Rhizosphere 9 (2019) 2–9

Contents lists available at ScienceDirect

Rhizosphere
journal homepage: www.elsevier.com/locate/rhisph

Phosphate solubilization potential of endophytic fungi isolated from Taxus T

wallichiana Zucc. roots
⁎
Priyanka Adhikari, Anita Pandey
Center for Environmental Assessment and Climate Change, G.B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora
263643, Uttarakhand, India

A R T I C LE I N FO A B S T R A C T

Keywords: Endophytic microorganisms live inside the host plant and contribute in various biological processes, without
Taxus wallichiana causing any harmful effect. Inorganic phosphate solubilization, through microorganisms, is one of the major
Fungal endophytes mechanisms involved in plant growth. The present study highlights the potential of endophytic fungi for their
Penicillium ability to solubilize insoluble phosphates in presence of tricalcium (TCP), aluminium (AlP), and iron phosphate
Aspergillus
(FeP) at different temperatures through production of phosphatases, phytases and organic acids. Five endophytic
Phosphate solubilization
fungi, isolated from the roots of Taxus wallichiana, were identified following their phenotypic and molecular
characters. Three fungal isolates showed maximum similarity with species of Penicillium (GBPI TWR_F1, GBPI
TWR_F2, and GBPI TWR_F3) and two with species of Aspergillus (GBPI TWR_F4 and GBPI TWR_F5). All the
endophytes solubilized phosphate by utilizing the substrates namely calcium, aluminium and iron phosphate
along with the production of phosphatase and phytase enzymes. Maximum phosphate solubilization and phytase
activity was recorded in case of the fungal isolate GBPI TWR_F2 (P. daleae) being 83.42 ± 3.41 µg/ml TCP,
57.63 ± 0.79 µg/ml AlP, and 57.76 ± 1.70 µg/ml FeP at 15 °C. GBPI TWR_F2 and GBPI TWR_F5 (Aspergillus
sp.) produced maximum calcium phytase at 25 and 15 °C, 10.33 ± 0.13 and 10.37 ± 0.37 µM/ml, respectively.
Phosphatase production was higher in acidic conditions in comparison to alkaline. In quantification of organic
acids through HPLC, malic and succinic acids were determined in maximum quantity 0.97 ± 0.003 and
0.92 ± 0.008 µg/ml, respectively, followed by oxalic (0.71 ± 0.006 µg/ml) and lactic acid (0.61 ± 0.005 µg/
ml). Citric acid was estimated in minimum quantity.

1. Introduction fixation, phosphate solubilization, etc (Santoyo et al., 2016).

Solubilization of inorganic insoluble phosphate salts by different
Recent advances in plant-microbe interaction research reveal that microorganisms depends on their ability to produce organic acids in the
different plant species are colonized by specific rhizosphere micro- respective environment. These organic acids decrease the pH of the soil
biome. This rhizosphere microbiome is crucial for the plant health as it or any medium, providing the facility to exchange the metal part of
contributes to the uptake of nutrients and extends protection to the host insoluble phosphates to potassium or sodium, resulting in the formation
plant against biotic and abiotic stresses (Bakker et al., 2018; Saleem of soluble phosphate salts. Inorganic forms of phosphorus (P) found in
et al., 2018). In this background, the endophytic microorganisms, that soil, in general, are a mixture of crystalline unstructured calcium, alu-
are known to colonize nearly all the plants, are being focused with minum, and iron phosphate. In the total P, almost 50–60% is in form of
respect to their structural as well as functional aspects. These micro- organic phosphates in soil, and in this phytate (myo-inositol hexakis
organisms produce plenty of secondary metabolites, possessing anti- phosphate phospho hydrolase) and phytic acid ((1r,2R,3S,4s,5R,6S)-
oxidants and antimicrobial activities, by which the host plants are cyclohexane-1,2,3,4,5,6-hexayl hexakis [dihydrogen (phosphate)])
benefitted with respect to protection against a range of pathogenic constitute the major organic sources. The enzyme phytase is required to
microorganisms and also withstand various stresses (Rodriguez et al., hydrolyze both phytate and phytic acid and release the inorganic
2009; Singh and Pandey, 2017; Wani et al., 2015). Endophytic micro- phosphate present in bound form. Phosphate solubilizing microorgan-
organisms contribute to several processes related to plant growth and isms are known to help in minimizing this problem that can result in
development similar to the rhizospheric microbes, such as, nitrogen enhancing the soil fertility (Rinu et al., 2012; Singh and Satyanarayana,

⁎
Corresponding author.
E-mail address: anita@gbpihed.nic.in (A. Pandey).

https://doi.org/10.1016/j.rhisph.2018.11.002
Received 7 September 2018; Received in revised form 2 November 2018; Accepted 3 November 2018
Available online 06 November 2018
2452-2198/ © 2018 Elsevier B.V. All rights reserved.
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

2011). 100 ml.

