culture was mixed with Salkowski's reagent (1 mL of 0.5 mol/L FeCl 3 in 50 mL of 35% HClO 4 ) in ratios of 1:2 (v/v), respectively, and incubated in the dark for 30 min. The pink to red color was considered positive for IAA production. Three replications were made.

Extraction of fungal indole metabolites
The fungal IAA supernatant was acidified to pH 4.0 by using 1 mol/L HCl and mixed twice with ethyl acetate at double the volume of the supernatant. The extracted ethyl acetate was dried using a rotary evaporator. The crude extract was dissolved in methanol and kept at -20˚C.

Detection and quantification of fungal IAA and related indole compounds
Thin layer chromatography. The crude ethyl acetate extract of fungal IAA was applied to of thin layer chromatography (TLC) plate (Silica gel G F257, thickness 0.25 mm, Merck, Germany). The chromatogram was performed in the mobile phase that contained n-hexane: ethyl acetate: isopropanol: acetic acid (40:20:5:1, v/v/v/v) following the study of Chung et al. [16].
High performance liquid chromatography. The quantification of fungal IAA was performed with high performance liquid chromatography (HPLC) according to the method described by Szkop and Bielawski [45] with some modifications. The Shimadzu Prominence UFLC system was used, equipped with an LC-20 AD pump, SIL20ACHT autosampler, CTO-20 AC column oven, CBM-20A system controller and SPD-20A photodiode array detector. Ten microliters of the sample were injected to the Mightysil RP-18 (250 × 4.6 mm, 5 μm) column heated at 40˚C. The gradient elution was applied. The solvent A consisted of 2.5 : 97.5% (v/v) acetic acid : deionzed water, pH 3.8 (the final pH was adjusted by 10 mol/L KOH) and solvent B consisted of 80 : 20% (v/v) acetonitrile : deionzed water. The mobile phase start with solvent A: solvent B at 80 : 20%, changing to 50 : 50%, 0 : 100% and 80 : 20% at 25, 31 and 33 min, respectively. The total run time was 40 min and a flow rate of mobile phase was set to 0.5 mL/min. The injection volume was 10 μL. The detection wavelengths were 280 and 350 nm. Retention times of the samples were determined by comparing them with the L-Trp, IAA, TOL, IPyA, IAM, TAM, ILA and IAN standards and further by co-injections. Quantification of fungal IAA and related indole compounds were carried out with the calibration curve constructed with various indole compounds.

Determination of IAA biosynthesis pathway of selected IAA producing isolate IAA production from intermediate indole compound cultivation.
To analyze the IAA biosynthetic pathway of the selected fungus, five mycelial discs were cultivated with 5 mL of basal liquid medium (2.0 g NaNO 3 , 0.5 g MgSO 4 �7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 �7H 2 O, 10.0 g glucose and 15.0 g agar per liter of distilled water, pH 6.0) supplemented with 2 mg/mL of each indole intermediate compound (IAM, IAN, IPyA and TAM) in 18 × 180 mm test tubes at 30˚C under shaking conditions at 120 rpm in the darkness for one week. Cultured filtrates were sampled every day and were then extracted. The crude extracts were examined in terms of the IAA and intermediate indole compound production by HPLC techniques, as has been described above. Three replications were made.
Tryptophan 2-monooxygenase activity. The fungus was cultivated in the basal liquid medium supplemented with 2 mg/mL of L-Trp at 30˚C under shaking conditions at 120 rpm in the darkness for five days. Mycelia were filtrated and washed twice with Tris-HCl buffer (50 mmol/L, pH 7.8) and resuspended in 4 mL of the same buffer. Mycelia were lysed by sonication with an Ultrasonic disruptor UD-201 (Tomy Seiko, Japan). After sonication, lysate was centrifuged (1250 × g, 10 min, 4˚C) and clear supernatant was used as a source of enzyme. Determination of tryptophan 2-monooxygenase activity followed the method described by Mujahid et al. [46] with some modifications. Enzyme assay was carried out in the final volume of 1 mL Tris buffer (50 mmol/L, pH 7.8) containing 0.5 mL of L-Trp and 0.5 mL of enzyme extract. Reaction mixture was incubated at 30˚C for 25 min and then stopped by addition of 100 μL of 5 mol/L HCl. Reaction mixture was centrifuged and the supernatant was collected. The formation of IAM was analyzed by HPLC techniques as mentioned above. Three replications were made.

