Auxin-producing fungal endophytes promote growth of sunchokeRhizosphere 16 (2020) 100271
Available online 28 October 2020 2452-2198/© 2020 Elsevier B.V. All rights reserved.
Auxin-producing fungal endophytes promote growth of sunchoke
Thanapat Suebrasri a,b, Hiroyuki Harada c, Sanun Jogloy d, Jindarat Ekprasert a, Sophon Boonlue a,*
a Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand b Graduate School, Khon Kaen University, Khon Kaen, 40002, Thailand c Department of Environmental Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Japan d Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand
A R T I C L E I N F O
Keywords: Plant growth promoting fungi Fungal endophytes IAA biosynthesis Pathway phosphate solubilization Rhizosphere
A B S T R A C T
Endophytic fungi were able to protect their host plants against pathogens and promote plant growth. No previous studies have been conducted on the growth promotion of sunchoke by endophytic fungi. This research was the first to characterize plant growth promoting properties of endophytic fungi including, Macrophomina phaseolina BUP2/3 and Diaporthe phaseolorum BUP3/1 isolated from sunchoke and Daldinia eschscholtzii 2NTYL11, Tricho- derma koningii ST-KKU1, Trichoderma erinaceum ST-KKU2, Macrophomina phaseolina SS1L10 and Macrophomina phaseolina SS1R10 from medicinal plants. Also, their plant growth promoting efficiency in artichoke plants under greenhouse condition was evaluated. The highest phosphate solubility and production of extracellular enzymes including amylase, protease, cellulase and xylanase were found after 5 and 21 days of incubation, respectively. Moreover, Indole-3-Acetic Acid (IAA) in the crude extracts of fungi isolated from Jerusalem artichoke was higher than that from medicinal plants (p ≤ 0.05). HPLC analysis suggested the putative IAA biosynthesis pathways in our fungi were via Indole-3-lactic acid (ILA) and Indole-3-acetamide (IAM). In the pot experiment, the values of height, diameter, chlorophyll content and leaf dry weight in sunchoke plants inoculated with fungal endophytes were significantly higher than those of un-inoculated plants. Therefore, this study suggested that fungal endo- phytes could be used as a biofertilizer for promoting growth of sunchoke.
1. Introduction
Helianthus tuberosus (Sunchoke or Jerusalem artichoke), also called sunchoke that belongs to the sunflower species (Asteraceae family), is a native plant in North America. It is a very famous vegetable for cooking in Europe and the United States. In terms of agricultural benefits, it was previously reported to have high growth rate, good tolerance to stress conditions (e.g., frost, drought and poor soil) and strong resistance to pests and plant diseases even when limited amounts of fertilizer was applied (Slimestad et al., 2010). There have been reports on the appli- cations of sunchoke in various industries. For example, it can be used for the production of biomass and animal feed. Its tubers can also be used for the production of human food (Bach et al., 2013) including func- tional food ingredients such as inulin oligofructose and fructose. Its leaves and stems consisted of bioactive compounds including antifungal agents, antioxidants and anticancer, all of which can be used in the
pharmaceutical sector (Yuan et al., 2012). Additionally, sunchoke tubers could be a cost effective feedstock for biofuels production and the chemicals fermentation (Song et al., 2017; Wang et al., 2015; Zhang et al., 2010).
Recently, the application of endophytic fungi for promoting plant growth has been of interest because the fungi play an important role in plant growth promotion leading to a higher yield and an increase in resistance to biotic and abiotic stresses (Domka et al., 2019). Endophytic fungi could symbiotically colonize the internal tissues of plants without causing any visible signs of infection (Pavithra et al., 2020). Moreover, they also produce various secondary metabolites which can be used as environmentally friendly products for promoting plant growth such as phytohormones, siderophore, hydrogen cyanide, phosphate solubilizing agents, and hydrolytic enzymes (Rana et al., 2020). There have been reports on endophytic fungi that used these metabolites to promote plant growth. For example, Aspergillus fumigatus TS1 and Fusarium
Abbreviations: L-tryptophan (L-Trp), Indole-3-acetic acid (IAA); Indole-3-acetamide (IAM), Tryptamine (TAM); Indole-3-acetonitrile (IAN), Indole-3-lactic acid (ILA).
* Corresponding author. E-mail address: bsopho@kku.ac.th (S. Boonlue).
