Insights into diversity and L-asparaginase activity of fungal endophytes associated with medicinal plant Grewia hirsuta

Abstract

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. The present work aimed to study the endophytic fungal diversity of Grewia hirsuta and their ability to produce L-asparaginase. A total of 1575 culturable fungal endophytes belonging to four classes, Agaricomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, were isolated. The isolates were grouped into twenty-one morphotypes based on their morphological characteristics. Representative species from each group were identified based on their microscopic characteristics and evaluation of the ITS and LSU rDNA sequences. Most of the fungal endophytes were recovered from the leaves compared to other plant parts. Diaporthe sp. was the predominant genus with a colonization frequency of 8.62%. Shannon-Wiener index for diversity ranged from 2.74 to 2.88. All the plant parts showed similar Simpson’s index values, indicating a uniform species diversity. Among the sixty-three fungal endophytes screened, thirty-two were identified as L-asparaginase-producing isolates. The enzyme activities of fungal endophytes estimated by the nesslerization method were found to be in the range of 4.65–0.27 IU/mL with Fusarium foetens showing maximum enzyme activity of 4.65 IU/mL. This study for the first time advocates the production of L-asparaginase from Fusarium foetens along with the endophytic fungal community composition of Grewia hirsuta. The results indicate that the fungal endophyte Fusarium foetens isolated in the present study could be a potent source of L-asparaginase.

Graphical Abstract

Introduction

Acute lymphoblastic leukemia (ALL) is a form of blood cancer characterized by the production of massive numbers of underdeveloped white blood cells (WBC) or lymphocytes in the bone marrow [1]. ALL mainly affects children, contributing to approximately 80% of childhood leukemia [2]. One of the chemotherapeutic drugs used in the therapy of ALL is L-asparaginase (E.C.3.5.1.1), a pyrimidine derivative of the amidohydrolase family which catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia [3].

The origin of L-asparaginase as a therapeutic agent began several decades ago when Kidd [4] reported the inhibition of some neoplasms in mice and rats by serum of guinea pig. Subsequently, Broome [5] showed it was the enzyme L-asparaginase present in the serum of guinea pigs that was responsible for the inhibition of the tumor. The next important breakthrough in the development of L-asparaginase as a chemotherapeutic drug was when Mashburn and Wriston [6] isolated L-asparaginase from Escherichia coli (E. coli) and demonstrated its effectiveness as an antitumor agent. L-asparagine is necessary for the growth and development of both normal and leukemic cells. The normal cells can self-synthesize L-asparagine, but the latter cannot. Therefore, the leukemic cells solely rely on exogenic supply of L-asparagine. Thus, when L-asparaginase is injected into the bloodstream, the serum asparagine is hydrolyzed making it unavailable for leukemic cells, eventually leading to the starvation and death of leukemic cells. Thus, it prevents the growth of malignant cells by reducing the L-asparagine which is an essential nutrient for the growth of tumor cells [7].

In recent years, L-asparaginase has gained much importance for its antineoplastic potential. Globally, it contributes to 40% of the overall enzyme demands [8]. The demand for the enzyme is estimated to increase by up to 420 million USD by 2025 which was 380 million USD in 2017 [9]. Apart from ALL, it is also employed in the treatment of acute myeloblastic leukemia (AML), Hodgkin’s disease, chronic lymphocytic leukemia (CLL), pancreatic carcinoma, non-Hodgkin’s lymphoma, reticulosarcoma, melanosarcoma, and bovine lymphosarcoma [10]. Further, L-asparaginase is used as one of the crucial food-processing agents to reduce the formation of carcinogenic acrylamide in fried food items [11].

L-Asparaginase is found in many sources such as plants, animals, and microorganisms [12]. However, L-asparaginase from microorganisms is studied extensively as they confer several advantages over other sources, such as easy optimization of culture conditions, can be grown using simple substrates, simplified extraction and purification, and can be genetically modified for higher yield [13]. Many bacteria like Proteus vulgaris [14], Corynebacterium glutamicum [15], Thermus thermophilus [16], Helicobacter pylori [17], Pseudomonas aeruginosa [18], Lactobacillus casei [19], and Pyrococcus furiosus [20] have been reported to produce L-asparaginase. Some of the fungi such as Fusarium tricinctum [21], Aspergillus niger [22], Penicillum digitatum [23], Mucor hiemalis [24], Aspergillus terreus [25], Fusarium culmorum [26], and Fusarium equisetti [27] have displayed great ability to produce L-asparaginase. Several actinomycetes including Thermoactinomyces vulgaris [28], Streptomyces albidoflavus [29], Streptomyces gulbargensis [30], Streptomyces ginsengisoli [31], and Streptomyces rochei [32] are known to produce L-asparaginase. Few algae like Chlamydomonas sp. [33] and Chlorella vulgaris [34] are also known to produce L-asparaginase.

However, the currently used L-asparaginase formulations for the treatment of ALL are produced industrially from E. coli and Erwinia chrysanthemi (E. chrysanthemi, now known as Dickeya dadanti) [35]. Until now, four main types of L-asparaginase, the native L-asparaginase obtained from E. coli, a PEGylated form (PEG asparaginase), L-asparaginase derived from E. chrysanthemi, and a recombinant E. coli-asparaginase preparation have been used. However, because of their prokaryotic origin, these L-asparaginase formulations cause hypersensitivity and many side effects [36-38]. In addition, its short half-life (early clearance from blood) requires repeated administration making the overall treatment price relatively high [39]. Whereas, L-asparaginase from fungi induces relatively less toxicity and a weak immune response [40] and has been studied in many fungal species.

