Fungal Communities in the Native New Zealand Medicinal Plant Pseudowintera colorata (Horopito) Are Determined by Plant Organ Type and Host Maturity with Key Members Promoting Plant Growth

Abstract

The plant Pseudowintera colorata is well known for its antimicrobial and medicinal properties and is endemic to New Zealand. Using PCR-Denaturing gradient gel electrophoresis (DGGE), we investigated the factors influencing the composition of endophytic fungal communities in P. colorata from ten distinct sites across New Zealand. Our results showed that plant organs of P. colorata influenced the diversity and richness of endophytic fungi (PERMANOVA, p < 0.05). In addition, plant maturity and its interactions revealed that endophytic fungal communities formed discrete clusters in leaves, stems, and roots of mature and immature P. colorata plants (PERMANOVA; p = 0.002, p = 0.001 and p = 0.039, respectively). For identifying isolates with biocontrol potential, dual culture tests were set up against four different phytopathogenic fungi. Isolates with high activity (zone of inhibition > 10 mm) were sequenced and identified as Trichoderma harzianum, Pezicula neosporulosa, Fusarium tricinctum, Metarhizium sp., and Chaetomium sp. Applying selected endophytic fungi (n = 7) as soil drenchers significantly increased the growth of P. colorata seedlings and produced more internodes. Seedling shoots treated with Trichoderma sp. PRY2BA21 were 2.2 × longer (8.36 cm) than the untreated controls (3.72 cm). Our results elucidate the main plant factors influencing fungal community composition and demonstrate a role for endophytic fungi in P. colorata growth and further demonstrate that medicinal plants are a rich source of endophytes with potential as biocontrol agents.

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. Sampling information.

Location	Coordinates
Taihape Scenic Reserve (North Island)	−39.67635° S 175.80560° E
Tongariro National Park (North Island)	−39.02237° S 175.71810° E
Kaimanawa Forest Park (North Island)	−38.94721° S 175.94370° E
Lake Rotopounamu Scenic Reserve (North Island)	−39.02656° S 175.73502° E
Kahurangi National Park (South Island)	−41.07224° S 172.59166° E
Paringa Forest (South Island)	−43.69379° S 169.40724° E
Arthur’s Pass National Park (South Island)	−42.94215° S 171.56414° E
Kaituna Valley Scenic Reserve (South Island)	−43.71655° S 172.7554° E
Peel Forest (South Island)	−43.91835° S 171.25934° E
Otago Peninsula Scenic Reserve (South Island)	−45.88184° S 170.58049° E

, 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.

3. Results

3.1. Analysis of the Structure and Richness of Endophytic Fungi Using DGGE

Plant organ, location, and the interaction between the two factors influenced the endophytic fungal communities in P. colorata (PERMANOVA, p ≤ 0.005) (

Table 2. Influence of location and plant organs on the similarity and richness of endophytic fungal communities in Pseudowintera colorata.

Treatment	Microbial Communities Similarity a	Microbial Richness a
Location	0.081	0.095
Plant organ	0.001 **	<0.001 **
Location vs. Plant organ	0.002 **	<0.001 **

^a Asterisk denotes levels of statistical significance of microbial community similarity based on PERMANOVA and microbial richness based on GLM. ** High significant difference (p ≤ 0.005).

). The DGGE patterns grouped the endophytic fungal communities in the stems and roots together, whereas the fungal communities in the leaves were more diverse (Figure 2). The fungal taxa (n) were richer in the stems (n = 18) and roots (n = 15) compared to leaves (n = 5) (LSD, p ≤ 0.005). Plant location did not influence the richness of the fungal communities in P. colorata (Table 2).

3.2. Influence of Plant Age on the Community Structure and Richness of Endophytic Fungi in P. colorata

As the sampling sites chosen for this study were restricted to sites conserved by the New Zealand Department of Conservation (DOC), collecting plants of different ages in the same vicinity (+/−10 m) was not always possible. Thus, for analyzing the influence of plant age on the community structure and richness of endophytic fungi in P. colorata, a subset of three sites (Kaituna Valley Forest Park, Paringa Forest, and Peel Forest) where plants of different maturities were present was selected. For this study, plant height was used as a classifier, and the plants were classified as fully mature/old plants (>3 m) and young plants (≤1 m).

