1. Introduction
Almost all land plants are inhabited by endophytic microorganisms, including fungi [
1]. Endophytes are microbes that colonize inner plant tissues without causing any apparent symptoms of disease, and in return for nutrients and habitat, some can confer beneficial traits to the host plant, which include growth promotion, tolerance to biotic and abiotic stress, and biological control of phytopathogens [
2,
3,
4]. Endophytic fungi have been identified for their potential to synthesize a wide variety of biologically active compounds, including the same or similar compounds for which the host plant is recognized [
5,
6,
7]. For example,
Taxomyces andreanae, an endophytic fungus, and its host,
Taxus brevifolia (Pacific yew tree), are both reported to produce paclitaxel (Taxol
®), an anticancer compound [
8]. Similarly, the anticancer drug camptothecin, the anticancer drug lead compound podophyllotoxin, and the natural insecticide azadirachtin are co-produced by the plants and their associated endophytic fungi [
9,
10,
11,
12].
In addition to being rich sources of bioactive compounds, native medicinal plants also host unique endophytic fungi with potential applications in treating infectious diseases, as biocontrol agents, and for several other applications, thus harnessing the potential of endophytic fungi in medicinal plants is important [
13].
Pseudowintera colorata (horopito) is a primitive endemic medicinal shrub that grows in the sub-alpine regions of New Zealand and has been an integral part of traditional Māori medicine (Rongoā). The leaves of
P. colorata have been used as a remedy for fever, toothache, skin infections, and gonorrhea, and contain two sesquiterpene dialdehydes, polygodial and 9-deoxymuzigadial [
14,
15,
16]. Polygodial has been reported to possess potent antifungal and antibacterial properties [
17,
18]. Previous studies on
P. colorata have assessed the community structure and functional potential of endophytic bacteria and actinobacteria, but did not investigate endophytic fungi, which may confer unique characteristics to the host with a key influence on plant growth, development, and disease resistance [
19,
20]. For example, the endophytic fungus
Piriformospora indica when colonizing the roots of
Prosopis juliflora (mesquite) and
Zizyphus mummularia conferred biotic (resistance against root pathogens) and abiotic resistance (salt stress) to its plant hosts [
21,
22]. Although there is increasing evidence highlighting the importance of endophytic fungi from medicinal plants, there is a significant knowledge gap regarding the endophytic fungi in native New Zealand medicinal plants.
While the use of medicinal plants as a source of biologically active compounds has been documented by ancient agricultural societies, the diversity of the endophytic fungi among the medicinal plants is poorly understood [
23,
24]. Endophytic fungi can directly or indirectly influence plant growth and productivity and, as with international research, the endophytic fungi of
P. colorata may function in enhancing the growth of the host plant and protect against phytopathogens. For example, the colonization of maize plants by the endophytic fungus
P. indica led to increased growth and systemic resistance to the root pathogen
Fusarium verticilloides by enhancing antioxidant defenses within the host plant [
25]. Other research has demonstrated that endophytic fungi promote the growth of plants by secreting plant growth regulators, enhancing hyphal growth and mycorrhizal colonization, by producing siderophores, and fixing nitrogen [
26,
27]. A study on the Indian medicinal plants
Withania somnifera and
Spilanthes calva demonstrated that inoculation with
P. indica significantly increased the growth and yield [
28]. Further research has elucidated that this molecular mechanism, via the activation of specific kinase proteins (MAPK3/6), is involved in plant growth [
29]. The leaves of
P. colorata are used for the extraction of polygodial to manufacture Kolorex
®, a treatment for candidiasis. However, as
P. colorata is a very slow growing plant, the endophytic fungal inoculants may offer a solution to promote the growth of
P. colorata, which could have a positive impact on the ecology of the host and thus offer a sustainable solution to the industry as such. As the first study investigating the importance of endophytic fungi in
P. colorata, this study has implications for future work on the ecology and biocontrol potential of endophytic fungi in native medicinal plants.