Endophytic fungi are a group of diversified fungi that inhabit the internal tissues of living plants without causing any noticeable infections to the host [41]. They are found in all plants aiding in many aspects including defense against phytopathogens [42], and survival in unfavorable conditions, such as high temperatures and nutrient scarcity [43]. In recent years, they have gained much attention because of their capacity to produce several bioactive compounds [44]. It has been speculated that endophytic fungi have the capacity to produce compounds found in the host plant or other compounds with medicinal properties [45]. Several anticancer compounds have been isolated from endophytic fungi, such as campthothecin from Nothapodytes foetida [46], podophyllotoxin from Fusarium oxysporum [47], and cajanol from Hypocrea lixii [45]. L-asparaginase is one such anticancer enzyme that can be produced from endophytic fungi. Many plants have been screened for L-asparaginase producing endophytic fungi, and several strains are reported to produce L-asparaginase, which include Fusarium verticillioides [48], Fusarium oxysporum, Penicillium simplicissimum [49], and Talaromyces pinophilus [50].

Grewia hirsuta is a small shrub from the Malvaceae family. The root and drupes of the plant are used as a folk medicine for the treatment of heart disease, cough, wounds, diarrhea dysentery, fever, and dyspnea [51]. The plant extract is used as an anticancer, anti-ulcer, aphrodisiac, and anti-fertility agent [52]. Ema et al. [53], reported antiproliferative activity of Grewia hirsuta. It has been hypothesized that endophytic fungi have the ability to produce compounds responsible for therapeutic activity of the host plant [45]. Hence, the current work is aimed to study the endophytic fungal diversity of medicinal plant Grewia hirsuta and their ability to produce L-asparaginase, a potential anticancer enzyme.

Materials and methods

Chemicals

Agar powder, PDB (potato dextrose broth), SDS (sodium dodecyl sulfate), CTAB (cetyl trimethyl ammonium bromide) were obtained from Himedia, India. L-asparagine, agarose, silica gel, chloramphenicol, RNAse, and proteinase K were acquired from Merck, Germany. KH₂PO₄, KCl, MgSO₄.7H₂O, CuNO₃.3H₂O, ZnSO₄.7H₂O, FeSO₄.7H₂O, NaCl, NaNO_3, Tris, EDTA and phenol red were obtained from SD Fine chemicals, India. Sodium hypochlorite, lactophenol cotton blue, HCl, mercaptoethanol, Nessler’s reagent, TCA, phenol, chloroform, isoamyl alcohol, isopropanol, and acetic acid were acquired from Nice chemicals, India.

Isolation of fungal endophytes

Fresh and healthy plant material of Grewia hirsuta was collected in a sterile polythene bag near Bandipur National Park region of Mysuru district, Karnataka, India (11° 52′ 34.1″ N 76° 20′ 31.1″ E). The plant material was first washed with slow-running tap water to remove unwanted debris. Further, plant material was cut into small segments of 0.5 cm² using a sterile scalpel blade. The surface microflora was eradicated through a surface sterilization process by washing initially in ethanol (70%) for 1 min then with sodium hypochlorite solution (4%) for 4 min, and finally by rinsing in sterile distilled water thrice. The tissue segments (leaf, stem, root, and fruit) were blot-dried and placed equidistantly on the surface of water agar plates (10 bits/plate) added with chloramphenicol (100 mg/L) to prevent bacterial contamination. The plates were incubated at 25 ± 2 °C for 2–3 days. Fungal hyphae emerging from the tissue segments were carefully transferred onto freshly prepared potato dextrose agar (PDA) plates and incubated at 25 ± 2 °C for 5–7 days to establish pure cultures [54]. The overall procedure is depicted in Fig. 1

Fig. 1.

Isolation, identification, and screening of fungal endophytes for L-asparaginase production.

Identification of fungal endophytes

Microscopic identification

The fungal mycelia were stained using lactophenol cotton blue, and the slides were examined under a light microscope (Olympus, USA). The endophytic fungi were identified by referring to standard fungal identification manuals [55, 56].

Molecular identification

Preparation of fungal material

The fungal endophytes were grown in PDB at 25 ± 2 °C for 7–10 days. The fungal mycelium was separated from the culture broth by filtering through a double-layered cheesecloth. Approximately 5 g of mycelium was freeze-dried by lyophilization (Sub-zero, India).

DNA extraction

The lyophilized fungal mycelium (0.5 g) was ground with cover glass and silica gel to make a fine powder using a pestle and mortar. This was taken in a 2-mL centrifugation tube and mixed with 500 μL of extraction buffer (20 mM EDTA, 1.4 M NaCl, 100 mM Tris-HCl) containing 2% CTAB and 0.5% SDS. Ten microliters of mercaptoethanol was added to the above mixture and kept in a hot water bath for 30 min. To this, 10 μL of RNAse and proteinase K solution was added and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and transferred into a fresh centrifugation tube, and an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture was added and centrifuged at 12,000 rpm for 10 min. The supernatant was collected in a new tube, and an equal volume of chloroform:isoamyl alcohol (1:1) mixture was added and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a fresh tube and added with an equal volume of ice-cold isopropanol. The mixture was gently mixed and kept at −20 °C for 30 min. This mixture was centrifuged at 12,000 rpm for 10 min; the resulting pellet was collected and washed with 70% ethanol and resuspended in 100 μL of sterile water [57]. The yield and quality of the DNA were assessed by a Nanodrop spectrophotometer (ThermoFisher Scientific, USA) and agarose gel electrophoresis (1% w/v).