Taxus wallichiana Zucc. (English name: Himalayan Yew; Hindi
name: Thuner; Family: Taxaceae) is a well-recognized medicinally im- Colour reagent: 125 ml of Reagent A and 37.5 ml of Reagent B were
portant evergreen tree that grows under the temperate locations in mixed thoroughly, and 75 ml of Reagent C and 12.5 ml of Reagent D
Himalaya (Poudel et al., 2013). The plant species has been best known solution were added.
for its anticancerous properties, antimicrobial potential along with
many other medicinal uses (Adhikari and Pandey, 2017, 2018; Juyal
2.3.1.2. Qualitative estimation. Qualitative estimations on fungal
et al., 2014). Taxus spp. have also been studied for colonization of
phosphate solubilization was performed on Pikovskaya's agar at four
microbial endophytes possessing abilities to produce a range of bioac-
temperatures (5, 15, 25 and 35 °C). Formation of clear zone of
tive compounds (Vasundhara et al., 2017; Xiong et al., 2013; Zhang
solubilization around the fungal colony on one week incubation was
et al., 2009; Zhao et al., 2009). Further, the species is placed in the
indicative of the positive result.
IUCN Red List of Threatened Species (Thomas and Farjon,2011) and,
therefore, is a priority species in biodiversity conservation. In view of
the regeneration of this species, understanding of the rhizosphere 2.3.1.3. Quantitative estimation. Quantitative estimations were
beneficial microbiome, endophytes in particular, associated with the performed in Pikovskaya's broth at 15 and 25 °C. The fungal cultures
host species will be a prerequisite. In this background, the present study were inoculated, separately, in Pikovskaya's broth providing three
focuses on the phosphate solubilization potential of five culturable substrate(s), separately, viz. tri calcium phosphate, aluminium
endophytic fungi, isolated from T. wallichiana roots. The fungal isolates phosphate and iron phosphate, following one week incubation at two
are studied with respect to their potential in solubilizing various source selected temperature. 0.1 ml of the enzyme extract plus 1.9 ml of double
of phosphates through production of phytases and phosphatases en- distilled water and 2 ml colour reagent were placed in a test tube.
zymes and the organic acids. Reference tube was maintained by replacing enzyme extract by
uninoculated broth. Blank, containing only water and colour reagent,
2. Experimental were used to set zero of spectrophotometer. The optical density was
recorded after allowing the reaction for 20 min at 882 nm.
2.1. Plant sample collection
2.3.2. Production of phosphatases
The plant root samples were collected from Jageshwar (29°35´-
2.3.2.1. Qualitative estimation. Qualitative estimation of alkaline and
29°39´ N and 79°59´-79°53´E) Forest area in dist. Almora, Uttarakhand,
acidic phosphatase activity was performed in Pikovskaya's broth. The
India. T. wallichiana grows at this site between 1790–1950 m amsl as an
fungal culture broth was incubated at 25 °C for 48 h and the enzyme
under canopy tree (Nadeem et al., 2002).
extract activity was recorded in both, alkaline (pH 9) and acidic (pH 5)
medium following the spectrophotometric procedure as described in
2.2. Isolation and identification of fungal endophytes
Rinu and Pandey (2010).
T. wallichiana roots were washed in running tap water for removing
excess soil and then surface sterilized following washing in 99% ethanol 2.3.2.2. Quantitative estimation. Quantitative estimation of alkaline and
(1 min), rinsing with sterilized water (1 min), immersing in 5% solution acidic phosphatase activity was done at 15 and 25 °C. For assaying
of sodium hypo chloride (5 min), and finally rinsing 4–5 times in ster- alkaline phosphatase, 1 ml of glycine-sodium hydroxide buffer (pH 9/
ilized water (Tayung and Jha, 2006). Then the roots were cut into small 0.1 M) was taken in a test tube and 0.4 ml enzyme extract was added to
pieces and checked for complete sterilization by plating on potato it. This was followed by addition of 0.5 ml p-nitrophenol phosphate
dextrose agar (PDA). These roots were further processed for isolation of substrate and 0.1 ml of MgCl2 to the reaction mixture. Same procedure
endophytic fungi on PDA, incubating at 25 °C for 7 days (Kumaran and was followed for acidic phosphatase assay, instead citrate buffer (pH 5/
Hur, 2009). The pure fungal cultures were identified on the basis of 0.1 M) was used in place of sodium hydroxide buffer. The tubes were
phenotypic (colony morphology and microscopy) and molecular char- incubated at 25 °C for 30 min. Following incubation, 100 µl of NaOH
acters (amplification of ITS region (ITS1–5.8S-ITS2)) using PCR fol- (5 M) was added and the release of p-nitrophenol was measured at
lowing sequencing of the amplified product. The fungal endophytes 400 nm. The optical density (OD) was converted to micromoles of p-
were maintained on PDA slants at 4 °C. nitrophenol released by extra-plotting against standard curve.

2.3. Fungal phosphate solubilization

2.3.3. Production of phytases
2.3.3.1. Qualitative estimation. Qualitative estimation on fungal
2.3.1. Enzyme extracts preparation
phytase production was done at four different temperatures 5, 15, 25
Enzyme extracts were prepared in Pikovskaya's and phytase
and 35 °C. The fungal isolates were screened for their ability to
screening broth. The broth was inoculated with the fungal endophytes,
hydrolyze phytate using phytase screening medium (PSM; 10 g/l D-
separately, and incubated for 8 days. Following incubation, the broth
glucose, 2 g/l CaCl2, 5 g/l NH4NO3, 0.5 g/l KCl, 0.5 g/l MgSO4.7H2O,
was centrifuged (10,000 rpm, 10 min) and the clear suspension was
0.01 g/l FeSO4.7H2O, 0.01 g/l MnSO4.H20, 4 g/l Sodium phytate/
collected for further analysis. The pH of the broth was recorded in each
Calcium phytate substrate), using two different substrates- calcium
case before and after the autoclave.
and sodium phytate. The isolates producing phytase enzyme formed a
clear hollow zone around the colony due to the hydrolysis of calcium
2.3.1.1. Colour reagent preparation.
and sodium phytate in one week incubation.
Reagent A: Sulphuric acid (5 N).
Reagent B: Ammonium molybdate. 20 g of ammonium molybdate 2.3.3.2. Quantitative estimation. The quantitative estimation was done
was dissolved in water and diluted to 500 ml. The solution was in two selected temperature- 15 and 25 °C. 150 µl of enzyme extract was
stored in a Pyres glass bottle. added in 600 µl enzyme substrate (2 mM sodium phytate in 0.2 M Tris
Reagent C: Ascorbic acid (0.1 M). buffer pH 6.5) and incubated for 30 min. After that, 750 µl of 5% TCA
Reagent D: Potassium antimonyl tartarate. 0.2743 g of potassium (Tri Chloro Acetic acid) and 1.5 ml of colour reagent were added. The
antimonyl tartrate was dissolved in distilled water and diluted to OD was taken at 700 nm.