All the factors and their interactions influenced the fungal communities in P. colorata (PERMANOVA, p ≤ 0.005) except for the interaction between location and plant maturity (

Table 3. Influence of location, plant organ, and plant age on the similarity and richness of endophytic fungal communities in Pseudowintera colorata.

Treatment	Microbial Communities Similarity a	Microbial Richness a
Location	0.002 **	0.121
Plant organ	0.001 **	0.015 *
Plant age	0.002 **	0.785
Location vs. Plant organ	0.001 **	<0.001 **
Location vs. Plant age	0.164	0.448
Plant organ vs. Plant age	0.001 **	0.393
Plant organ vs. Location vs. Plant age	0.001 **	0.060

^a Asterisk denotes levels of statistical significance of microbial community similarity based on PERMANOVA and microbial richness based on GLM. * Significantly different (p ≤ 0.05), ** High significant difference (p ≤ 0.005).

). For the combined tissue data, the endophytic fungal communities were not distinguishable between mature and immature plants (Figure 3A). For individual organs, the endophytic fungal communities within the roots, stems, and leaves of the young plants formed discrete clusters (PERMANOVA; p = 0.002, p = 0.001 and p = 0.039, respectively), while the tissues of older plants were more diverse (Figure 3B–D).

3.3. Isolation, Sequencing Data, and Identity of Culturable Endophytic Fungi from P. colorata Tissues

A total of 200 endophytic fungi were isolated from the surface-sterilized P. colorata organs, which included 98 (49%), 80 (40%), and 22 (11%) isolates from leaves, stems, and root sections, respectively. The fungi (n = 200) were grouped into a representative set of 50 morphotypes (26, 18, and 6 from stem, roots, and leaves, respectively) based on their cultural characteristics on PDA, and were identified by sequencing the ITS gene (550–750 bp). The representative set (n = 50) was also used for bioactivity testing in dual culture assays. Following ITS gene sequencing analysis, 47 isolates were categorized to the genus level, whereas two isolates were identified to family level and one to order due to low sequence homology (

Table 4. Identity of culturable endophytic fungi isolated from Pseudowintera colorata based on ITS sequencing.