PCR amplification

Polymerase chain reaction (PCR) was carried out to amplify internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal RNA. The ITS regions were amplified using the universal primers ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′-TCCGTAGGTGAACCTGCGG-3′) with the following PCR cycling protocol: initial denaturation at 95 °C for 60 s, followed by 30 cycles of 94 °C for 60 s, 57.6 °C for 60 s, 72 °C for 90 s and a final extension at 72 °C for 10 min [58]. Amplification of LSU region was carried out using the primers LROR (5′-ACCCGCTGAACTTAAGC-3′) and LR7 (5′-TACTACCACCAAGATCT-3′) with the following PCR cycling protocol: pre-heating at 95 °C for 60 s, 30 cycles 95 °C for 60 s, 52 °C for 60 s, 72 °C for 90 s, and then at 72 °C for 10 min [59]. The amplification was performed in a total volume of 50 μL with 25 μL of Taq DNA Polymerase 2x Master Mix RED (Amplicon, Denmark), 16 μL of Milli Q water, 3 μL of each primer (forward and reverse), and 3 μL of template DNA.

Electrophoresis of PCR products

The amplified samples were examined by electrophoresis using 1.8% agarose gel (w/v). Electrophoresis was conducted in a horizontal electrophoresis system (MT-108 Mini Horizontal Gel Electrophoresis System, Major science, USA) for 60 min at 60 V and 145 mA using 1x TAE (Tris-Acetate-EDTA) running buffer. Vilber Lourmat™ Docprint VX2 Gel Documentation System (Vilber, France) was used to capture images.

Sequence analysis of PCR products

The amplified samples were sequenced by Eurofins Genomics India Pvt. Ltd. The sequences were then analyzed and edited using the BioEdit 7.2 software. The edited sequence data was subjected to BLASTn search on the NCBI (National Center for Biotechnology Information) website and deposited in NCBI’s GenBank Database. Sequences similar to the isolated fungal endophytes were acquired from the GenBank database using the BLASTn search analysis. These sequences were subjected to multiple sequence alignment by using Clustal Omega web-based tool. MEGA 5.2 software was used for the construction of a phylogenetic tree by maximum likelihood analysis with a bootstrap analysis of 1000 replicates.

Data analysis of fungal endophytes

The endophytic fungal diversity of Grewia hirsuta was quantitatively analyzed in terms of colonization frequency (CF), isolation rate (IR), species richness (S), species evenness (E) along with Shannon-Wiener (H′), and Simpson diversities (Ds). The total number of segments inhabited by fungal endophytes divided by the number of segments plated was calculated as the colonization frequency [60]. The total number of isolates obtained divided by the number of segments plated was calculated as the isolation rate [60]. The total number of fungal species obtained is described as species richness [61]. Species evenness was described as the relative contribution of each species to the total biomass [62]. The comparison of endophytic fungal diversity from four different tissue segments was analyzed using principal component analysis (PCA). PCA was performed with ORIGINPRO software version 2018. An online diversity calculator (http://www.changbioscience.com/genetics/shannon.html) was used to calculate species evenness, Shannon-Wiener, and Simpson diversity index. The calculations are as follows

$$H^{\prime }=-\sum \text{Pi}\times \text{Ln}\left(\text{Pi}\right)$$

$$\text{Ds}=1-\sum {\text{Pi}}^2$$

$$\text{E}={\text{H}}^{\prime }/\text{Ln}\left(\text{S}\right)$$

Pi = ni/N, is the relative abundance of the endophytic fungal species, where ni is the number of isolates of one species, and N is the total number of species [63]. S is the total number of genera present in the individual sample [64].

Screening of fungal endophytes for L-asparaginase production

Primary screening of fungal endophytes for L-asparaginase production was done using Modified Czapek Dox (MCD) agar medium with phenol red indicator. MCD agar medium was composed of L-asparagine (10.0 g/L), glucose (2.0 g/L), KH₂PO₄ (1.52 g/L), KCl (0.52 g/L), MgSO₄.7H₂O (0.52 g/L), CuNO₃.3H₂O (0.001 g/L), ZnSO₄.7H₂O (0.001 g/L), FeSO₄.7H₂O (0.001 g/L), and agar powder (20.0 g/L) supplemented with 0.009% (w/v) phenol red. The pH of the medium was adjusted to 7. The prepared culture medium was sterilized by autoclaving and poured into sterilized petri plates. Sodium nitrate (NaNO₃) was used as a substitute nitrogen source for the preparation of control plates. A 5-mm endophytic fungal mycelial plug was inoculated onto these MCD agar medium plates and incubated at 25 ± 2 °C for 7 days. After incubation, the diameter of the pink zone around the colonies was measured [65]. The enzyme index was determined using the formula:

$$\text{Enzyme}\text{index}=\text{Pink}\text{zone}\left(\text{mm}\right)/\text{Diameter}\text{of}\text{the}\text{colony}\left(\text{mm}\right)$$