2. Materials and Methods
2.1. Sample Collection and Processing
Pseudowintera colorata plants were sampled between March and August 2014 from ten distinct sites across New Zealand, and a total of 87 individual plants (leaves, stems, and roots) were collected and processed as described previously (
Table 1,
Figure 1) [
19]. Leaves sampled were fully open, mature, and free of herbivory or disease damage. Woody stems and lateral branches, including green succulent growth, were selected and cut using sterile secateurs. Roots were collected by excavating soil close to the
P. colorata plant being sampled, and putative lateral roots along with root hair were traced back to the
P. colorata plant and cut using sterile secateurs. To check the effectiveness of the sterilization process, leaves were imprinted onto synthetic nutrient deficient agar (SNA, SIFIN, Berlin, Germany) and R2A (Difco laboratories, Detroit, MI, USA) plates, and an aliquot of the final rinse water was plated onto SNA and R2A agar prior to sectioning the tissues. The plates were incubated at 25 °C for 24–48 h, and the absence of growth on plates indicated that surface sterilization was successful. The surface-sterilized tissues were then cut into 1-mm portions and plated onto SNA amended with ampicillin (100 µg/mL) to isolate fungi selectively. The plates were incubated at 20 °C for 5–7 d in 12 h light/12 h dark cycle, and the emerging mycelium was transferred to sterile potato dextrose agar (PDA, Difco laboratories, Detroit, MI, USA). Portions of each sterilized plant organ were stored in sterile glycerol at −80 °C to extract DNA for DGGE analysis.
2.2. Diversity Analysis of the Endophytic Fungi in P. colorata Using DGGE
Before extracting DNA, surface-sterilized
P. colorata organs were treated with 1.25 µL of 20 mM propidium monoazide (PMA, Biotium, Fremont, CA, USA) to avoid amplification of epiphytic DNA by PCR [
20,
30]. DNA was extracted using a CTAB method and amplified using a nested PCR approach with group-specific primers AU2-AU4 and FF390-FR1GC [
31,
32]. The amplified PCR products were separated in 8% (
w/
v) polyacrylamide gel (acrylamide/bis solution, 37.5:1) with a linear gradient of 25–55% denaturant using a Cipher DGGE Electrophoresis system (CBS Scientific, Del Mar, CA, USA) [
33]. Phoretix 1D Pro Gel Analysis (Totallab, Newcastle upon Tyne, UK) and Primer version 7 (Primer-E Ltd., Plymouth Marine Laboratory, Plymouth, UK) were used to analyze the endophytic fungal communities as previously described [
34]. Each band in the DGGE gel was considered as one fungal taxon. Fungal diversity was assessed based on the presence or absence of the same bands in different samples in DGGE gels. At the same time, fungal richness was assessed based on the number of bands per lane in the DGGE gel.
2.3. Isolation and Identification of Cultured Endophytic Fungi
Emerging fungal hyphae from
P. colorata tissue sections plated on SNA plates were sub-cultured onto PDA plates by hyphal tipping using a sterile needle. The DNA for each isolate was extracted using the PureGene kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions, and the ITS gene was amplified using the primer pair ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [
35]. The PCR-amplified ITS regions were sequenced, and the sequences were trimmed using DNAMAN v4 (Lynnon Biosoft, San Ramon, CA, USA). The final sequences were compared against known sequences in NCBI BLAST and were deposited in the GenBank database. Sequence alignment was performed using MUSCLE, distance matrices, and phylogenetic trees were calculated by neighbor-joining algorithms with 1000 bootstrap replication in Geneious prime software (Biomatters, Auckland, New Zealand).
2.4. Functional Activity of Endophytic Fungi Isolated from P. colorata
2.4.1. Activity against Phytopathogenic Fungi
For the functionality testing, the endophytic fungi (
n = 50) isolated from
P. colorata were screened against phytopathogenic fungi
Neofusicoccum luteum isolate ICMP 16678,
Neofusicoccum parvum isolate MM562,
Ilyonectria liriodendri isolate WPa1c, and
Neonectria ditissima isolate ICMP 14417.