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proliferatum BRL1 could produce gibberellins and also regulate plant endogenous hormones (Lubna Bilal et al., 2018). Also, a previous work from our group suggested that the co-inoculation between phosphate solubilizing bacteria (PSB) and arbuscular mycorrhizal fungi (AMF) could significantly enhance the growth of artichoke plants (Nacoon et al., 2020). The co-inoculation of PSB and AMF to promote plant growth does not easily work in general due to their antagonism. It, however, would be more interesting if we can have any microbes which have both the plant root colonization ability and the plant growth pro- moting (PGP) ability. Another advantage of having such microbes is that they can supply PGP substances directly into the plant cells. To the best of our knowledge, there was still no report on plant growth promoting activities of fungal endophytes isolated from sunchoke plants and on how they promote growth of sunchoke. Therefore, the aims of this study were to investigate plant growth promoting properties of the fungal endophytes isolated from sunchoke and some medicinal plants. More- over, in order to investigate how these fungal endophytes promote plant growth, the production of plant hormone, especially indole-3-acetic acid was quantified, and its biosynthesis pathway was elucidated. Finally, the effect of fungal endophytes to promote growth of sunchoke plants under greenhouse conditions was assessed.
2. Materials and methods
2.1. Plant materials collection
Siam weed (Chromolaena odorata) and Ginger (Zingiber officinale) were collected from Khon Kaen province in Northeastern of Thailand with GPS coordinates as (1627′38′′N, 10249′04′′E) and (1627′31′′N, 10249′11′′E), respectively. The samples were kept in the sterile plastic zip bag and stored at 4 C until further use.
2.2. Isolation and identification of endophytic fungi
The plant organs including leaves, stem and root were isolated by surface sterilization method. Subsequently, the fungal endophytes were identified by morphological characteristics and ITS rRNA gene se- quences analysis as described by Suebrasri et al. (2020). Four isolates of endophytic fungi used in this study were obtained from the Myco- technology laboratory, Department of Microbiology, Faculty of Science, Khon Kaen University. Two of them which are Macrophomina phaseolina BUP2/3 LC536686 and Diaporthe phaseolorum BUP3/1 LC536686 were isolated from sunchoke. The other 2 endophytic fungi including Daldinia eschscholtzii 2NTYL11 LC536685 and Trichoderma erinaceum ST-KKU2 LC536684 were isolated from Stemona and ginger, respectively. These fungal endophytes have been reported for the control of pathogenic fungus Sclerotium rolfsii (Suebrasri et al., 2020). All fungal endophytes were cultured onto potato dextrose agar (PDA) plates and then incu- bated at 28 C for 7 days. Stock cultures were maintained on PDA slants and stored at 4 C for use in further experiments.
2.3. Enzymatic activity
The enzymatic activities of amylase, xylanase, protease and cellulase were determined using starch, beech wood xylan, gelatin and carbox- ymethyl cellulose, respectively, as substrates. The amount of reducing sugar released due to those enzyme activities was determined by the Somogyi-Nelson method. The fungal endophytes were cultured in potato dextrose broth (PDB) medium for 7, 14, 21 and 30 days under static condition. Then, 20 mL of each cultured broth were centrifuged at 4 C, 15,000×g for 20 min. A 1% of the substrates including starch, beech wood xylan, gelatin and carboxymethyl cellulose were dissolved by McIlvane (pH 7.0). Then, 500 μL of culture supernatants was transferred into microcentrifuge tubes, which were separated into two sets, the sample and the control. The control set was boiled at 100 C for 10 min by soaking in a water bath. After that, 500 μL of inactivated enzymes was
added in both sets and incubated at 30 C for 10 min. The enzyme re- action was stopped by heating a 100 C for 10 min prior to centrifuga- tion at 15,000×g for 5 min. Then, 500 μL of supernatant was transferred into the new tubes and the Nelson-Somogyi method was carried out to quantify the amount of reducing sugars (Somogyi, 1952). Briefly, the 500 μL of enzyme reaction was mixed with 500 μL of copper reagent (KNa tartrate: Na2CO3: Na2SO4: NaHCO3 = 1:2:12:1.3) and 500 μL of the CuSO4.5H2O:Na2SO4 (1:9) solution. The mixtures were boiled for 10 min in a water bath. After cooling at room temperature, 500 μL of Nelson arsenomolybdate reagent, containing 25 g ammonium molybdate in 450 mL H2O, 21 mL H2SO4, 3 g Na2HAsO4.7H2O dissolved in 25 mL H2O, then stored at 37 C for 24 h before use, was added. The solution was shaken thoroughly and incubated for 10 min at room temperature. After incubation, the colorimetric measurements were carried out by transmitted light at a wavelength of 500 nm using the spectrophotom- eter (HITACHI, Japan). A standard curve was a plot between absorbance values against known concentrations of glucose and tyrosine. A unit of enzyme activity is defined as 1 μmol of reducing sugar liberated per unit of time under the specified conditions (Green et al., 1989; Gusakov et al., 2011).