3
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

2.4. Organic acid production 57.11 ± 1.83 µg/ml for TCP, AlP and FeP, respectively. Among all the
five phosphate solubilizing fungi, the fungal isolate GBPI TWR_F2
Production of organic acids (oxalic acid, malic acid, succinic acid, (Penicillium daleae) was found to be the best solubilizer of (p < 0.05)
citric acid and lactic acid) was done using high performance liquid TCP (83.42 ± 3.41 µg/ml), (p < 0.001) FeP (57.76 ± 1.70 µg/ml),
chromatography (Shimadzu LC solution) equipped with Photodiode and (p < 0.005) AlP (57.63 ± 0.79 µg/ml) at 15 °C. Overall, P. daleae
Array Detector (PDA). The culture fluid of the fungal isolates was fil- (GBPITWR_F2) and Aspergillus sp. (GBPITWR_F5) were found to be the
tered through 0.2 µm Whatman membrane filter. The organic acids in best fungal solubilizers. Increase in phosphate solubilization coincided
the filtrate samples were determined by HPLC in 220 nm, column C18, with the decline in pH of the medium in case of P. daleae (GBPITWR_F2)
flow rate 1 ml/min, eluent 1 mM H2SO4 in 1 mM Na2SO4, pH 2.8. and Penicillium sp. (GBPITWR_F3) in case of all three substrates.
Deionized water was taken as control and was used as blank mobile However, in case of GBPITWR_F5, phosphate solubilization was re-
phase. corded higher with lesser decline in pH. Results are presented in Fig. 2
and Table S1.
2.5. Statistical analysis
3.4. Production of alkaline and acidic phosphatases
Multiple analysis of variance (MANOVA) was carried out using
STATISTICA 8 for analyzing the homogenous grouping between dif- The endophytic fungi, under study, produced phosphatases, both
ferent endophytic fungal species using duncan test. Homogenous alkaline and acidic, at 15 as well as 25 °C. In comparison, the produc-
grouping was done in all the five fungal species by comparing their tion of phosphatases was higher in acidic condition. The phosphatase
phosphate solubization and phytase and phosphatase enzyme activity at production varied with respect to the substrates used, in the order
both the temperature (15 and 25 °C), and with different substrates. calcium > iron > aluminium phosphate in acidic medium and iron >
Mean values were considered at 95% significance level (p < 0.05). calcium > aluminium phosphate in alkaline medium. Among five
fungal endophytes, GBPI TWR_F2 (P. daleae) produced maximum acidic
3. Results phosphatase in presence of tri calcium phosphate (4.14 ± 0.37 µM/ml)
at 25 °C. Maximum alkaline phosphatase activity was recorded in GBPI
3.1. Description of fungal endophytes TWR_F2 and GBPI TWR_F2 (P. daleae and A. versicolor, respectively), at
15 °C; the differences being statistically significant (p < 0.0001).
The endophytic fungi were identified on the basis of their pheno- Results are summarized in Fig. 3 and Table S2.
typic (colony morphology and microscopic features) and molecular
characters (ITS region analysis). The code numbers given to the five 3.5. Production of phytases
fungal isolates and their respective identification are as follows: GBPI
TWR_F1 (Penicillium sp.), GBPI TWR_F2 (P. daleae), GBPI TWR_F3 The fungal isolates produced phytases in presence of calcium and
(Penicillium sp.), GBPI TWR_F4 (Aspergillus versicolor) and GBPI TWR_F5 sodium phytate in the medium, being higher in case of calcium phytate,
(Aspergillus sp.). The maximum similarity indices along with their ac- at both the temperatures. Phytase production, in presence of sodium
cession numbers, given to these endophytic fungi by NCBI, are pre- phytate ranged from 7.66 ± 0.20 to 8.71 ± 0.66 µM/ml at 15 °C
sented in Table 1. (p < 0.005) and from 5.58 ± 0.15 to 7.13 ± 0.42 µM/ml at 25 °C
(p < 0.01). In presence of calcium phytate, the phytase production
3.2. Qualitative estimations (Plate based bioassays) ranged between 6.69 ± 0.6 to 10.37 ± 0.3 µM/ml at 25 °C
(p < 0.05) and between 6.17 ± 0.7 to 10.33 ± 0.1 at 15 °C
All the fungal isolates were positive for phosphate solubilization and (p < 0.001). The fungal isolate GBPI TWR_F2 (P. daleae) and GBPI
production of phytases with all the three substrates (TCP, AlP and FeP) TWR_F2 (Aspergillus sp.) produced maximum phytase (10.33 ± 0.13
at a wide range of temperature (5 to 35 °C). Further, the fungal cultures and 10.37 ± 0.37 µM/ml) at 25 and 15 °C, respectively (p < 0.05) (
were positive for production of phosphatases (alkaline and acidic) at Fig. 4 and Table S2).
25 °C in spectrophotometric procedure. All these results are presented
in Fig. 1 and Table 2. On the basis of these results, the quantitative 3.6. Organic acid production
estimations were performed at two best temperatures viz. 15 and 25 °C.
Out of five organic acids (malic acid, citric acid, lactic acid, succinic
3.3. Quantitative estimation of fungal phosphate solubilization acid and oxalic acid), malic and succinic acids were determined in
maximum quantity (0.97 ± 0.003 and 0.92 ± 0.008 µg/ml;
The fungal isolates were positive for phosphate solubilization with p < 0.05, respectively), followed by oxalic (0.71 ± 0.006 µg/ml;
all the three substrates, in an order of calcium > iron > aluminum p < 0.05) and lactic acid (0.61 ± 0.005 µg/ml; p < 0.01). Citric acid
phosphate, at both the temperatures. The phosphate solubilization po- was estimated in minimum quantity. Almost all the fungi produced
tential of these fungi ranged from 46.58 ± 0.79 to 83.42 ± 3.41 µg/ higher amount of organic acids at 15 °C. At both the temperatures (15
ml, 35.92 ± 3.54 to 57.63 ± 0.79 µg/ml and 42.50 ± 4.06 to and 25 °C), organic acid production was maximum in presence of TCP,
59.34 ± 0.39 µg/ml for TCP, AlP and FeP, respectively, at 15 °C. At followed by FeP and AlP, respectively, pH reaching up to 2.98 ± 0.06
25 °C, it ranged between 44.08 ± 2.49 to 61.18 ± 0.66 µg/ml, in case of FeP. Citric acid was produced in presence of FeP and AlP, in
31.84 ± 1.57 to 53.03 ± 4.32 µg/ml, and 41.84 ± 0.26 to most cases. However, GBPITWR_F1 (Penicillium sp.) produced this acid

Table 1
Maximum similarity of the fungal isolates to their corresponding reference strains and accession nos. given by NCBI.
S. No. Fungal isolate Reference strain Similarity index (%) Nucleotide accession number