Neofusicoccum luteum ICMP 16678 and
Neonectria ditissima ICMP 14417 were obtained from the International Collection of Microorganisms from Plants (ICMP, Maanaki Whenua-Landcare Research, Lincoln, New Zealand), and
N. parvum MM562 and
Ilyonectria liriodendri WPa1c were obtained from Lincoln University Plant Microbiology culture collection. Activity against phytopathogenic fungi was tested on a dual culture on Waksman agar (WA) plates [
19]. The antagonistic potential of the endophytic fungi was classified based on the zone of inhibition where high activity (zone of inhibition > 10 mm), moderate activity (zone of inhibition < 10 mm but >/=5 mm) and low activity (zone of inhibition < 5 mm but >2 mm).
2.4.2. Activity against Opportunistic Human Pathogens
To assess the potential of endophytic fungi to produce antimicrobial compounds, potentially including polygodial like the host P. colorata, the endophytic fungi (n = 50) were tested against the opportunistic human pathogens Staphylococcus aureus, Escherichia coli, and Candida albicans in dual culture assays. The test isolates were obtained from the Institute of Environmental Science and Research (ESR, Porirua, New Zealand). GelAir Cellophane (Biorad) membranes were cut with a scalpel using a petri dish lid as a reference. The membranes were autoclaved prior to usage, and each membrane was carefully transferred onto individual WA plates using sterile forceps. The membrane was gently pressed onto the agar using a sterile spreader to ensure that the membrane adhered to the agar. Using a sterile cork borer, a 6 mm plug from the margins of a 3–5 d old test fungal colony was transferred onto the center of the cellophane membrane on the WA plates. The plates were sealed and incubated for 7 d at 25 °C in a 12 h light/12 h dark cycle. After 7 d, the cellophane membranes with the fungal mycelium were carefully lifted using sterile forceps and discarded. Using a sterile spreader, 100 µL of the overnight cultures of S. aureus, E. coli, and C. albicans grown in nutrient broth (NB, Difco laboratories, Detroit, MI, USA) were spread onto their respective test plates. A 100 µL aliquot of S. aureus, E. coli, and C. albicans was plated onto individual Waksman agar plates without any fungi growth on it as controls. The plates were sealed with parafilm and incubated at 25 °C for 24–48 h. The presence of a clear zone of inhibition in the plates was noted as being positive for inhibitory activity, and the results were recorded compared to control plates.
2.5. Influence of Endophytic Fungi on the Growth of P. colorata Seedlings in the Glasshouse
To assess the influence of endophytic fungi on the growth of P. colorata seedlings, selected isolates were applied as root/soil drenches to six-week-old P. colorata seedlings (Southern Woods Plant Nursery, Christchurch, New Zealand). The seedlings lacked a fully developed root system at the time of purchase and were acclimatized in the shade house for 3–4 weeks in February 2017 before the experiment. For inoculation, the 5–7 d old culture was grown on PDA, and when the fungal mycelia covered more than half of the agar surface, 1–2 mL SDW (sterile distilled water) was added to the plates. Using a sterile glass spreader, the spores were dislodged and the spore suspension decanted into a 50 mL tube. The spore concentration of the suspension was determined using a hemocytometer and adjusted to 1 × 105 spores/mL in a total volume of 1 L of SDW in sterile aluminum trays (270 mm W × 95 mm H × 325 mm L). Prior to inoculation with the test fungal cultures, the seedlings were not watered for 24–48 h, and the length of the shoots was measured using a digital caliper. For treatment with the fungal spore suspensions, the P. colorata seedlings with their soil-plugs intact were carefully soaked in the spore suspensions overnight in the aluminum trays, with the trays being covered with cling wrap overlaid with aluminum foil to avoid evaporation and cross-contamination. The following day, the seedlings with the soil-plugs were repotted into 1 L pots containing a potting mix medium of [20% pumice, 80% composted bark, 2 kg/m3 Osmocote® standard 3–4 months gradual release fertilizer (NPK 16-3.5-10 plus trace elements)], 1 kg/m3 agricultural lime, and 500 g/m3 Hydraflo® 2 (granular wetting agent, Scott Australia Pty Ltd., Auckland, New Zealand). Each treatment was replicated 10 times, and 10 uninoculated control P. colorata seedlings soaked in SDW were also set up. The trial design in a randomized complete block design and the plants were watered once daily and regularly observed to see if there were any dead or diseased plants following the treatments. The plants were grown under a 12 h daylight and 12 h dark regime using an HPS lighting system (high pressure sodium) at 100 lumens light intensity.