2.4. Quantitative phosphate solubilization activity
The ability of the isolated endophytic fungi to solubilize phosphate was determined by inoculating fungal endophytes in 100 mL of Pikov- skaya’s broth (g/L): 0.5 g (NH4)2SO4, 0.1 g MgSO4⋅7H2O, 0.02 g NaCl, 0.02 g KCl, 0.003 g FeSO4⋅7H2O, 0.003 g MnSO4⋅H2O, 10.0 g glucose, 0.5 g yeast extract, and 1000 mL of distilled water. Then, the solution was supplemented with 0.5% tricalcium phosphate (Adnan et al., 2018). Ten agar mycelial plugs of each endophytic fungi isolate were inoculated into 100 mL of Pikovskaya’s broth medium and then incubated with shaking at 150 rpm, 28 C for 7 days. The control was also prepared without inoculation of endophytic fungi. The control and culture sam- ples were aliquoted after 2, 5 and 7 days of incubation. The culture was centrifuged at 15,000×g for 5 min in order to collect supernatant. Subsequently, the pH of the supernatant was measured. A 2 mL of the supernatant was mixed with 1 mL of 2.5% ascorbic acid, 5 mL of 2% boric acid, 2 mL of Murphy’s reagent and 15 mL of deionized water. The solution was incubated at room temperature for 30 min prior to the determination of the amount of soluble phosphorus in the cultured su- pernatant, which was measured at a wavelength of 820 nm. The results were compared with the standard curve of potassium dihydrogen phosphate, KH2PO4 (Murphy & Riley, 1962).
2.5. Production of ammonia and hydrogen cyanic acid (HCN)
Ammonia production was measured in the 7-day-old culture broth of endophytic fungi grown in peptone broth incubated at 28 C with shaking at 150 rpm. The supernatant was collected by centrifugation and then was added with 0.5 mL of Nessler’s reagent. The development of a brown to yellow color is an indicative of ammonia production. The absorbance at a wavelength of 530 nm was measured using a spectro- photometer. The ammonia production was recorded in mg/mL when compared with the standard curve of (NH4)2SO4.
Hydrogen cyanide (HCN) production was determined by following the methods of Lorck (1948). The fungal isolates were cultured in Bennett agar (per liter: 1 g of yeast extract; 1 g of beef extract; 2 g of casein enzyme hydrolysate; 10 g of dextrose and 18 g of agar) supple- mented with 4.4 g/L of glycine. Then, the Whatman filter paper No. 1 was soaked with a mixture of 0.5% picric acid and 2% of sodium car- bonate for 1 min prior to adhere beneath the Petri dish lids. The plates were sealed with parafilm and incubated at 28 C for 7 days. After in- cubation, a change in color of the Whatman filter papers from white to orange or red was an indicative of positive HCN production (Passari et al., 2016).
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2.6. Siderophore production
Endophytic fungal isolates were determined for siderophore pro- duction by following the modification method of Schwyn and Neilands (1987). The endophytic fungal isolates were grown on potato dextrose agar (PDA) at 28 C for 3–5 days. Then, the mycelial tips were cut and inoculated on chrome azurol S (CAS) agar and incubated at 28 C for 3–5 days under a dark condition. The CAS reaction rate was determined by measuring the color change around a fungal colony on the CAS agar. The colonies surrounded by the developed orange zones were considered as siderophore-producing isolates. Uninoculated plates of CAS agar were set up as described above as a control. The experiment was carried out in four replications (Kandel et al., 2017; Milagres et al., 1999; Schwyn & Neilands, 1987; Verma et al., 2011).
2.7. Indole acetic acid production
2.7.1. Quantification of IAA produced by endophytic fungi The quantity of IAA produced by endophytic fungal isolates was
determined. Two fungal mycelium discs of 5 mm diameter were inoc- ulated into 20 mL of PDB supplemented with 0.2% of L-tryptophan and then incubated with shaking at 150 rpm, 28 C for 7 days. The experi- ment was carried out in triplicates. The 5 mL culture solution was centrifuged at 15,000×g for 10 min to retrieve supernatant. One milli- liter of the supernatant was mixed with 4 mL of Salkowski reagent, comprising 0.5 M FeCl3 solution and 35% perchloric acid (Platt & Thi- mann, 1956). The mixtures were incubated for 30 min under the dark condition. IAA production was positive if a pink color developed. Then, the absorbance of the solution was measured at a wavelength of 530 nm and compared to the standard curve of IAA.
2.7.2. Elucidation of a fungal IAA biosynthesis pathway In order to extract metabolites from fungal cultures, the supernatant
was acidified to pH 4.0 by using 1 M HCl prior to extraction with an equal volume of ethyl acetate. The ethyl acetate extract was dried using a rotary evaporator. The crude extract was dissolved in methanol and kept at − 20 C until used. In order to screen for the metabolites in the IAA synthesis pathway of the endophytic fungi, thin layer chromatog- raphy (TLC) analysis was carried out. The crude ethyl acetate extracts were spotted onto the TLC plate. The chromatogram was performed in the mobile phase at the concentration of n-Hexane: Ethyl acetate: Iso- propanol: Acetic acid, 40:20:5:1 v/v. Spots were sprayed with Salkow- sky’s reagent and then visualized under UV light. In order to predetermine the types of compounds, Rf values of spots were compared to those of the standards L-tryptophan (L-Trp), Indole-3-acetic acid (IAA), Indole-3-acetamide (IAM), Tryptamine (TAM), Indole-3- acetonitrile (IAN) and Indole-3-lactic acid (ILA).