Optimal conditions for fungal IAA production
One fungal isolate (CMU-A109) showing the ability to produce IAA was obtained and used to identify the optimal conditions for IAA production, including L-Trp concentration, temperature and cultivation period. The effect of L-Trp concentration on IAA produced by isolate CMU-A109 was studied by inoculating fungal disc (5 mm in diameter) into 18 × 180 mm test tubes containing 5 mL PDB supplemented with different concentrations of L-Trp (0, 2, 4, 6, 8 and 10 mg/mL) and shaken in the darkness at room temperature (25±2ºC) for one week. The fungal supernatants were then harvested and the estimated levels of IAA production were measured by HPLC. Three replications were made. The concentration of L-Trp that gave the highest level of IAA was selected for further experiments.
Different temperatures for fungal IAA production were examined. Cultivation was performed in the darkness at various temperatures (22ºC, room temperature (25±2ºC), 30ºC and 37˚C) on a shaker at 120 rpm. The fungal supernatants were harvested and the amount of IAA was determined after one week of cultivation. This treatment was carried out in triplicate.
The effects of the cultivation period on IAA production were studied. The fungal discs were cultivated in liquid medium and incubated in the dark on a shaker with 120 rpm at 30˚C for 30 days. The amount of fungal IAA was evaluated every 2 days. Three replications were made.

Biological activities of fungal IAA
Biological activity of fungal IAA produced by isolate CMU-A109 was determined with the coleoptile elongation of corn (Zea mays L.), rice (Oryza sativa L.) and rye (Secale cereal L.) [17,20]. Crude fungal IAA was dissolved in 0.5 mL of 0.1 mol/L NaOH, and then diluted with sterile distilled water. Crude fungal IAA solution (containing 20 and 30 μg/mL of IAA and IAM, respectively) was used in this experiment. The positive control consisted of the stock solution of 20 μg/mL IAA, 30 μg/mL , IAM and IAA mixed with IAM (20 μg/mL IAA mixed with 30 μg/mL IAM), and sterile distilled water was used as the negative control. The seed were surface disinfested in a mixture of 0.2% Tween 80 and 2% sodium hypochlorite for 3 min, followed by rinsing three times in sterile water. The surface disinfested seeds were placed on 1.5% water agar and incubated at 25ºC. After 3 days, 1.5−2.0 mm tips of the coleoptiles from seedlings non-colonized by fungi and bacteria were removed and adjusted to 10 mm lengths. Sections of coleoptiles were floated in distilled water for 2 h before being used. For each treatment, ten coleoptile segments were floated in a 9 cm Petri dish containing 20 mL of each solution and were then incubated in the dark at 25ºC. After 24 h, the length of the coleoptile segments was measured. Each treatment was conducted with five replicates.

Statistical analysis
Statistical analyses were carried out by one-way analysis of variance (ANOVA) using SPSS program version 20 for Windows. Tukey's range test was used to determine significant differences (P<0.05) between the mean values of each treatment.

Fungal isolation and identification of IAA producing isolate
Twenty-seven endophytic fungi isolates were obtained from arabica coffee. Ten isolates were obtained from stem tissues and 17 isolates were obtained from leaf tissues. Only one isolate (CMU-A109) displayed positive IAA production when tested by colorimetric assay of Salkowski's reagent as indicated by red color formation, while the uncultivated medium displayed a negative reaction.
Colonies of isolate CMU-A109 on PDA grew to 80−85 mm at 25˚C in the darkness after one week (Fig 1A). Colonies were cottony, white to pale grey and the reverse side was white to pale yellow in color. Sclerotia were absent. Mycelia were superficial and immersed. Hyphae were branched, septate, and hyaline to dark brown in color and 2−3 μm wide. Appressoria were 4.0−9.5 × 3.5−7.5 μm, mostly formed from mycelia, they were brown to dark brown in color, ovoid, clavate and slightly irregular to irregular in shape while often becoming complex with age ( Fig 1B). Conidiophores were reduced to conidiogenous cells. Conidiogenous cells were cylindrical to ampulliform, hyaline, smooth, straight to curved and wider at the base, 7 −18 × 1−3 μm (Fig 1C). Conidia were 9.7−14 × 3−4.5 μm (n = 50), commonly one-celled, smooth-walled with a large guttule at the centre, hyaline, cylindrical with obtuse to slightly rounded ends that were sometimes oblong (Fig 1D). Based on morphological observations, this fungal isolate was initially identified as belonging to the Colletotrichum gloeosporioides species complex [47,48].
Molecular methods were used to confirm the identity of the isolate. The ITS, ACT, TUB2, CHS-1 and GAPDH sequences of isolate CMU-A109 were deposited in GenBank database under accession KT378065, MG717492, MG717493, MG717494 and MG717495, respectively. A phylogram of the combined five-gene loci (ITS, ACT, TUB2, CHS-1 and GAPDH) is presented in Fig 1E. Our phylogenetic results assigned isolate CMU-A109 to C. fructicola species. The species was clearly distinguished from other Colletotrichum species and formed a sister taxon to C. nupharicola with BS support of 82% and PP value support of 0.86. Therefore, isolate CMU-A109 was identified as C. fructicola based on molecular characteristics. A pure culture was deposited in the Culture Collection of the SDBR Laboratory, Faculty of Science, Chiang Mai University and Thailand Bioresource Research Center, Thailand under number SDBR-CMU-A109 and TBRC9265, respectively.