After 3 months of growth (March 2017 to May 2017), the potting mix around the root region of each treatment of P. colorata seedlings was reinoculated by drenching with 50 mL of freshly prepared spore suspensions (1 × 105 spores/mL) of their respective treatments. Four weeks after the second inoculation (June 2017), the seedlings were destructively harvested. At harvest, the shoot height was measured from the stem base (at the soil level) to the top leaf using a ruler. The number of internodes was measured for each plant stopping at the top two leaves. The difference in heights pre-treatment (X) and post-treatment (Y) was calculated (Y − X). The shoot and root portions were weighed after drying in an oven at 60 °C for 2 d. The data were analyzed using a general analysis of variance (ANOVA). Fisher’s protected least significant difference (LSD) was used to test the mean difference between shoot lengths, shoot weights, root weights, and the number of internodes of treated plants with untreated controls. The analyses were performed in Minitab 17 (Minitab LLC, State College, PA, USA).
2.6. Confirmation of Endophytic Colonization Using DGGE
To confirm whether the endophytic fungi were able to colonize the roots of P. colorata seedlings, after harvest a small section of the roots from treated and untreated control P. colorata seedlings were surface-sterilized as previously described and plated on PDA amended with ampicillin (100 µg/mL) to re-isolate the inoculated fungi. In addition, small sections of the roots were set aside for DGGE. PCRs and DGGE were performed as described previously. Pure DNA of endophytic fungal isolates used for inoculation experiments were also amplified using the same primer pairs and were used as reference markers in DGGE gels. The presence of the corresponding band in treatments indicated successful colonization by the endophytic fungi.
4. Discussion
This study analyzed the diversity and functional potential of endophytic fungi in a primitive and important native New Zealand medicinal plant. It was the first study to comprehensively examine the community structure and diversity of endophytic fungi in
P. colorata using a combination of culture-dependent and culture-independent techniques. The molecular tool DGGE, targeting the ITS subunit as a taxonomic marker, was used to analyze the structure and diversity of the endophytic fungal communities in
P. colorata tissues [
36]. Plant organ type was the main factor influencing the composition and richness of endophytic fungi in
P. colorata and indicated that it is an overriding factor in the formation of endophytic fungal communities in
P. colorata tissues. Similar findings were reported in other studies where tissue type primarily influenced the diversity and community structure of endophytic fungi [
34,
37].
Interactions between organ type and plant age of
P. colorata influenced the diversity of endophytic fungal communities. The endophytic fungal communities in the roots, stems, and leaves of young
P. colorata plants grouped together, while those in older plants were more diverse, suggesting a shift in the fungal communities as the plants matured. These results are consistent with a study on
Pinus taeda, where a difference in richness and diversity of the endophyte community between seedlings and adult plants was observed [
38]. Chemotype variability in
P. colorata was demonstrated, which could account for the differences in the endophytic fungal communities across various locations [
39]. In this study, all the tissues of
P. colorata sampled (root, stem, and leaves) hosted at least one culturable endophytic fungus. These results support the theory that all individual plants on earth are colonized by one or more endophytes [
3]. In this study, the fungal taxa in the stems was higher than in the roots and leaves. Similar results were reported from the Indian medicinal plant
Madhuca indica, where a greater diversity of fungi was observed in stems [
40]. However, a similar study on the Chinese medicinal plant
Stellera chamaejasmae reported a higher number of endophytic fungi in roots compared to above ground tissues (stems and leaves) [
41]. This difference in fungal communities in
P. colorata can be attributed to some extent to the presence of antifungal compounds in the leaves and stems, which may place high selection pressure on the fungal communities and requires further investigation.