The quantification and confirmation of the types of fungal metabo- lites were analyzed using a high-performance liquid chromatography (HPLC pump GL-7410, FL detector GL-7453 A and Column oven CO 631 A were obtained from GL Sciences, Japan). The types of compounds consisting in the samples were determined by comparing the sample chromatogram with that of the standards L-Trp, IAA, IAM, TAM, ILA and IAN. Further determination of the compounds was confirmed by co- injecting samples with individual standards. Concentrations of IAA and other indole-related metabolites were carried out by extrapolating with the calibration curves of the corresponding compounds (Num- ponsak et al., 2018). The mobile phase was methanol: 0.5 M sodium acetate buffer (30: 70, v/v) and the pH was adjusted to 4.8 with 0.5 M acetic acid. Prior to use, the mobile phase was filtered through a hy- drophilic PTFE membrane filter (0.5 μm, ADVANTEC, Toyo Roshi Kai- sha, Ltd. Tokyo, Japan). The HPLC condition was as follows: the flow rate was 0.6 mL/min; column temperature, 40 C; monitoring wave- length at 280 and 350 nm using a UV absorbance detector; column, InertSustain™ C18 (250 × 4.6 mm) column of 5 μm particle size.
2.8. Pot experiment
This experiment was conducted in order to investigate the efficiency of endophytic fungi to promote growth of sunchoke plants. The exper- imental design was a randomized complete block design (RCBD) with 8 treatments in 3 replications as follows: T1, control (Non-inoculated); T2, M. phaseolina BUP2/3; T3, D. phaseolorum BUP3/1; T4, D. eschscholtzii 2NTYL11; T5, T. koningii ST-KKU1; T6, T. erinaceum ST-KKU2; T7, M. phaseolina SS1L10 and T8, M. phaseolina SS1R10. Fungal endophytes were inoculated on PDA and then incubated for 5 days. Then, PDA containing fungal endophytes were cut by a 0.5 mm-diameter cork borer. The discs of mycelium tip were transferred to a tube containing 30 g of sterilized sorghum seeds. The fungal endophytes were incubated under static condition for 2 weeks or until full colonization on sorghum seeds was observed. Tubers of sunchoke HEL 65 were pre-germinated by incubating with charred rice husks for 7 days. The tubers were trans- ferred to plastic trays containing 2 endophytic fungi-infected sorghum seeds and then incubated for 1 week until the first 2 leaves were fully expanded. Germinated tubers were transferred to the 12.7 cm diameter pots containing sterilized soil. After 1 month of transplantation, 2 endophytic fungi-infected sorghum seeds were inoculated again into the wound which was cut at the root of each plant. Then, a cotton wool was used to cover infested sorghum seeds for maintaining moisture. The plant was watered regularly to avoid drought stresses. After 2 months of plant growth, the growth parameters including height, stem diameter and the chlorophyll contents were measured. Moreover, dry weights of shoots and roots were measured after they were dried at 80 C for 72 h.
2.9. Statistical analysis
Analysis of variance (ANOVA) was conducted using SPSS version 26 (IBM). Data were analyzed by least significant difference (LSD) test at a probability of 0.05 to identify significant effects among treatments.
3. Results
3.1. Isolation and identification of endophytic fungi
In the present study, fungal endophyte strains were isolated from Siam weed (Chromolaena odorata) and Ginger (Zingiber officinale). In this, we found three fungi that having properties to promote plant growth and inhibit growth of pathogenic fungi Sclerotium rolfsii and Fusarium oxysporum. Therefore, they were identified based on morphology characteristics and molecular identification. Based on the identities from blast search of ITS rRNA gene sequences of the fungal endophytes to the corresponding reference sequences in the GenBank. A total 3 fungi were isolated out of which two fungal strains isolated from Siam weed belong to Macrophomina phaseolina SS1L10 and Macro- phomina phaseolina SS1R10, one belongs to Trichoderma koningii ST- KKU1 which was isolated from Ginger root (Table 1).
3.2. Enzymatic activity
Activities of several enzymes of the fungal endophytes isolated from sunchoke and some medicinal plants were investigated (Table 2). Among all the endophytic fungi tested in this study, T. erinaceum ST- KKU2 showed the maximum xylanase and cellulase activities of 3.272 ± 0.207 and 0.665 ± 0.023 unit/mL, respectively. The maximum pro- tease activity of 0.046 ± 0.010 unit/mL was found in M. phaseolina SS1L10. In the case of amylase, the highest activity of 1.361 ± 0.137 unit/mL was found in M. phaseolina BUP2/3.