1 GBPITWR_F1 Penicillium sp. KP747701.1 99% MH166337

2 GBPITWR_F2 Penicillium daleae KF313087.1 99% MH191158
3 GBPITWR_F3 Penicillium sp. MF588872.1 99% MH191159
4 GBPITWR_F4 Aspergillus versicolor LN898740.1 100% MH191160
5 GBPITWR_F5 Aspergillus sp. MG022169.1 100% MH191161

4
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

Fig. 1. Phosphate solubilization by fungal isolates. A. phytase production, B. efficiency of endophytic fungi from T. wallichianaZucc. A1 (GBPITWR_F1), A2
(GBPITWR_F2), A3 (GBPITWR_F4), B1 (GBPITWR_F2), B2 (sodium phytate), B3 (calcium phytate) (GBPITWR_F4).

only in presence of TCP. In general, the fungi were more efficient in been extensively researched due to their ability to produce taxol; the
producing succinic and malic acids in comparison to other acids. In drug was also detected in the endophyte (Taxomyces andreanae) that
phytase screening medium, the production of organic acids was rela- was isolated from Taxus brevifolia (Stierle et al., 1993). Later, several
tively poor at 25 °C, and it was higher in presence of sodium phytate as endophytic fungi have been reported from various species of Taxus for
compared to calcium phytate. Results on production of organic acids their taxol producing trait (Somjaipeng et al., 2015; Xiong et al., 2013;
are presented in Table 3. Zhang et al., 2009). While species of Taxus including its endophytes
have attracted attention for its taxol producing trait, these endophytes
4. Discussion did not receive due attention in their ecological perspective. The pre-
sent study, was focused on the culturable endophytic fungi, isolated
Bioprospection of fungal endophytes is increasingly becoming re- from T. wallichiana roots, with respect to their phosphate solubilization
levant on account of their ability to produce a range of secondary potential.
metabolites and the associated biological activities (Corrêa et al., 2014; The culturable fungal endophytes, isolated from the T. wallichiana
Zheng et al., 2016). The beneficial relationship between endophytic roots, belonged to the species of Penicillium and Aspergillus. These spe-
fungi and the host species, particularly in medicinal plants, has also cies were referred as psychro, pH and salt tolerants due to their ability
been reviewed (Jia et al., 2016; Rai et al., 2014). Species of Taxus have to grow at wide temperature (5 to 25 °C), pH (0.5–12.0) and salt

Table 2
Qualitative estimation of fungal isolates for phosphate solubilization and phytase production at different temperatures.
Zone of solubilization (mm)
Phosphate solubilization
Fungal isolate Tri calcium phosphate Aluminiumphosphate Iron phosphate
5 °C 15 °C 25 °C 35 °C 5 °C 15 °C 25 °C 35 °C 5 °C 15 °C 25 °C 35 °C
GBPITWR_F1 + ++ ++ ++ + + ++ + + ++ ++ ++
GBPITWR_F2 ++ ++++ ++++ +++ + +++ +++ ++ + +++ +++ +++
GBPITWR_F3 + +++ +++ ++ + ++ +++ ++ + ++ ++ ++
GBPITWR_F4 ++ +++ +++ +++ + +++ +++ +++ + +++ +++ +++
GBPITWR_F5 ++ +++ +++ ++ + ++ ++ ++ + ++ +++ +
Zone of solubilization (mm) Colorimetric method (O.D.)
Phytase production Phosphatase production 25 °C
Fungal isolate Sodium phytate calcium phytate TCP IP AP
5 °C 15 °C 25 °C 35 °C 5 °C 15 °C 25 °C 35 °C AcP AlkP AcP AlkP AcP AlkP
GBPITWR_F1 + ++ ++ ++ ++ ++ ++ ++ + + + + + +
GBPITWR_F2 +++ ++++ ++++ +++ ++ ++++ ++++ ++ + + + + + +
GBPITWR_F3 ++ +++ +++ ++ + ++ +++ ++ + + + + + +
GBPITWR_F4 ++ +++ ++++ +++ ++ ++++ ++++ +++ + + + + + +
GBPITWR_F5 + ++ ++ ++ + +++ ++ ++ + + + + + +
Zone of solubilization: 0.1-0.3 +, 0.4-0.6 ++,0.7-0.9+++,1.0-1.2++++;
Ac P=Acidic phosphatase, Alk P=alkaline phosphatase; in phosphatase production: + present, - absent

5
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

Fig. 2. Phosphate solubilizing efficiency of fungal isolates.

(12–14%) tolerance (unpublished records). These specific characters 2012). Enhanced phosphate solubilization at suboptimal conditions has
are indicative of the resilience prevailed in the symbiotic plant-microbe been considered as a strategy for survival of these cold adapted fungi
relationship existing in mountain ecosystems. Occurrence of wide pH under low temperature environment (Rinu and Pandey, 2011). The
tolerance in microbes isolated from the extreme environments (Dhakar present study reveals that the ascomycetes fungi while colonize the low
and Pandey, 2016) and dominance of ascomycetes fungi including temperature soils up to sub zero levels, also involve in beneficial
species of Penicillium and Aspergillus in colder regions of Himalaya partnership with the plant species, such as T. wallichiana in the present
(Pandey et al., 2018) has been reported in recent years. Species of Pe- study. Ascomycetes fungi have been known for their potential in colo-
nicillium and Aspergillus, isolated from high altitude soils, have also been nizing the internal plant tissues including species of Taxus (Nicoletti
investigated for their phosphate solubilization potential (Rinu et al., et al., 2014; Wu et al., 2013; Xiang et al., 2003).

Fig. 3. Acidic (A) and alkaline (B) phosphatase production by fungal isolates.

6
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

Fig. 4. Phytase production by fungal isolates.