Estimation of L-asparaginase activity in selected fungal endophytes

The fermentation process for quantitative estimation of L-asparaginase was carried out in liquid broth. One milliliter spore suspension (10⁶ spore/mL) of each selected endophytic fungal culture grown on PDB at 25 ± 2 °C for 7 days was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of MCD medium (without phenol red indicator, pH 7) and then incubated at 25 °C for 7 days. After incubation, the culture broth was filtered and centrifuged at 10,000 rpm for 15 min at 4 °C and the resulting supernatant (crude enzyme) was used for the estimation of L-asparaginase. The reaction mixture consisted of 0.5 mL of the above crude enzyme and 0.5 mL of L-asparagine (0.04 M) 0.5 mL of Tris-HCl buffer (0.1 M, pH 8) and 0.5 mL of distilled water. The mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding 0.5 mL of trichloroacetic acid (1.5 M). The obtained mixture was centrifuged to remove the precipitates at 10,000 rpm for 15 min at 4 °C. Two hundred microliters of Nessler’s reagent was added to 100 μL of the above mixture diluted with 3.7 mL of distilled water. Blank tubes were prepared by adding the crude enzyme after the addition of trichloroacetic acid. After incubating the mixture at 20 °C for 20 min, the absorbance was recorded at 450 nm, and the amount of ammonia released was determined. The amount of enzyme that catalyzes the formation of 1 μmol of ammonia per minute at 37 °C is expressed as one unit of L-asparaginase [66].

Statistical analysis

All the values reported for enzyme activity are mean ± standard deviation (SD) of three replicates. Significant differences (p < 0.05) between enzyme activities were determined by one-way analysis of variance (ANOVA) and the Tukey test. Statistical analyses were performed using the IBM SPSS software (version 27).

Results

Isolation and identification of fungal endophytes

A total of 1575 culturable fungal endophytes were isolated from Grewia hirsuta. The highest number of fungal endophytes were recovered from leaves. The isolates were grouped into twenty-one morphotypes based on their morphological characteristics. Representative species from each group were identified based on their microscopic characteristics (spore morphology) and evaluation of the ITS and LSU rDNA sequences. Most of the fungal endophytes belonged to Ascomycota, followed by Basidiomycota. Ten different orders were observed, of which five represented Sordariomycetes, three represented Dothideomycetes, and one order each represented by Agaricomycetes and Eurotiomycetes. Most of the endophytic fungal isolates were from the order Pleosporales, grouped under five different genera. The most dominant class was Sordariomycetes with colonization frequency of 41.87%. Diaporthe sp. was the predominant genus with a colonization frequency of 8.62%, followed by Neopestalotiopsis sp. Thus, the host plant Grewia hirsuta has the resilience to establish a symbiotic association with Diaporthe (Sordariomycetes). Alternaria sp. was the most prevailing genus among the Dothideomycetes and third dominant overall with a colonization frequency of 6.5%. Isolates of the most genera inhabited more than one plant part, i.e., the genera of Lasidiplodia sp., Macrophomina sp., Cladosporium sp., Acrocalyma sp., Alternaria sp., Bipolaris sp., Curvularia sp., Aspergillus sp., Penicillium sp., Neopestalotiopsis sp., Colletotrichum sp., Pestalotiopsis sp., Diaporthe sp., Fusarium sp., and Xylariaceae sp. were recovered from the all plant parts. The two principal components (1 and 2) obtained by PCA explained 62.96% of total variation (Fig. 2). The PCA analysis revealed that fungal endophytes of some genera have tissue specificity. The isolates of Porostereum sp., Corynespora sp., and Monosporascus sp. were isolated respectively from fruits, leaves, and roots only. Thus, among the eighteen endophytic fungal genera analyzed, three taxa were extensively restricted to one definite tissue, whereas fifteen genera were found to occur in more than one tissue (Table 1).

Fig. 2.

Principal component analysis (PCA) of fungal endophytes isolated from different tissues of Grewia hirsuta

Table 1.

Diversity and distribution of fungal endophytes from Grewia hirsuta

Sl. no.	Class	Order	Family	Fungi	Leaf	Stem	Fruit	Root	Total	CF (%)
1	Agaricomycetes	Polyporales	Phanerochaetaceae	Phanerochaete sp.	13	12	–	17	42	2.62	3.81
2				Porostereum sp.	–	–	19	–	19	1.18
3	Dothideomycetes	Botryosphaeriales	Botryosphaeriaceae	Lasidiplodia sp.	30	26	11	18	85	5.31	38.43
4				Macrophomina sp.	18	14	10	27	69	4.31
5		Capnodiales	Cladosporiaceae	Cladosporium sp.	33	23	13	28	97	6.06
6		Pleosporales	Acrocalymmaceae	Acrocalyma sp.	23	16	12	25	76	4.75
7			Corynesporascaceae	Corynespora sp.	14	–	–	–	14	0.87
8			Pleosporaceae	Alternaria sp.	31	41	18	14	104	6.5
9				Bipolaris sp.	24	9	16	37	86	5.37
10				Curvularia sp.	29	20	13	22	84	5.25
11	Eurotiomycetes	Eurotiales	Aspergillaceae	Aspergillus sp.	17	13	9	14	53	3.31	7.31
12				Penicillium sp.	18	13	14	19	64	4
13	Sordariomycetes	Amphisphaeriales	Sporocadaceae	Neopestalotiopsis sp.	37	25	18	28	108	6.75	41.87
14				Pestalotiopsis sp.	25	23	26	33	107	6.68
15		Diaporthales	Diaporthaceae	Diaporthe sp.	38	29	25	46	138	8.62
16		Glomerellales	Glomerellaceae	Colletotrichum sp.	37	18	12	9	76	4.75
17		Hypocreales	Nectriaceae	Campylocarpon sp.	12	–	–	26	38	2.37
18				Fusarium sp.	44	7	17	36	104	6.5
19		Xylariales	Diatrypaceae	Monosporascus sp.	–	–	–	23	23	1.43
20			Xylariaceae	Xylaria sp.	33	17	11	15	76	4.75
21	Unrecogonized	Mycelia sterilia		Morpho sp.	32	31	22	27	112	7	7
Total/Mean					508	337	266	464	1575	4.68	19.68

