Transcript of Auxin-producing fungal endophytes promote growth of sunchoke
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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Rhizosphere
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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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Rhizosphere 16 (2020) 100271
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
T. Suebrasri et al.
Rhizosphere 16 (2020) 100271
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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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Rhizosphere 16 (2020) 100271
5
(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.)
T. Suebrasri et al.
Rhizosphere 16 (2020) 100271
6
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.
T. Suebrasri et al.
Rhizosphere 16 (2020) 100271
7
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.
T. Suebrasri et al.
Rhizosphere 16 (2020) 100271
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