The qualitative estimations performed through agar and broth based due to the wide range of activity obtained in the different pH condi-
bioassays exhibited the potential of these fungal endophytes for phos- tions. Production of phosphatases by the fungal isolates in the present
phate solubilization, while the quantitative estimations showed the study was higher at 15 °C. Frankena et al. (1985) suggested a link be-
involvement of various mechanisms including production of enzymes tween the enzyme uptake, synthesis and energy metabolism which is
(phosphatases and phytases) and organic acids. Increasing fungal controlled by temperature. In plants phytic acid and phytate form a
phosphate solubilization with declining pH is in accordance with the major storehouse of phosphorus. Aspergillus niger has been reported for
earlier studies (Pandey et al., 2008; Rinu and Pandey, 2010). The fungal the production of extracellular phytase (Casey and Walsh 2003). In the
endophytes have revealed their potential in solubilizing all the three present study, GBBPITWR_F2 (Penicillium daleae) and GBBPITWR_F4
substrates, TCP, AlP and FeP. Calcium phosphate is known as the pre- (Aspergillus versicolor) produced maximum phytase enzyme ranging
dominant source of phosphate followed by AlP and FeP, respectively. from 4.14 ± 0.13 and 3.50 ± 0.08 µM/ml, respectively. Studies on
In a recent study, Spagnoletti et al. (2016) demonstrated the phos- the role of phytases are limited and needs attention in future studies
phate solubilization potential of dark septate endophytic fungi in pre- mainly in plant-endophytes symbiosis.
sence of calcium, iron and aluminium. The cited study reported max- Organic acids can form the complexes with calcium, iron and alu-
imum P solubilized in calcium phosphate ranging from 42.87 ± 5.37to minium, thus help in release of phosphate into the soil by chelation and
51.33 ± 1.87 µg/ml followed by P solubilized in aluminium phosphate exchange reaction. Organic acids also increase the P accessibility in the
ranging from 5.56 ± 0.14 to 20.14 ± 0.01 µg/ml and in case of iron soil by jamming P assimilation sites on soil particles or by forming
phosphate from 0.80 ± 0.31to 10.59 ± 0.11 µg/ml. These results are complexes with cation on the soil mineral surface (Behera et al., 2017).
in accordance with the findings of the present study. However, the In the present study, HPLC analysis of fungal isolates, grown in both
phosphate solubilization efficiency, recorded in the present study, is Pikovskaya and phytase screening media, indicated the involvement of
lower in comparison to the previous reports on the psychrotolerant organic acids in phosphate solubilization. Phosphate soulbilization,
species of Aspergillus and Penicillum that were isolated from the soil along with the production of organic acids, was higher in presence of tri
samples collected from the high altitudes in IHR (Pandey et al., 2008; calcium phosphate as a substrate. Production of organic acids, in fact,
Rinu and Pandey 2010; Rinu et al., 2013). This appears to be an in- causes the lowering in pH and enhances the P solubilization. While the
dicative of the influence of prevalent climatic conditions on the fungal isolates GBPI TWR_F2 and GBPI TWR_F5 indicated that organic
bioactivities performed by the microorganisms. Species of ascomycetes acid production play major role in phosphate solubilization, results on
fungi that dominate the low temperature soil environments exhibit other isolates (GBPI TWR_F1, GBPI TWR_F3 and GBPI TWR_F4)
variable (lower) efficiency when follow endophytic symbiosis in the showing relatively lesser decline in pH, indicated the involvement of
plant tissues. Interestingly, in both cases, soil as well as the plant tis- other mechanisms in solubilization of phosphates.
sues, the fungal phosphate soulbilization was favoured at suboptimal Microbial phosphate solubilization is one of the fundamental pro-
temperature. cesses that contributes to the plant growth. Release of organic acids,
Enzymatic mineralization of organophosphorus compounds through such as citric, gluconic and ketogluconic acids, has been known as the
phosphatases (primarily acidic phosphatases) has been proposed as one major biochemical process to mobilize the insoluble phosphorus com-
of the mechanisms in phosphate solubilization (Tarafdar and Gharu, pounds. Besides, the enzymes phosphatases and phytases have also
2006). Several studies, carried out on cold tolerant ascomycetes fungi, been studied for their contribution in this process through catalyzing
have demonstrated the involvement of phosphatases in phosphate so- the hydrolysis of phosphatic compounds (Tarafdar and Gharu, 2006;
lubilization. As in previous studies on cold tolerant ascomycetes fungi Velazquez and Rodriguez, 2007). Besides these components, role of
(Rinu et al., 2012), production of phosphatases was higher in acidic temperature and pH has been specifically studied with respect to low
medium that can be due to the acidity of the medium caused by the temperature environments under mountain ecosystems such as IHR and
organic acid production. Phosphatases are classified into two groups High Andes mountains (Rinu et al., 2012; Yarzábal, 2014). Positive

7
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

Table 3
Production of organic acids in Pikovskaya's broth (PVK) and Phytase screening media (PSM) at 15 and 25 °C.
Fungal isolate Temperature Organic acids PVK (µg/ml) PSM (µg/ml)

Tri calcium phosphate Aluminium phosphate Iron phosphate Calcium phytate Sodium phytate

GBPITWR_F1 15 °C Oxalic acid 0.32 ± 0.02 ND 0.07 ± 0.36 0.04 ± 0.005 0.02 ± 0.001
Malic acid 0.53 ± 0.04 ND 0.03 ± 0.67 0.02 ± 0.002 0.05 ± 0.004
Lactic acid 0.05 ± 0.05 0.006 ± 0.005 0.01 ± 0.46 0.001 ± 0.0002 0.06 ± 0.0001
Succinic acid 0.70 ± 0.006 ND ND 0.009 ± 0.0007 ND
Citric acid 0.08 ± 0.007 0.03 ± 0.006 0.07 ± 0.006 0.07 ± 0.03 0.04 ± 0.02
25 °C Oxalic acid 0.30 ± 0.003 ND 0.05 ± 0.006 ND 0.01 ± 0.002
Malic acid 0.27 ± 0.004 ND 0.04 ± 0.002 ND 0.02 ± 0.001
Lactic acid 0.31 ± 0.0006 0.02 ± 0.001 0.01 ± 0.003 ND 0.05 ± 0.04
Succinic acid 0.71 ± 0.005 ND ND ND 0.01 ± 0.0002
Citric acid 0.01 ± 0.006 0.06 ± 0.002 0.05 ± 0.004 ND 0.02 ± 0.01