Detection and quantification of IAA production by Colletotrichum fructicola CMU-A109
A combination of TLC and chromogenic reagents can provide another means for the verification of indole derivatives. The R f values of each indole compound, crude fungal culture and crude uncultivated medium, which were applied in UV and chemical reagents, are presented in Fig 2 and Table 2 (Fig 3A). The ethyl acetate extract of C. fructicola CMU-A109 showed a peak that corresponded to the IAA standard, with a maximum absorption value of 279 nm (Fig 3B). The identification of fungal IAA was confirmed by a co-injection with the IAA standard. The fungal IAA levels were also quantified by HPLC. Colletotrichum fructicola isolate CMU-A109 produced IAA at the level of 662.96±56.18 μg/mL. The HPLC chromatogram presented fungal IAM in crude fungal culture with a maximum absorption value of IAM at 230 nm. Identification of fungal IAM was confirmed by co-injection with the IAM standard. No correspondence of the IAA and IAM peaks was presented in the uncultivated medium extract (Fig 3C).

Determination of IAA biosynthesis pathway of Colletotrichum fructicola CMU-A109
IAA production from intermediate indole compound. The IAA production by C. fructicola CMU-A109 by IAM, TAM, IPyA and IAN was examined by HPLC techniques. Fungal   IAA was presented in the crude fungal culture with IAM (S1 Fig). No correspondence of the IAA peak was presented in the crude fungal culture with TAM, IPyA and IAN. Therefore, C. fructicola CMU-A109 produced IAA from IAM. Tryptophan 2-monooxygenase activity. HPLC analysis of the enzyme reaction mixture indicated the formation of IAM and IAA at retention times of 13.9 and 18.7 min, respectively. This corresponds to the IAA and IAA standards (Fig 4), which indicates that the crude lysate of C. fructicola CMU-A109 has tryptophan 2-monooxygenase activity.

Optimal conditions for fungal IAA production by Colletotrichum fructicola CMU-A109
Fungal IAA production was estimated with different concentrations of L-Trp by C. fructicola CMU-A109. The amount of fungal IAA is shown in Fig 5A. Maximum IAA production (799.18±65.00 μg/mL) was observed in the liquid medium supplemented with 8 mg/mL L-Trp when shaken in the darkness at room temperature for 7 days.
The IAA production by C. fructicola CMU-A109 at different temperatures is shown in Fig  5B. The results indicated that this fungus could produce IAA at all of the tested temperatures in this study and the maximum IAA yield (862.26±28.03 μg/mL) was observed at 30ºC, followed by that at room temperature (752.75±62.30 μg/mL). The lowest IAA yield (70.39 ±7.23 μg/mL) was recorded at 37ºC.
The effects of the cultivation period on the IAA production of C. fructicola CMU-A109 are shown in Fig 5C. The results indicated that the IAA yield increased from 0 to 26 days. The highest IAA yield was found (1205.58±151.89 μg/mL) at 26 days after cultivation.

Biological activity of fungal IAA
Elongation of rice, corn and rye coleoptile segments induced by crude IAA extract of C. fructicola CMU-A109 is shown in Fig 6. The lengths of all coleoptiles treated with fungal IAA were not statistically different from those of the IAA, IAM and IAA mixed IAM treatments. However, the values were significantly higher than the distilled water treatment.