3.3. Quantitative phosphate solubilization activity
Tricalcium phosphate supplemented in Pikovskaya’s medium was examined for the ability of fungi to solubilize insoluble phosphate. The
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results are shown in Fig. 1. All endophytic fungi could solubilize tri- calcium phosphate within 5 days of incubation, except strain M. phaseolina BUP 2/3 which took 7 days to solubilize tricalcium phosphate.
3.4. Ammonia, hydrogen cyanide and siderophore production
All 7 endophytic fungal strains were able to produce ammonia in the range from 28.72 to 74.63 ppm. Strain D. eschscholtzii 2NTYL11 pro- duced the highest amount of ammonia of 74.63 ppm followed by strain M. phaseolina SS1R10 (69.90 ppm) and strain T. erinaceum ST-KKU2 (69.59 ppm), all of which were significantly different to that of other strains (P ≤ 0.05) (Fig. 2). None of the endophytic fungal strains could produce HCN.
To determine the possible ability of endophytic fungi to assist iron uptake by plants, siderophore production using CAS-blue agar assay was carried out. The color change of agar from blue to orange was observed in cases of endophytic fungal strains T. koningii ST-KKU1, M. phaseolina SS1L10 and M. phaseolina SS1R10 (Fig. 3), indicating that these strains were able to produce siderophores.
3.5. IAA production and elucidation of a fungal IAA biosynthesis pathway
Fig. 4 showed the amount of IAA produced by 7 endophytic fungi when grown on medium supplemented with 0.2% L-tryptophan. The highest concentration of IAA was found in D. phaseolorum BUP 3/1 and M. phaseolina BUP2/3 which were isolated from the sunchoke plants. In
comparison, the concentrations of IAA produced by fungi isolated from sunchoke were significantly higher (P ≤ 0.05) than those of the fungi isolated from medicinal plants (Fig. 2).
In order to determine possible pathways for IAA biosynthesis by endophytic fungi, HPLC analysis was carried out to identify IAA and other indole-related compounds. According to Fig. 5A, the retention times of the standards L-Tryp, TAM, ILA, IAM, IAA and IAN were 15.783, 27.682, 33.125, 41.489, 62.390 and 74.374 min, respectively (Fig. 4A). None of the peaks corresponding to IAA and other indole compounds was found in the medium without inoculation of fungal endophytes
Table 1 Identification of endophytic fungi isolated from two medicinal plants, based on ITS rDNA sequences and endophytic fungi in this study.
Isolate code
Host plant
odorata SS1R10 Macrophomina phaseolina LC585243 Chromolaena
odorata a Diaporthe phaseolorum
BUP 3/1 LC536686 Helianthus
tuberosus a Macrophomina phaseolina
BUP 2/3 LC536687 Helianthus
tuberosus a Daldinia eschscholtzii
2NTYL11 LC536685 Stemona tuberosa
a Trichoderma erinaceum ST-KKU2
LC536684 Zingiber officinale
a Endophytic fungi in this study were obtained from Mycotechnology labo- ratory, Department of Microbiology, Faculty of Science, Khon Kaen University.
Table 2 Enzymatic activities of endophytic fungi isolated from sunchoke and some medicinal plants.
Enzyme Incubation time
D. eschscholtzii 2NTYL11
M. phaseolina SS1L10
M. phaseolina SS1R10
Xylanase 7 days 0.000 ± 0.000d 0.079 ± 0.015d 0.015 ± 0.025d 0.017 ± 0.011d 0.733 ± 0.129b 0.323 ± 0.200a 1.234 ± 0.050c
14 days 1.473 ± 0.210b 0.390 ± 0.090cd 0.328 ± 0.004d 0.331 ± 0.100d 0.528 ± 0.110c 1.453 ± 0.130b 2.694 ± 0.106a
21 days 1.192 ± 0.104d 1.014 ± 0.097d 1.897 ± 0.280c 3.272 ± 0.207a 0.496 ± 0.082e 2.803 ± 0.089b 3.045 ± 0.100ab
Amylase 7 days 0.000 ± 0.000c 0.000 ± 0.000c 0.455 ± 0.046a 0.034 ± 0.006c 0.016 ± 0.003c 0.158 ± 0.028b 0.000 ± 0.000c
14 days 0.234 ± 0.022c 0.000 ± 0.000d 0.529 ± 0.053a 0.244 ± 0.047c 0.347 ± 0.043b 0.319 ± 0.018b 0.374 ± 0.016b
21 days 1.361 ± 0.137a 0.553 ± 0.032c 0.742 ± 0.115b 0.467 ± 0.004cd 0.206 ± 0.010e 0.763 ± 0.060b 0.424 ± 0.028d
Cellulase 7 days 0.078 ± 0.019b 0.242 ± 0.027a 0.021 ± 0.036c 0.258 ± 0.020a 0.000 ± 0.000c 0.000 ± 0.000c 0.000 ± 0.000c
14 days 0.396 ± 0.083a 0.338 ± 0.029ab 0.273 ± 0.021b 0.361 ± 0.018ab 0.029 ± 0.016d 0.168 ± 0.083c 0.432 ± 0.070a
21 days 0.260 ± 0.030c 0.326 ± 0.016b 0.334 ± 0.021b 0.665 ± 0.023a 0.019 ± 0.032d 0.367 ± 0.037b 0.269 ± 0.021c
Protease 7 days 0.000 ± 0.000a 0.000 ± 0.000a 0.000 ± 0.000a 0.003 ± 0.005a 0.001 ± 0.001a 0.000 ± 0.000a 0.000 ± 0.000a
14 days 0.022 ± 0.007 ab 0.000 ± 0.000b 0.000 ± 0.000b 0.027 ± 0.003a 0.024 ± 0.034ab 0.014 ± 0.001ab 0.007 ± 0.002ab
21 days 0.021 ± 0.005c 0.019 ± 0.010c 0.014 ± 0.005c 0.029 ± 0.008bc 0.038 ± 0.009ab 0.046 ± 0.010a 0.017 ± 0.002c
Different letters indicate significant differences among values (P ≤ 0.05) by LSD test. Values are means ± SE (n = 6).