GBPITWR_F2 15 °C Oxalic acid 0.41 ± 0.006 ND 0.01 ± 0.004 0.02 ± 0.001 0.01 ± 0.002
Malic acid 0.92 ± 0.006 ND ND 0.06 ± 0.003 0.06 ± 0.003
Lactic acid 0.07 ± 0.0008 0.03 ± 0.008 0.007 ± 0.001 0.01 ± 0.0002 0.07 ± 0.0002
Succinic acid 0.83 ± 0.006 ND ND 0.08 ± 0.0007 ND
Citric acid ND 0.02 ± 0.005 0.14 ± 0.006 0.09 ± 0.03 0.05 ± 0.01
25 °C Oxalic acid 0.20 ± 0.007 ND 0.01 ± 0.004 ND 0.02 ± 0.003
Malic acid 0.82 ± 0.007 ND ND ND 0.03 ± 0.002
Lactic acid 0.07 ± 0.0006 0.01 ± 0.003 0.007 ± 0.001 ND 0.03 ± 0.03
Succinic acid 0.80 ± 0.008 ND ND ND 0.03 ± 0.0001
Citric acid ND 0.27 ± 0.002 0.14 ± 0.006 ND 0.01 ± 0.01

GBPITWR_F3 15 °C Oxalic acid 0.37 ± 0.006 ND ND 0.03 ± 0.001 0.023 ± 0.001

Malic acid 0.97 ± 0.003 0.29 ± 0.02 0.45 ± 0.005 0.05 ± 0.004 0.05 ± 0.004
Lactic acid 0.06 ± 0.0006 0.006 ± 0.0001 0.008 ± 0.007 0.003 ± 0.002 0.002 ± 0.0001
Succinic acid 0.51 ± 0.008 ND ND 0.009 ± 0.0003 ND
Citric acid ND 0.06 ± 10.02 0.02 ± 0.005 0.07 ± 0.05 0.03 ± 0.02
25 °C Oxalic acid 0.62 ± 0.004 ND ND ND 0.02 ± 0.002
Malic acid 0.66 ± 0.006 0.72 ± 0.06 0.49 ± 0.003 ND 0.02 ± 0.001
Lactic acid 0.08 ± 0.003 0.02 ± 0.0015 0.009 ± 0.005 ND 0.03 ± 0.04
Succinic acid 0.71 ± 0.004 ND ND ND 0.003 ± 0.0002
Citric acid ND 0.2 ± 0.04 0.07 ± 0.002 ND 0.05 ± 0.02

GBPITWR_F4 15 °C Oxalic acid 0.71 ± 0.006 0.1 ± 0.009 ND 0.01 ± 0.021 0.03 ± 0.021
Malic acid 0.64 ± 0.007 0.2 ± 0.01 0.6 ± 0.009 0.07 ± 0.003 0.05 ± 0.005
Lactic acid 0.07 ± 0.006 0.08 ± 0.007 0.04 ± 0.004 0.04 ± 0.003 0.02 ± 0.002
Succinic acid 0.61 ± 0.007 ND 0.2 ± 0.001 0.06 ± 0.0001 0.08 ± 0.0001
Citric acid ND 0.01 ± 0.01 0.05 ± 0.002 0.08 ± 0.01 0.03 ± 0.01
25 °C Oxalic acid 0.32 ± 0.004 0.03 ± 0.001 ND ND ND
Malic acid 0.84 ± 0.006 0.09 ± 0.009 0.4 ± 0.004 ND ND
Lactic acid 0.61 ± 0.005 0.05 ± 0.004 0.04 ± 0.004 ND ND
Succinic acid 0.71 ± 0.005 0.68 ± 0.01 0.3 ± 0.006 ND ND
Citric acid ND ND 0.6 ± 0.006 ND ND

GBPITWR_F5 15 °C Oxalic acid 0.23 ± 0.005 0.42 ± 0.006 ND 0.32 ± 0.002 0.22 ± 0.02
Malic acid 0.87 ± 0.004 0.45 ± 0.009 0.4 ± 0.005 0.51 ± 0.003 0.44 ± 0.005
Lactic acid 0.05 ± 0.006 0.03 ± 0.0004 0.02 ± 0.006 0.02 ± 0.002 0.07 ± 0.003
Succinic acid 0.92 ± 0.008 0.62 ± 90.01 ND 0.06 ± 0.001 0.08 ± 0.0002
Citric acid ND ND ND 0.02 ± 0.02 0.41 ± 0.02
25 °C Oxalic acid 0.36 ± 0.006 0.33 ± 0.001 ND ND ND
Malic acid 0.95 ± 0.006 0.67 ± 0.009 0.62 ± 0.02 ND ND
Lactic acid 0.04 ± 0.007 0.08 ± 0.0004 0.14 ± 0.06 ND ND
Succinic acid 0.62 ± 0.006 0.71 ± 0.01 ND ND ND
Citric acid ND ND ND ND ND

ND (peak not detected)

response of inoculation with endophytic plant growth microorganisms, natural ecosystems. They are likely to play a significant role in plant
including bacteria and fungi, has been demonstrated in previous studies biogeography, evolution and community structure (Rodriguez et al.,
(Pandey et al., 2014; Bi et al., 2018). The endophytes and their host 2009). The microbial communities associated with the host influence
plants establish a special relationship which is likely to significantly the ecophysiology with respect to nutrition, growth, resistance to biotic
influence the formation of metabolic products in plants (Jia et al., and abiotic stresses and the survival and distribution of the plant spe-
2016). Although these reports are evident in favour of the plant growth cies (Rey and Schornack, 2013; Wani et al., 2015). For better under-
activities associated with the endophytic microorganisms and their standing of the symbiotic systems genome-centric approaches have also
expression under field conditions in an indirect manner, this aspect been recommended (Kumar and Blaxter, 2011). The endophytic fungi
needs to be strengthened further by using advance techniques. isolated from T. wallichiana roots growing in colder regions of IHR
Research on rhizosphere microbiome composition is becoming more showed different ability to solubilize insoluble calcium, aluminum and
relevant in view of understanding the plant-microbe interactions based iron phosphates through various mechanisms. Bioformulation of these
ecosystem services and plant adaptation under stress environments in fungi should be useful in the developing the environment friendly
climate change and food security scenario (Adl, 2016; Ahkami et al., technology supporting the conservation of Himalayan yew.
2017). The fungal endophytes that vary in symbiotic and ecological
functions can influence the survival and fitness of plants in all the