A phylogenetic tree obtained based on the maximum likelihood method revealed that the isolates can be assembled into various clades. The isolates PJGHROP4145 and PJGHROP1123 were grouped under Nectriaceae with 88% bootstrap support. Although the isolates PJGHLFP454 and PJGHFRP283 initially clustered under the same branch, subsequently divided into two branches indicating that they are two different species. The remaining fungal endophytes assembled into clades of identified sequences with a significant bootstrap rating. The details of GenBank accession number and the percentage of identity of fungal endophytes are given in Tables 2 and 3. The phylogenetic tree showing the evolutionary relationship among the isolated fungal endophytes is depicted in Figs. 3 and 4.

Table 2.

Molecular identification of fungal endophytes from Grewia hirsuta based on ITS rDNA sequences

Sl. no.	Isolate code	Fungi	Genbank accession no.	Closest match	Identity (%)
1	PJGHFRP475	Acrocalyma vagum	OL304940	Acrocalymma vagum MT299683	95.15
2	PJGHLFP319	Aspergillus nomius	OL539331	Aspergillus nomiae LC638671	99.10
3	PJGHROP1123	Campylocarpon fasciculare	OL539373	Campylocarpon fasciculare MK330702	99.07
4	PJGHLFP5181	Corynespora cassiicola	OL304972	Corynespora cassiicola MK571389	99.61
5	PJGHFRP683	Diaporthe miriciae	OL305015	Diaporthe miriciae MH220211	99.41
6	PJGHLFP454	Diaporthe sp.	OL533645	Diaporthe sp. GU066697	99.62
7	PJGHROP3145	Fusarium sp.	OL533642	Fusarium solani MG775558	99.25
8	PJGHROP4124	Fusarium foetens	OL305021	Fusarium foetens MT406749	100.00
9	PJGHLFP325	Lasidiplodia sp.	OL304939	Lasiodiplodia pseudotheobromae MT332314	100.00
10	PJGHSTP6137	Macrophomina sp.	OL687536	Macrophomina phaseolina MT127373	90.54
11	PJGHROP8125	Monosporascus sp.	OL305682	Monosporascus caatinguensis MG735227	97.87
12	PJGHLFP9193	Neopestalotiopsis sp.	OL305057	Neopestalotiopsis sp. MT626602	100.00
13	PJGHROP7188	Pestalotiopsis sp.	OL305055	Pestalotiopsis oryzae KM199304	100.00
14	PJGHSTP1130	Phanerochaete sp.	OL305684	Phanerochaete sp. MT084047	97.83
15	PJGHROP3171	Porostereum sp.	OL305685	Porostereum fulvum MW081306	97.45
16	PJGHROP3160	Xylariaceae sp	OL305688	Xylaria sp. HM044132	99.22

Table 3.

Molecular identification of fungal endophytes from Grewia hirsuta based on LSU rDNA sequences

Sl. no.	Isolate code	Fungi	Genbank accession no.	Closest match	Identity (%)
1	PJGHFRP475	Acrocalyma vagum	OL687556	Acrocalymma vagum MT299683	95.15
2	PJGHLFP319	Aspergillus nomius	OL687557	Aspergillus nomiae MZ664040	100.00
3	PJGHROP1123	Campylocarpon fasciculare	OL687562	Campylocarpon fasciculare HM364313	100.00
4	PJGHLFP5181	Corynespora cassiicola	OL687563	Corynespora cassiicola MH869486	99.20
5	PJGHFRP683	Diaporthe miriciae	OL687918	Diaporthe ampelina MK749854	97.54
6	PJGHLFP454	Diaporthe sp.	OL687919	Diaporthe eres MT378367	98.34
7	PJGHROP3145	Fusarium sp.	OL688388	Fusarium solani MW811461	96.25
8	PJGHROP4124	Fusarium foetens	OL687921	Fusarium foetens NG069868	99.54
9	PJGHLFP325	Lasidiplodia sp.	OL688340	Lasiodiplodia theobromae MN181372	95.62
10	PJGHSTP6137	Macrophomina sp.	OL688472	Macrophomina phaseolina MK968808	97.54
11	PJGHROP8125	Monosporascus sp.	OL688616	Monosporascus eutypoides MH877468	97.92
12	PJGHLFP9193	Neopestalotiopsis sp.	OL688617	Neopestalotiopsis sp. OK087303	97.47
13	PJGHROP7188	Pestalotiopsis sp.	OL688619	Pestalotiopsis sp. MH398533	98.64
14	PJGHSTP1130	Phanerochaete sp.	OL688634	Phanerochaete velutina MT248162	93.83
15	PJGHROP3171	Porostereum sp.	OL944438	Porostereum sp. KY440768	91.86
16	PJGHROP3160	Xylariaceae sp	OL688663	Xylaria longipes MK408619	96.59

Fig. 3.

Phylogenetic tree of fungal endophytes from Grewia hirsuta based on ITS rDNA sequences. The isolates obtained in this study are highlighted in bold.

Fig. 4.

Phylogenetic tree of fungal endophytes from Grewia hirsuta based on LSU rDNA sequences. The isolates obtained in this study are highlighted in bold.