Discussion
IAA production by plant-associated microorganisms have been broadly studied and reported [7,12,20,33]. In this study, C. fructicola CMU-A109 isolated from arabica coffee produced IAA in liquid medium supplemented with L-Trp at the IAA level 662.96 μg/mL. The results are similar to those of previous studies that found that the pure culture of endophytic fungi (e.g. Alternaria alternata, Aspergillus fumigatus, Chaetomium globosum, Chrysosporium pseudomerdarium, C. acutatum, C. gloeosporioides, Fusarium spp., M. cinnamomi, Paecilomyces spp., Penicillium spp., Phoma spp. and Tulasnella sp.) could produce IAA after being cultured in liquid medium supplemented with L-Trp [7, 10-13, 16, 17, 19, 49]. However, the present study provides the first report of IAA produced by C. fructicola. Indole derivatives as intermediate compounds give clues for identification of the IAA biosynthetic pathway. Reports on the IAA biosynthetic pathway of microbes are limited [22] and there is a need to more clearly understand the IAA production pathway of microorganisms. Consequently, a different biosynthesis pathway of IAA has been proposed for various microbial species [21,22,50] and it has been determined that a single species contained more than one pathway [22,51]. Our study found that C. fructicola produced IAA from IAM, which was confirmed by the fungal IAA production from IAM and tryptophan 2-monooxygenease activities. Similarly, previous studies have reported synthesis of IAA via the IAM pathway in C. gloeosporioides f. sp. aeschynomene, F. proliferatum, F. verticillioides, F. fujikuroi and F. oxysporum [25,31,52]. However, Chung et al. [16] and Shilts et al. [53] reported synthesis of IAA by multiple pathways in C. acutatum (IAM and IPyA pathways). IAA production through the IPyA pathway was found in Piriformospora indica, Rhodosporidium paludigenum, Rhizoctonia cerealis, R. solani, Ustilago maydis and U. esculenta [26,27,30,33,54]. L-tryptophan concentration, temperature and cultivation period affected microbial IAA production. IAA produced by C. fructicola CMU-A109 increased when L-Trp concentration was increased to 8 μg/mL, IAA levels decreased when high concentration was used. This result is similar to those of previous reports, which indicated that the L-Trp concentration significantly affects microbial IAA synthesis. For example, Chutima and Lumyong [17] reported that the IAA level of C. fructicola CMU-AU 006 and Tulasnella sp. CMU-SLP 007 increased along with increased L-Trp concentrations but decreased after the L-Trp concentration reached 6 mg/mL. Bose et al. [55] found that the optimum L-Trp concentration for IAA produced by Pleurotus ostreatus was 1 mg/mL. Kumla et al. [20] reported that the maximum IAA level of different ectomycorrhizal fungi was found at levels of 2 mg/mL (Astraeus odoratus, Pisolithus albus, and Scleroderma sinnamariense) and 4 mg/mL (Phlebopus portentosus), which were Biosynthesis pathway of indole-3-acetic production by Colletotrichum fructicola CMU-A109 optimal L-Trp concentrations. However, the concentration values decreased after adding L-Trp to the culture medium in higher amounts than the optimal L-Trp concentration. In this study, the optimal temperature for IAA production by C. fructicola CMU-A109 was 30ºC. This is in accordance with findings of previous studies, which reported that endophytic fungi normally produced IAA at temperatures between 25 to 30ºC [7, 11-13, 17, 50]. Moreover, 30ºC was the optimal temperature for IAA synthesis of a wide range of microbes such as Lentinus sajor-caju [56], Streptomyces sp. CMU-H009 [57], Pantoea agglomerans strain PVM [58] and some ectomycorrhizal fungi; A. odoratus, Ph. portentosus, Pi. albus and S. sinnamariense [20]. Our results indicated that the cultivation period affected the IAA production of C. fructicola CMU-A109. This result is supported by previous studies that have indicated that IAA production varies greatly among different microbial species, and our result also correlates with the finding that L-Trp availability in the culture medium is associated with microbial growth [20,55,57]. Moreover, the highest level of IAA was recorded when microbes were grown in the stationary phase [12,20,43]. Under the optimal conditions for in vitro IAA production, bacteria producing IAA ranged from 26.63 to 6100 μg/mL, respectively [57][58][59][60][61]. Previous reports have shown that the in vitro IAA level produced by fungi under the optimal conditions ranged from 40.76 to 563.80 μg/mL [12,20,32,33,55,56,62]. Interestingly, C. fructicola CMU-A109 Biosynthesis pathway of indole-3-acetic production by Colletotrichum fructicola CMU-A109 produced IAA 1205.58±151.89 μg/mL under optimal conditions and its ability to produce IAA was highest when compared with previous reports on IAA produced by fungi.

Conclusion
The endophytic fungus, C. fructicola CMU-A109 isolated from leaf tissues of coffee plant produced IAA in vitro via the IAM pathway. The highest IAA yield was obtained after 26 days of cultivation in liquid medium supplemented with 8 mg/mL L-Trp at 30˚C. Crude fungal IAA stimulated coleoptile elongation as plays an important role in plant-growth promotion. Further study of this fungal isolate is required to evaluate its plant growth promoting abilities for the purposes of developing fungal inoculum. In addition, any pathogenicity needs to be tested.