8
P. Adhikari, A. Pandey Rhizosphere 9 (2019) 2–9

Conflict of interest statement Rai, M., Rathod, D., Agarkar, G., Dar, M., Brestic, M., Pastore, G.M., Junior, M.R.M., 2014.
Fungal growth promotor endophytes: a pragmatic approach towards sustainable food
and agriculture. Symbiosis 62, 63–79.
We declare that we have no conflict of interest. Rey, T., Schornack, S., 2013. Interactions of beneficial and detrimental root-colonizing
filamentous microbes with plant hosts. Genome Biol. 14, 121.
Appendix A. Supporting information Rinu, K., Pandey, A., 2010. Temperature-dependent phosphate solubilization by old tol-
erant species of Aspergillus isolated from Himalayan Soil. Mycoscience 51, 263–271.
Rinu, K., Pandey, A., 2011. Slow and steady phosphate solubilization by psychrotolerant
Supplementary data associated with this article can be found in the strain of Paecilomyces hepiali (MTCC 9621). World J. Microbiol. Biotechnol. 27,
online version at doi:10.1016/j.rhisph.2018.11.002. 1055–1062.
Rinu, K., Pandey, A., Palni, L.M.S., 2012. Utilization of psychrotolerant phosphate solu-
bilizing fungi under low temperature conditions of the mountain ecosystem. In:
References Satyanarayana, T., Johri, B.,N., Prakash, A. (Eds.), Microorganisms in Sustainable
Agriculture and Biotechnology. Springer Science + Buisiness Media, Dordrecht
Heidelberg London New York, pp. 77–90.
Adhikari, P., Pandey, A., 2017. Taxus wallichiana Zucc. (Himalayan Yew) in antimicrobial
Rinu, K., Malviya, M.K., Sati, P., Tiwari, S.C., Pandey, A., 2013. Response of cold-tolerant
perspective. Adv. Biotechnol. Microbiol. 4 (5), 555–650.
Aspergillus spp. to solubilization of Fe and Al phosphate in presence of different nu-
Adhikari, P., Pandey, A., Agnihotri, V., Pandey, V., 2018. Selection of solvent and ex-
tritional sources. ISRN Soil Sci. https://doi.org/10.1155/2013/598541.
traction method for determination of antimicrobial potential of Taxus wallichiana
Rodriguez, R.J., White Jr., J.F., Arnold, A.E., Redman, R.S., 2009. Fungal endophytes:
Zucc. Res. Pharm. 8, 1–9.
diversity and functional roles. New Phytol. 182, 314–330.
Adl, S.M., 2016. Rhizosphere, food security, and climate change: A critical role for plant-
Saleem, M., Law, A.D., Sahib, M.R., Pervaiz, Z.H., Zhang, Q., 2018. Impact of root system
soil research. Rhizosphere 1, 1-3.
architecture on rhizosphere and root microbiome. Rhizosphere 6, 47–51.
Ahkami, H.A., White, R.A., Handakumbura, P.P., Jansson, C., 2017. Rhizosphere en-
Santoyo, G., Hagelsieb, M.G., Mosqueda, O.C.M., 2016. Plant growth-promoting bacterial
gineering: enhancing sustainable plant ecosystem productivity. Rhizosphere
endophytes. Microbiol. Res. 183, 92–99.
233–243.
Singh, B., Satyanarayana, T., 2011. Microbial phytases in phosphorus acquisition and
Bakker, P.A.H.M., Pieterse, C.M.J., Jonge, R.D., Berendsen, R.L., 2018. The soil-borne
plant growth promotion. Physiol. Mol. Biol. Plants 17 (2), 93–103.
legacy. Cell 172, 1178–1180.
Singh, S., Pandey, A., 2017. Plant-associated microbial endophytes: promising source for
Behera, B.C., Yadav, H., Singh, S.K., Mishra, R.R., Sethi, B.K., Dutta, S.K., Thatoi, H.N.,
bioprospecting. In: Kalia, V.C., Shouche, Y., Purohit, H., Rahi, P. (Eds.), Mining of
2017. Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated
Microbial Wealth and Meta Genomics. Springer, Singapore, pp. 249–265.
from mangrove soil of Mahanadi river delta, Odisha, India. J. Genet. Eng. Biotechnol.
Somjaipeng, S., Medina, A., Kwaśna, H., Ortiz, J.O., Magan, N., 2015. Isolation, identi-
15, 169–178.
fication, and ecology of growth and taxol production by an endophytic strain of
Bi, Y., Zhang, Y., Zou, H., 2018. Plant growth and their root development after in-
Paraconiothyrium variabile from English yew trees (Taxus baccata). Fungal Biol. 119,
oculation of arbuscular mycorrhizal fungi in coal mine subsided areas. Int. J. Coal Sci.
1022–1031.
Technol. 5 (1), 47–53.
Spagnoletti, F.N., Tobar, N.E., Fernandez, A.P.D., Chiocchio, V.M., Lavado, R.S., 2016.
Casey, A., Walsh, G., 2003. Purification and characterization of extracellular phytase from
Dark septate endophytes present different potential to solubilize calcium, iron and
Aspergillus niger ATCC 9142. Biores. Technol. 86, 183–188.
aluminium phosphates. Appl. Soil. Ecol. 111, 25–32.
Corrêa, R.C., Rhoden, S.A., Mota, T.R., Azevedo, J.L., Pamphile, J.A., de Souza, C.G.,
Stierle, A., Strobel, G.A., Stierle, D., 1993. Taxol and taxane production by Taxomyces
Polizeli,Mde, L., Bracht, A., Peralta, R.M., 2014. Endophytic fungi: expanding the
andreanae, anendophytic fungus of Pacific yew. Science 260, 214–216.
arsenal of industrial enzyme producers. J. Ind. Microbiol. Biotechnol. 41 (10),
Tarafdar, J.C., Gharu, A., 2006. Mobilization of organic acid and poorly soluble phos-
1467–1478.
phatase by Chaetomium globosum. Appl. Soil Ecol. 32, 273–283.