Data analyses of fungal endophytes

Colonization frequency of fungal endophytes varied from one plant part to another with leaf segments harboring 32.25% of total endophytic fungi. The isolation rate for leaves was higher when compared to other plant parts as most of the fungal endophytes were recovered from leaf segments. The diversity of the isolated fungal endophytes, as per the Shannon-Wiener index (H), ranged from 2.74 to 2.88. Leaf and fruit had higher Shannon-wiener indexes whereas low indices were recorded for stem and root parts. All the plant parts showed similar Simpson’s index values, indicating a uniform species diversity. The tissue-specificity was higher in the leaves and root indicating high species richness when compared to stem and fruit parts. Species evenness was slightly lower in stem than in leaves, roots, and fruits. Diversity indices of isolated fungal endophytes are given in Table 4.

Table 4.

Diversity indices of fungal endophytes from Grewia hirsuta

	Leaf	Stem	Root	Fruit	Total
No. of segments	400	400	400	400	1600
No. of segments yielding endophytic fungi	334	267	302	211	1114
No. of isolates	508	337	464	266	1575
Isolation rate (%)	127	84.25	116	66.75	98.43
Colonization frequency (%)	83.5	66.75	75.5	52.75	69.62
Shannon-Wiener diversity index (H)	2.88	2.74	2.87	2.78	2.93
Species richness (S)	19.0	17.0	19.0	17.0	21.0
Simpson diversity index: 1-D	0.94	0.93	0.94	0.93	0.94
Evenness	0.97	0.96	0.97	0.98	0.96

Screening of fungal endophytes for L-asparaginase production

Representative fungal endophytes were screened for extracellular production of L-asparaginase by rapid qualitative plate assay. Among the sixty-three fungal endophytes screened, thirty-two were identified as L-asparaginase positive isolates. These positive isolates exhibited a pink zone encircling the colonies on MCD agar medium suggesting that they had the ability to use the substrate (L-asparagine) by producing L-asparaginase that catalyzes the breakdown of the L-asparagine into aspartic acid and ammonia, thus converting the media color from yellow to pink. Among the total isolates screened, the percentage of L-asparaginase producing endophytic fungi was 48.48%. The enzyme index varied from 1.15 to 6.07 mm (Table 5). Campylocarpon sp. designated as PJGHROP1123 showed a maximum enzyme index of 6.07. This was followed by Fusarium sp. (5.31). All three species of Fusarium, Aspergillus, and Campylocarpon screened were able to produce L-asparaginase. Thirty-one isolates were negative for L-asparaginase production. All the isolates of Acrocalyma sp., Alternaria sp., Curvularia sp., Phanerochaete sp., Porostereum sp., and Monosporascus sp. did not produce L-asparaginase. Fifteen isolates showing prominent enzyme index were selected for secondary screening.

Table 5.

Enzyme index of selected fungal endophytes from Grewia hirsuta. Significant differences between enzyme activities were determined by one-way ANOVA and the Tukey test. Means without a common superscript letter are significantly different (p <0.05)

Sl. no.	Fungi	Isolate code	Enzyme index (mm)	Sl. no.	Fungi	Isolate code	Enzyme index (mm)
1	Campylocarpon fasciculare	PJGHROP1123	6.07 ± 0.011a	17	Penicillium sp.	PJGHFRP332	3.14 ± 0.000 k
2	Fusarium foetens	PJGHROP4124	5.31 ± 0.000 b	18	Campylocarpon sp.	PJGHROP6786	3.13 ± 0.011 k
3	Aspergillus nomius	PJGHLFP319	5.00 ± 0.005 c	19	Colletotrichum sp.	PJGHROP2974	2.92 ± 0.005 l
4	Colletotrichum sp.	PJGHFRP71501	4.72 ± 0.011 d	20	Neopestalotiopsis sp.	PJGHLFP3156	2.82 ± 0.011m
5	Fusarium sp.	PJGHROP3145	4.46 ± 0.011 e	21	Corynespora sp.	PJGHFRP7198	2.07 ± 0.000 n
6	Penicillium sp.	PJGHFRP8921	4.25 ± 0.000 f	22	Xylariaceae sp.	PJGHROP3160	2.00 ± 0.005 o
7	Corynespora cassiicola	PJGHLFP5181	3.86 ± 0.000 g	23	Aspergillus sp.	PJGHFRP51346	1.73 ± 0.010 p
8	Aspergillus sp.	PJGHROP1543	3.85 ± 0.005 g	24	Xylariaceae sp.	PJGHLFP6325	1.50 ± 0.011 q
9	Morpho sp.	PJGHSTP8762	3.85 ± 0.005 g	25	Pestalotiopsis sp.	PJGHROP7188	1.31 ± 0.011 r
10	Morpho sp.	PJGHLFP91121	3.84 ± 0.011 g	26	Bipolaris sp.	PJGHSTP91412	1.30 ± 0.000 r
11	Diaporthe sp.	PJGHLFP454	3.68 ± 0.005 h	27	Campylocarpon sp.	PJGHLFP21160	1.24 ± 0.011 s
12	Penicillium sp.	PJGHROP5837	3.67 ± 0.011 h	28	Diaporthe miriciae	PJGHFRP683	1.22 ± 0.005 s
13	Corynespora sp.	PJGHLFP7643	3.40 ± 0.005 i	29	Diaporthe sp.	PJGHSTP9231	1.18 ± 0.005 t
14	Fusarium sp.	PJGHSTP61232	3.26 ± 0.000 j	30	Macrophomina sp.	PJGHFRP71225	1.17 ± 0.005 tu
15	Morpho sp.	PJGHLFP1275	3.25 ± 0.005 j	31	Cladosporium sp.	PJGHFRP273	1.16 ± 0.000 tu
16	Colletotrichum sp.	PJGHLFP21421	3.25 ± 0.017 j	32	Lasidiplodia sp.	PJGHLFP4119	1.15 ± 0.011 u