Fig. 1. Available P released by the endophytic fungi isolated from sunchoke and medicinal plants.
Fig. 2. IAA and ammonia production by fungal endophytes isolated from sunchoke and some medicinal plants. Different letters indicate significant dif- ferences among the mean values (P ≤ 0.05) by LSD test.
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(Fig. 4B and J) The ethyl acetate extract obtained from all fungal en- dophytes was identified for the intermediates in the IAA biosynthesis pathway compared to the standards. The results showed that IAM and IAN were present in the extract of M. phaseolina BUP2/3. This suggested that this fungal strain could produce IAA through those two metabolites. Likewise, the presence of IAN, IAM and ILA in the M. phaseolina SS1L10 suggested the strain could produce IAA through those indole-related compounds. Similar IAA biosynthesis pathway might be used by M. phaseolina SS1R10 because IAN and ILA were found in its extract. In the case of T. erinaceum ST-KKU2, only IAM was found as a metabolite during biosynthesis of IAA. In contrast, a different synthesis pathway was observed in D. phaseolorum BUP3/1, D. eschscholtzii 2NTYL11 and T. koningii ST-KKU1 due to the presence of not only IAM and/or ILA but also TAM. All of these results suggested that IAM and ILA were the main metabolites for IAA synthesis in our endophytic fungi. The identification of fungal IAA was confirmed by a co-injection with each standard (Fig. 4). In addition, the fungal endophytes cultured in PDB without L- tryptophan were also observed for detection of Trp-independent biosynthesis pathway.
3.6. Pot experiment
After 2 months of transplantation, there were significant differences (P ≤ 0.05) with respect to diameter, leaves dry weight and chlorophyll content between the inoculated and the uninoculated sunchoke plants (Fig. 5). In contrast, there was no significant difference with the shoot and root dry weights of the inoculated and the uninoculated plants. The sunchoke plants inoculated with endophytic fungus T. koningii ST-KKU1 had the maximum root dry weight (Table 3).
4. Discussion
All 7 strains of endophytic fungi have the ability to produce xyla- nolytic and cellulolytic enzymes, which are involved in the degradation of beech wood xylan and carboxymethyl cellulose. The highest xylanase and cellulase activities of 3.272 ± 0.207 and 0.665 ± 0.023 unit/mL, respectively were found in an endophytic fungus T. erinaceum ST-KKU2. These values were much higher than other previous reports, for example, in the case of endophytic fungi Catharanthus roseus, isolated from medicinal plant, showed the maximum xylanase and cellulase enzyme activity of 0.262 ± 0.0172 and 0.239 ± 0.0061 unit/mL, respectively (Yopi et al., 2017). In addition, Uzma et al. (2016) reported no activity of amylase and cellulase enzymes in the Trichoderma genus which was isolated from medicinal plants (Tinospora cordifolia and Piper
nigrum L.). The benefits of fungal xylanase and cellulase are that they can act as bioactive compounds against plant pathogens (Jampala et al., 2017; Legodi et al., 2019). In our study, M. phaseolina SS1L10 and D. eschscholtzii 2NTYL11 exhibited the maximum protease activities of 0.046 ± 0.010 and 0.038 ± 0.009 unit/mL, respectively, which were higher than that of other endophytes such as Nigrospora spp. Fusarium chlamydosporum, Penicillum citrinum and Cylindrocephalum spp. (Shubha & Srinivas, 2017). Production of hydrolytic enzymes by endophytes enables effective plant colonization (Bezerra et al., 2012). Moreover, we investigated phosphate-solubilizing ability of our fungi, and found that all of them could effectively solubilize insoluble phosphate within 5 days of incubation. This result is similar to a previous study that suggested an optimal P-solubilizing rate in the culture condition at day 5 (Chen & Liu, 2019). Phosphate solubilizing fungi mainly belong to Trichoderma (Lei & Zhang, 2015), Macrophomina (Jain2014), Diaporthe (Ye et al., 2019) were able to solubilize rock phosphate. In this study, all endophytic fungi displayed higher P-dissolving capacity than the fungi in other previously mentioned reports. However, many types of soils contain insoluble phosphates which obstruct phosphate uptake by plants, so there were also studies on the inoculation of phosphate-solubilizing microorganisms to enhance soluble phosphate availability for plant growth (Granada et al., 2018; Oteino et al., 2015). The mechanism in- volves releasing of organic acids by fungal endophytes into the medium or soil which solubilize the phosphate complexes and then convert them into ortho-phosphate, which is available for plant uptake and utilization. Therefore, the phosphate solubilizing activity of our fungal endophytes is one important mechanism that can enhance growth of sunchoke plants.