Dhakar, K., Pandey, A., 2016. Wide pH range tolerance in extremophiles: towards un-
Tayung, K., Jha, D.K., 2006. Antimicrobial evaluation of some fungal endophytes isolated
derstanding an important phenomenon for future biotechnology. Appl. Microbiol.
from the bark of Himalayan yew. World J Agric. Sci. 2, 489–494.
Biotechnol. 100, 2499–2510.
Thomas, P., Farjon, A., 2011. Taxus wallichiana. The IUCN Red List of Threatened Species
Frankena, J., Van, H.W.V., Stouthamer, A.H., 1985. A countinious culture study of
2011: e.T46171879A9730085.
bioenergetics aspect of growth and production of exocellular protease in Bacillus li-
Vasundhara, M., Baranwal, M., Sivaramaiah, N., Kumar, A., 2017. Isolation and char-
cheniformis. Appl. Microbiol. Biotechnol. 22, 169–176.
acterization of trichalasin-producing endophytic fungus from Taxus baccata. Ann.
Jia, M., Chen, L., Xin, H.L., Zheng, C.J., Rahman, K., Han, T., Qin, L.P., 2016. A friendly
Microbiol. 67, 255–261.
relationship between endophytic fungi and medicinal plants: a systematic review.
Velazquez, E., Rodríguez, C., 2007. In: First international meeting on microbial phosphate
Front. Microbiol. 7, 906. https://doi.org/10.3389/fmicb.2016.00906.
solubilization. In: Rodriguez, B.C. (Ed.), Developments in Plant and Soil Sciences 102
Juyal, D., Thawani, V., Thaledi, S., Joshi, M., 2014. Ethnomedical properties of Taxus
Springer, Salamanca.
wallichiana Zucc. (Himalayan Yew). J. Tradit. Complement. Med. 4 (3), 159–161.
Wani, Z.A., Ashraf, N., Mohiuddin, T., Riyaz-Ul-Hassan, S., 2015. Plant-endophyte sym-
Kumar, S., Blaxter, M.L., 2011. Simultaneous genome sequencing of symbionts and their
biosis, an ecological perspective. Appl. Microbiol. Biotechnol. 99 (7), 2955–2965.
hosts. Symbiosis 55, 119–126.
Wani, Z.A., Ashraf, N., Mohiuddin, T., Hassan, R.U.S., 2015. Plant-endophyte symbiosis,
Kumaran, R.S., Hur, B.K., 2009. Screening of species of the endophytic fungus Phomopsis
an ecological perspective. Appl. Microbiol. Biotechnol. 99 (7), 2955–2965.
for the production of the anticancer drug taxol. Biotechnol. Appl. Biochem. 54 (1),
Wu, L., Han, T., Li, W., Jia, M., Xue, L., Rahman, K., Qin, L., 2013. Geographic and tissue
21–30.
influences on endophytic fungal communities of Taxus chinensis var. mairei in China.
Nadeem, M., Rikhari, H.C., Kumar, A., Palni, L.M.S., Nandi, S.K., 2002. Taxol content in
Curr. Microbiol. 66 (1), 40–48.
the bark of Himalayan Yew in relation to tree age and sex. Phytochemistry 60,
Xiang, Y., Lu, A., Wu, W., 2003. Identification of Taxus cuspidata Sieb. et Zucc. endophytic
627–631.
fungi new species, species known and their metabolite. J. For. Res. 14, 290–294.
Nicoletti, R., Fiorentino, A., Scognamiglio, M., 2014. Endophytism of Penicillium species
Xiong, Z., Yang, Y., Zhao, N., Wang, Y., 2013. Diversity of endophytic fungi and screening
in woody plants. Open Mycol. J. 8, 1–26.
of fungal paclitaxel producer from Anglojap yew, Taxus x media. BMC Microbiol.
Pandey, A., Das, N., Kumar, B., Rinu, K., Trivedi, P., 2008. Phosphate solubilization by
13, 71.
Penicillium spp. isolated from soil sample of Indian Himalayan Region. World J.
Yarzábal, L.A., 2014. Cold-Tolerant phosphate-solubilizing microorganisms and agri-
Microbiol. Biotechnol. 24, 97–102.
culture development in mountainous regions of the World. In: Khan, M.S., Zaidi, A.,
Pandey, A., Sati, P., Malviya, M.K., Singh, S., Kumar, A., 2014. Use of endophytic bac-
Musarrat, J. (Eds.), Phosphate Solubilizing Microorganisms. Springer International
terium (Pseudomonas sp., MTCC9476) in propagation and conservation of Ginkgo
Publishing, Switzerland, pp. 113–136.
biloba L.: a living fossil. Curr. Sci. 106, 25.
Zhang, P., Zhou, P., Yu, L., 2009. An endophytictaxol-producing fungus from Taxus x
Pandey, A., Dhakar, K., Jain, R., Pandey, N., Gupta, V.K., Kooliyottil, R., Dhyani, A.,
media, Aspergillus candidus MD3. FEMS Microbiol. Lett. 293 (2), 155–159.
Malviya, M.K., Adhikari, P., 2018. Cold Adapted Fungi from Indian Himalaya: un-
Zhao, K., Ping, W., Li, Q., Hao, S., Zhao, L., Gao, T., Zhou, D., 2009. Aspergillusniger
tapped Source for Bioprospecting. Proc. Natl. Acad. Sci. India, Sect. B Biol. Sci.
subsp. taxi, a new species variant of taxol producing fungus isolated from Taxus
https://doi.org/10.1007/s40011-018-1002-0.
cuspidata in China. J. Appl. Microbiol. 107, 1202–1207.
Poudel, R.C., Goa, L.M., Moeller, M., Baral, S.R., Uprety, Y., Liu, J., Li, D.Z., 2013. Yews
Zheng, Y.K., Qiao, X.G., Miao, C.P., Liu, K., Chen, Y.W., Xu, L.H., Zhao, L.X., 2016.
(Taxus) along the Hindu Kush-Himalayan region: exploring the ethnopharmacolo-
Diversity, distribution and biotechnological potential of endophytic fungi. Ann.
gical relevance among communities of Mongol and Caucasian origins. J.
Microbiol. 66, 529–542.
Ethnopharmacol. 147 (1), 190–203.