Table 5 Enzyme index of selected fungal endophytes from Grewia hirsuta. Significant differences between enzyme activities were determined by one-way ANOVA and the Tukey test. Means without a common superscript letter are significantly different ( < 0.05)

Estimation of L-asparaginase activities in selected fungal endophytes

The amount of ammonia in the reaction mixture is indicated by the development of a yellowish-orange colored solution. The L-asparaginase activities of fungal endophytes were found to be in the range of 4.65–0.27 IU/mL (Table 6). Three of these were found to have relatively high L-asparaginase activity. Fusarium foetens designated as PJGHLFP319 showed the highest enzyme activity with mean asparaginase activity of 4.65 IU/mL. This was followed by Aspergillus nomius and Penicillium sp. Corynespora cassiicola, and Corynespora fasciculare showed average enzyme activity, while Cladosporium sp. exhibited the least enzyme activity of 0.27 IU/mL.

Table 6.

L-Asparaginase activities of selected fungal endophytes form Grewia hirsuta. Significant differences between enzyme activities were determined by one-way ANOVA and the Tukey test. Means without a common superscript letter are significantly different (p < 0.05)

Sl. no.	Fungi	Isolate code	Enzyme activity (IU/mL)
1	Fusarium foetens	PJGHROP4124	4.65 ± 0.121a
2	Aspergillus nomius	PJGHLFP319	4.30 ± 0.127b
3	Penicillium sp.	PJGHFRP8921	4.23 ± 0.127b
4	Campylocarpon fasciculare	PJGHROP1123	2.72 ± 0.000c
5	Corynespora cassiicola	PJGHLFP5181	2.42 ± 0.132cd
6	Fusarium sp.	PJGHROP3145	2.34 ± 0.132d
7	Aspergillus sp.	PJGHROP1543	1.73 ± 0.121e
8	Morpho sp.	PJGHLFP1275	1.59 ± 0.121fg
9	Penicillium sp.	PJGHROP5837	1.31 ± 0.115gh
10	Colletotrichum sp.	PJGHFRP71501	1.25 ± 0.000hi
11	Morpho sp.	PJGHLFP91121	0.97 ± 0.121ij
12	Morpho sp.	PJGHSTP8762	0.76 ± 0.121jk
13	Corynespora sp.	PJGHLFP7643	0.62 ± 0.000k
14	Fusarium sp.	PJGHSTP61232	0.55 ± 0.121kl
15	Diaporthe sp.	PJGHLFP454	0.27 ± 0.121l

Discussion

Many studies have shown that medicinal plants are associated with a high diversity of endophytic fungi [67-69]. These fungi can modulate the morphological and physiological activities of host plants through different processes [70]. They are present in almost every tissue studied [71] and the host-endophyte interaction has always given rise to the production of several novel bioactive metabolites [63]. In the present study, Grewia hirsuta, a medicinal plant was harnessed for the isolation of fungal endophytes and these fungal endophytes were assessed for their ability to produce L-asparaginase, an essential drug used in anti-leukemic therapy.

A total of 1575 fungal endophytes belonging to four classes, Agaricomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, were isolated from different tissues of Grewia hirsuta. Most of the fungal endophytes belonged to Ascomycota. It is noted that Ascomycota comprises around 8% of the Earth’s land and is amongst the most dominant and widespread phyla of eukaryotes [72, 73]. Among Ascomycota, Sordariomycetes was the predominant class, which included Neopestalotiopsis sp., Pestalotiopsis sp., Diaporthe sp., Colletotrichum sp., Campylocarpon sp., Fusarium sp., Monosporascus sp., and Xylaria sp. This substantiates earlier studies carried out, in which endophytic Sordariomycetes such as Colletotrichum sp., Xylaria sp., Glomerella sp., and Nodulisporium sp. were obtained from Huperzia serrata by Chen et al. [74]. However, in a study carried out by Shipunov et al. [75], most of the fungal endophytes from Catharanthus roseus belonged to the class Dothideomycetes. The other two genera belonged to Basidiomycota. Fungal endophytes from this phylum are reported to be dependent on the method of culture [76]; this corroborates the less number of isolates belonging to Basidiomycota in the present study.

Most of the fungal endophytes were recovered from the leaves in comparison to other plant parts. The difference in endophytic fungal colonization could be correlated to both biotic and abiotic factors. Further, the endophytic fungal composition is affected by plant physiology [77]. The lowest number of fungal endophytes were obtained from the fruit segments. This may be due to the short lifetime of fruit compared to roots, stems, and leaves [78]. In addition, the difference in the area of plant collection might be a probable cause for the difference in the isolated endophytic fungal community [79]. The fungal endophytes occupied different plant parts to varying degrees. This variation in colonizing ability of the fungal endophytes confirms that each fungal endophyte has a different range of associations for different parts of the plant. The total colonization frequency of leaves was above 100% as more than one fungal endophyte originated from some leaf segments indicating a higher density of colonization in the leaves. Earlier research has also shown high colonization frequency in leaf tissues [67, 80, 81]. In contrast, Gautam et al. reported higher endophytic fungal colonization in stem tissues [82]. Leaves are ideal for endophyte colonization as they have a higher surface area, high nutrient content, and their thin-walled cells make endophyte colonization easy [77, 83]. Many factors such as moisture and age of the host plant affect endophyte colonization. Moisture increases the dispersal of endophytes and their colonization [84], while older plant parts have sizeable organs to occupy more endophytes [85]. Further, it has been stated that the host species plays a vital role in determining the endophyte community structure [86, 87].