The bioactive potential of a randomly selected representative (
n = 50) endophytic fungi isolated from
P. colorata was tested against phytopathogenic fungi as well as opportunistic human pathogens, including the yeast
C. albicans, the target of polygodial. In this study, several endophytic fungi demonstrated very strong antagonistic activity against phytopathogenic fungi such as
Neofusicoccum luteum,
N. parvum, and
Neonectria ditissima, and also against opportunistic human pathogens such as
S. aureus and
C. albicans. Sequencing the ITS2 subunit revealed that the bioactive fungi belong to the genera
Trichoderma,
Pezicula,
Fusarium,
Metarhizium,
Chaetomium, and
Xylariaceae sp. Several studies have demonstrated the potential of endophytic
Trichoderma sp. and
Chaetomium sp. as biocontrol agents against phytopathogenic fungi such as
Fusarium solani and
Phytophthora nicotianae [
42,
43].
Of the strains tested, 18% (
n = 9) of isolates showed high activity (zone of inhibition > 10 mm) against
C. albicans. Similar results were reported for the Brazilian medicinal plant
Bauhinia forficate, where 34.3% of the endophytic fungi isolates showed activity against
S. aureus,
E. coli, and
Streptococcus pyogenes [
7]. In addition, endophytic fungal isolates from
B. forficata showed activity against pathogens such as
Aspergillus,
Cladosporium,
Cryptococcus,
Candida, and
Salmonella [
44]. In this study, the fungi identified as belonging to the genus
Pezicula exhibited strong activity against all the phytopathogenic fungi tested and the yeast
C. albicans strain 3395. Previous studies have reported that certain species of
Pezicula produce one or more lipopeptide antimycotics known as pneumocandins, and several other compounds such as (R)-Mellein Echinocandin A, which are recognized for their activity against
C. albicans,
S. aureus, and
Ustilago violacea [
45,
46].
This study is the first to demonstrate that endophytic fungi isolated from
P. colorata can positively influence the growth of
P. colorata seedlings. Treating
P. colorata seedlings with endophytic fungi resulted in increased plant heights and quantity of internodes for six and five treatments, respectively, compared to the control. These results are consistent with similar research where inoculation of the root zone of 4-week saplings of
Boswellia sacra with the endophytic fungus
Preussia sp. BSL 10 increased shoot length and internodes compared to the untreated control [
47].
P. colorata seedling treated with
Trichoderma sp. PRY2BA21 increased the height of seedlings but did not affect the root and shoot dry weight. Similar results were reported in
Miscanthus x
giganteus, where shoot lengths of plants treated with an endophytic
Trichoderma sp. were longer than the controls but were not different in terms of root and shoot biomass [
48]. The increased plant growth in similar research following inoculation with endophytic fungi has been attributed to the production of growth hormones such as auxins, which are produced by several fungi including
Trichoderma sp., and transportation of nutrients by organic matter mineralization [
49,
50,
51]. However, in this study the exact mechanisms responsible for the increase in plant growth were not studied.
Although endophytic colonization was not confirmed by re-isolation for any of the endophytic inoculants, pure cultures of the fungal inoculants were included as reference markers in DGGE gels along with the treatments and compared to uninoculated control to check for the presence or absence of the marker bands. Out of seven endophytic fungal inoculants used in this study, marker bands of three isolates viz.
Metarhizium sp. PR1SB1,
Xylariaceae sp. P4LA3, and
Fusarium sp. P4LC2 were detected in their respective treatments. Whereas the marker bands of
Trichoderma sp. PRY2BA21,
Trichoderma sp. PRY3BC1, and
Chaetomium sp. PR1BC2 were not detected in DGGE. As these isolates were isolated from mature
P. colorata plants, this variation in the age may have also affected successful colonization and re-isolation in this case. Although some of the isolates did colonize the plants, they still affected the growth of
P. colorata seedlings. These findings indicated that the effect of these treatments could be due to increased nutrient availability in the root zone, especially as these fungi are also commonly known to exist as free-living saprophytes or associated with the rhizosphere [
52]. However, the precise mechanism of how these endophytic fungi improve the growth of
P. colorata merits further investigation. The expected outcomes of such future studies will help develop efficient strategies for harnessing the full potential of endophytic fungi, and also advance understanding of the ecology of endemic native medicinal plants.