Production of ammonia is responsible for the indirect growth of plants and can serve as a triggering factor in the suppression of plant pathogens (Minaxi et al., 2012). Here, all endophytic fungi can produce ammonia that probably accumulate and supply nitrogen and also pro- mote shoot and root elongation, which subsequently increases plant biomass (Marques et al., 2010). On the other hand, siderophores are small, high-affinity iron-chelating compounds secreted by some micro- organism to bind the rhizosphere’s ferric ion. The formation of siderophore-iron complex increases the possibility of iron uptake by the root of certain plants, thus resulting in increased plant growth (Marquez et al., 2020). In this study, the endophytic fungi T. koningii ST-KKU1, M. phaseolina SS1L10 and M. phaseolina SS1R10 are siderophore pro- ducers, which may support its use as a possible growth promoter of sunchoke plant.
Although the IAA production by plant-associated bacteria has been widely reported (Bunsangiam et al., 2019; Suwannarach et al., 2015), it
Fig. 3. In vitro detection of siderophore production on CAS-blue agar plate by endophytic fungi. A, M. phaseolina BUP2/3. B, D. phaseolorum BUP3/1. C, D. eschscholtzii 2NTYL11. D, T. koningii ST-KKU1. E, T. erinaceum ST-KKU2. F, M. phaseolina SS1L10. G, M. phaseolina SS1R10. and H, CAS-blue agar plate. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. Identification of indole-related compounds produced by endophytic fungi using high performance liquid chromatography technique. HPLC chromatograms are indicated as follows: A, Indole compound standard; B, PDB extracted with 0.2% L-tryptophan; C, crude extract of M. phaseolina BUP2/3; D, crude extract of D. phaseolorum BUP3/1; E, crude extract of D. eschscholtzii 2NTYL11; F, crude extract of T. koningii ST-KKU1; G, crude extract of T. erinaceum ST-KKU2; H, crude extract of M. phaseolina SS1L10; I, Crude extract of M. phaseolina SS1R10; J, PDB extracted without 0.2% L-tryptophan. Indole-related compounds are abbreviated as follows: Trp, L-tryptophan; TAM, Tryptamine; ILA, Indole-3-lactic acid; IAM, Indole-3-acetamide; IAA, Indole-3-acetic acid; IAN, Indole-3-acetonitrile. Each chro- matogram is a representative of triplicate analysis.
Fig. 5. Effects of fungal endophytes on the growth characteristics of sunchoke plants.
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is less commonly known in fungi. Interestingly, our endophytic fungi isolated from sunchoke, Siam weed, Ginger and Stemona could produce IAA in PDB supplemented with 0.2% L-tryptophan. We are the first to report IAA production by the fungal endophytes which are M. phaseolina BUP2/3, D. phaseolorum BUP3/1, D. eschscholtzii 2NTYL11, T. koningii ST-KKU1, T. erinaceum ST-KKU2, M. phaseolina SS1L10 and M. phaseolina SS1R10. The IAA synthesis pathways in our endophytic fungi were also elucidated by monitoring the presence of indole-related metabolites using HPLC analysis. Our study revealed that the putative IAA synthesis pathway in the endophytic fungi isolated from sunchoke and medicinal plants was via IAM and ILA (Fig. 6).
A similar pathway in fungi was found in members of the genus Col- letotrichum such as Colletotrichum fructicola, Colletotrichum gloeospor- ioides f. sp. aeschynomene. and Colletotrichum acutatum (Robinson et al., 1998; Chung et al., 2003). In addition, a strain of Fusarium sp. was also found to synthesize IAA via IAM and ILA (Tsavkelova et al., 2012). Note that different fungal species have different pathways to synthesize IAA (Pedraza et al., 2004). Some previous literature suggested that a single microbial species can have more than one IAA synthesis pathway (Patten & Glick, 1996; Spaepen et al., 2007). Spaepen & Vanderleyden, 2011 suggested that the study on the microbial IAA biosynthesis path- ways are still limited. Therefore, the putative IAA synthesis pathway(s) for fungal endophytes elucidated in our work would provide more in-depth evidence on how endophytic fungi produce IAA.