Diaporthe sp. was the predominant genus isolated with a colonization frequency of 8.62%. Thus, the host has a strong affinity to establish a symbiotic association with the fungi belonging to the genus Diaporthe. The genera are reported as the most commonly occurring fungal endophytes in previous findings from the leaves of Schinus terebinthifolius [88]. They are generally considered multi-host fungal endophytes as they are found to occur frequently in varied tropical tree species of diverse environmental locations [89]. Most of the genera inhabited more than one plant part which suggests the ability of fungal endophytes to enter from one part to another part of a plant [48]. PCA indicated that there is an increased incidence and frequency by some species on specific tissues. Isolates belonging to the genera such as Porostereum sp., Corynespora sp., and Monosporascus sp. were isolated respectively from fruit, leaf, and root only. Much research has shown that endophytic fungal colonization of a few genera tends to be restricted to specific tissues [90-92].

The Shannon-Wiener and Simpson diversity indices exhibited minor variations among different tissues. This variation in species diversity is due to the difference in the number of isolates obtained, frequency of isolation, and species richness. Generally, if the Shannon-Wiener diversity index is greater and the Simpson’s diversity index closer to 1, there is more inherited variation and a higher adaptive ability for environmental changes [93]. The results showed that the fungal endophyte isolated in the study has a high heritable variation and a more adaptive ability for environmental changes as the values of the Shannon-Wiener diversity index was in the range of 2.74 to 2.93 and the Simpson diversity index was between 0.96 and 0.98. However, in a study conducted by Uzma et al., higher Shannon-Wiener diversity index was observed in the stem and there was no considerable variation in the values of Simpson’s diversity index [94].

Studies on L-asparaginase production from fungi are very limited, especially from endophytic fungi. Kumar et al., isolated thirty-eight L-asparaginase producing fungal endophytes from Ocimum tenuiflorum [66]. Thirteen isolates showing L-asparaginase activity were recovered from Myracrodruon urundeuva by Paduva et al. [95]. In the present study, a total of thirty-two, that is 48.48% of the total isolates could produce L-asparaginase. Fungal endophytes positive for L-asparaginase production with significant enzyme activity were mostly from the genus Fusarium. Some studies have emphasized that species of Fusarium have a great potential to produce many important bioactive compounds [96], along with anticancer drugs [97, 98]. Chow and Ting [49] screened fungal endophytes isolated from Murraya koenigii for L-asparaginase production and showed that Fusarium species were the predominant genera to produce L-asparaginase. Other genera, such as Aspergillus and Camylocarpon also showed great potential to produce L-asparaginase. L-asparaginase production by several Aspergillus sp. is reported in previous literature [99-101].

In the quantitative assay for extracellular L-asparaginase production, Fusarium foetens showed the highest enzyme activity of 4.65 IU/mL. This is greater than the earlier report of enzyme activities from endophytic Fusarium oxysporum [49] and Pleospora allii [102]. Although Campylocarpon fasciculare showed the highest enzyme index in the agar plate assay, enzyme activity was low in the quantitative analysis. This may be due to variations in the capability of the fungal endophyte to produce L-asparaginase in solid and liquid media [103]. Penicillium sp. also showed high L-asparaginase activity of 4.23 IU/mL. A previous study reported Penicillium sp. as a great source of L-asparaginase [104].

The importance of the enzyme makes it very necessary to isolate novel sources for L-asparaginase production. Thus, a new fungal source of L-asparaginase with high productivity can contribute for enhanced application in the medicinal field. Fusarium foetens obtained in the present study showed great potential to produce L-asparaginase. Thus, it could be a potent candidate for the bioprocessing of L-asparaginase at a large scale. Further studies on optimization of culture conditions could be effective in improving L-asparaginase production by Fusarium foetens. Accordingly, statistical methods have been proven to be very effective for optimizing the production of L-asparaginase from different sources. Moreover, new modifications at the molecular level through enzyme engineering technology can lead to the development of L-asparaginase biobetter.

Conclusion

The present work provides understandings on the diversity of fungal endophytes from medicinal plant Grewia hirsuta and ability of these endophytic fungi to produce anticancer L-asparaginase. The study is the first report on community composition, species distribution, and L-asparaginase production by fungal endophytes associated with Grewia hirsuta. The results have clearly shown that the study plant has great potential to host diversified endophytes that are capable of producing L-asparaginase. Fungal endophyte Fusarium foetens obtained in the study showed prominent enzyme activity, thus indicating it could be a potent source for L-asparaginase production. Further, optimizing the production parameters could aid in maximizing L-asparaginase production. However, purification and characterization of the enzyme along with evaluation of its cytotoxicity is required to establish it as a potent anticancer agent for efficient applications in the clinical field.

Funding

The study was funded by CSIR (Council of Scientific and Industrial Research) under Junior Research Fellowship (File No:09/119(0218)/2019-EMR-I).

Declarations

Conflict of interest

The authors declare no competing interests.