Endophytic bacteria improve growth of sunchoke have been
investigated by Namwongsa et al. (2019) and Khamwan et al., 2018. However, we firstly reported fungal endophytes isolated from sunchoke plants for promoting their growth. It is likely that an increase in plant growth parameters was a result of multifunctional properties of our fungal endophytes. In order to optimize suitable conditions to use our fungal endophytes as plant growth promoters, field trials with sunchoke plants would be carried out in the future.
5. Conclusion
This work is the first to report plant growth promoting properties of endophytic fungi isolated from sunchoke and some medicinal plants. The results strongly confirm that two fungal endophytes Macrophomina phaseolina BUP2/3 and Diaporthe phaseolorum BUP3/1 isolated from sunchoke and the other five endophytic fungi including Daldinia eschscholtzii 2NTYL11, Trichoderma koningii ST-KKU1, Trichoderma eri- naceum ST-KKU2, Macrophomina phaseolina SS1L10 and Macrophomina phaseolina SS1R10 isolated from medicinal plants can be a good alter- native plant growth promoting endophytic fungi for sunchoke crops. They enhanced several parameters such as height, diameter, chlorophyll content, leaf dry weight and root dry weight under the greenhouse condition without the addition of chemical fertilizer. The IAA biosyn- thesis pathways of the endophytic fungi were first reported here. We found that IAM and ILA were the main metabolites of the IAA synthesis pathway. Further experiments will be carried out to investigate how it
Table 3 Effect of fungal endophyte inoculations on the growth of sunchoke plants.
Treatments Height (cm) Diameter (cm) Chlorophyll dry weight (g)
Content Leaf Shoot Root
Control 25.250 ± 3.41b 0.284 ± 0.02c 29.683 ± 1.28b 0.442 ± 0.07c 0.255 ± 0.04ab 0.272 ± 0.09b
M. phaseolina BUP2/3 29.625 ± 4.76a 0.349 ± 0.03b 33.417 ± 1.44a 0.663 ± 0.19b 0.230 ± 0.04bc 0.340 ± 0.06ab
D. phaseolorum BUP3/1 30.125 ± 5.12a 0.358 ± 0.03ab 33.183 ± 1.61a 0.863 ± 0.23a 0.255 ± 0.06ab 0.392 ± 0.13ab
D. eschscholtzii 2NTYL11 30.333 ± 4.18a 0.354 ± 0.02ab 33.350 ± 1.83a 0.700 ± 0.11ab 0.252 ± 0.06ab 0.395 ± 0.14ab
T. koningii ST-KKU1 28.000 ± 2.86ab 0.348 ± 0.03b 33.567 ± 1.77a 0.635 ± 0.13bc 0.177 ± 0.06c 0.448 ± 0.14a
T. erinaceum ST-KKU2 31.083 ± 3.87a 0.368 ± 0.05ab 32.933 ± 2.13a 0.803 ± 0.15ab 0.265 ± 0.04ab 0.402 ± 0.11ab
M. phaseolina SS1L10 30.250 ± 4.15a 0.382 ± 0.03a 33.283 ± 2.21a 0.708 ± 0.21ab 0.280 ± 0.06ab 0.322 ± 0.12ab
M. phaseolina SS1R10 30.167 ± 3.01a 0.357 ± 0.03ab 34.233 ± 2.41a 0.725 ± 0.03ab 0.302 ± 0.03a 0.355 ± 0.05ab
Different letters indicate significant differences among values within the same column (P ≤ 0.05) by LSD test. Values are means ± SE (n = 9). Control is the plant without fungal inoculation.
Fig. 6. The putative pathway for IAA biosynthesis in the endophytic fungi isolated from sunchoke and some medicinal plants.
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promotes plant growth in the fields for increasing sunchoke yield.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
We are grateful to the TRF for providing financial support through the Senior Research Scholar Project of Prof. Dr. Sanun Jogloy (Project No. RTA 6180002). We would also like to thank Faculty of Medical Science, Nakhon Ratchasima College for partial financial supports. We extend my sincere thanks to the Supporting Scholar for Graduate Stu- dents for doing Research Activities in Abroad FY 2019, Faculty of Sci- ence, Khon Kaen University.
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T. Suebrasri et al.
1 Introduction
2.3 Enzymatic activity
2.5 Production of ammonia and hydrogen cyanic acid (HCN)
2.6 Siderophore production
2.7.1 Quantification of IAA produced by endophytic fungi
2.7.2 Elucidation of a fungal IAA biosynthesis pathway
2.8 Pot experiment
2.9 Statistical analysis
3.2 Enzymatic activity
3.4 Ammonia, hydrogen cyanide and siderophore production
3.5 IAA production and elucidation of a fungal IAA biosynthesis pathway
3.6 Pot experiment