Location	Plant Organ	NCBI Match	Identity (%)	Classification
Kahurangi Nat. Park	Stem	Acrocalymma vagum strain 29T (1) (KP784427)	100	Acrocalymma sp.
Kaituna valley scenic reserve	Root	Alternaria tenuissima isolate F293.82.1 (MZ678531)	100	Alternaria sp.
Kaituna valley scenic reserve	Root	Chaetomium globosum isolate 1 (FJ791145)	99	Chaetomium sp.
Kaituna valley scenic reserve	Stem	Paraconiothyrium sp. isolate MBD_4091 (MK595563)	98	Paraconiothyrium sp.
Kaituna valley scenic reserve	Stem	Fungal sp. isolate 81 (MT820053)	100	Ascomycota
Kaituna valley scenic reserve	Stem	Aspergillius arcoverdensis strain CBS DTO_316-F9 (KY808749)	100	Aspergillus sp.
Kaituna valley scenic reserve	Root	Aspergillus parvulus culture IBT:22045 (KX423655)	100	Aspergillus sp.
Arthur’s Pass nat. park	Root	Myceliophthora verrucosa (KM527251)	100	Myceliophthora sp.
Arthur’s Pass nat. park	Stem	Chaetomium cupreum (KM357332)	100	Chaetomium sp.
Tongariro nat. park	Root	Chaetomium globosum strain SYP-F8042 (MN960568)	100	Chaetomium sp.
Lake Rotopounamu	Root	Chaetomium globosum isolate UWR_157 (MN654349)	99	Chaetomium sp.
Kaituna valley scenic reserve	Root	Cadophora sp. BESC103j (KC007139)	100	Cadophora sp.
Kahurangi Nat. Park	Stem	Cadophora sp. BESC103j (KC007139)	100	Cadophora sp.
Kahurangi Nat. Park	Stem	Cadophora sp. isolate 500-G (MK163779)	100	Cadophora sp.
Kahurangi Nat. Park	Leaf	Paraconiothyrium variabile isolate BL (KR909137)	99	Paraconiothyrium sp.
Taihape scenic reseve	Root	Paraphoma chrysanthemicola strain HLP7 (MG025864)	100	Paraphoma sp.
Lake Rotopounamu	Stem	Penicillium sp. strain UNIJAG.PL.202 (MT357207)	100	Penicillium sp.
Lake Rotopounamu	Stem	Penicillium brefeldianum strain G26 (MT601953)	100	Penicillium sp.
Lake Rotopounamu	Stem	Penicillium sp. E/As/10/7 (JX238733)	100	Penicillium sp.
Kaimanawa forest park	Stem	Periconia macrospinosa strain ZMXR37 (MT446142)	100	Periconia sp.
Arthur’s Pass nat. park	Stem	Pezicula ericae isolate ARSL_190907.7	100	Pezicula sp.
Arthur’s Pass nat. park	Stem	Pezicula ericae (NR_155653)	99	Pezicula sp.
Taihape scenic reseve	Root	Phoma sp. NRRL 54108 (HM751088)	99	Phoma sp.
Tongariro nat. park	Root	Phomopsis sp. isolate 5(1)b (MT278345)	100	Phomopsis sp.
Kaimanawa forest park	Root	Diaporthe columnaris (MN540315)	99	Phomopsis sp.
Arthur’s Pass nat. park	Root	Fusarium sp. (MH550484)	100	Fusarium sp.
Taihape scenic reseve	Root	Fusarium acuminatum isolate N-51-1 (MT566456)	100	Fusarium sp.
Taihape scenic reseve	Root	Fusarium tricinctum strain ME4 (MK559443)	100	Fusarium sp.
Taihape scenic reseve	Stem	Fusarium solani isolate SY1 (MT605584)	100	Fusarium sp.
Tongariro nat. park	Stem	Fusarium solani isolate N-54-1 (MT560379)	100	Fusarium sp.
Taihape scenic reseve	Leaf	Diplogelasinospora grovesii CBS 340.73 (NR_077164)	94	Diplogelasinospora sp.
Taihape scenic reseve	Root	Diplogelasinospora grovesii CBS 340.73 (NR_077164)	94	Diplogelasinospora sp.
Peel forest	Leaf	Diplogelasinospora grovesii CBS 340.73 (NR_077164)	94	Diplogelasinospora sp.
Peel forest	Stem	Clonostachys sp. isolate RL478 (MT557564)	100	Clonostachys sp.
Peel forest	Stem	Clonostachys rosea isolate SRRB-171 (MT210883)	100	Clonostachys sp.
Peel forest	Stem	Trichoderma sp. (MT557215)	100	Trichoderma sp.
Peel forest	Stem	Trichoderma koningiopsis strain Tk1 (MT111912)	100	Trichoderma sp.
Lake Rotopounamu	Root	Trichoderma viride isolate CTs9 (MK290390)	100	Trichoderma sp.
Tongariro nat. park	Root	Trichoderma spirale isolate ELF14 (v)	100	Trichoderma sp.
Paringa forest	Stem	Chaetomium G7 (MG548563)	100	Chaetomium sp.
Peel forest	Leaf	Fusarium tricinctum (MH931273)	100	Fusarium sp.
Paringa forest	Stem	Metarhizium sp. (DQ385622)	100	Metarhizium sp.
Arthur’s Pass nat. park	Stem	Pezicula neosporulosa (LC206659)	100	Pezicula sp.
Paringa forest	Stem	Pezicula sp. 1 ICMP 18831 (JN225940)	100	Pezicula sp.
Peel forest	Leaf	Phoma sp. H39 (GU566295)	100	Phoma sp.
Arthur’s Pass nat. park	Root	Hypocrea lixii DAOM 229978 (EF191298)	100	Trichoderma sp.
Paringa forest	Stem	Trichoderma sp. XY24 (KX856006)	100	Trichoderma sp.
Paringa forest	Stem	Trichoderma harzianum strain I 33 (KT351798)	100	Trichoderma sp.
Peel forest	Stem	Xylariaceae sp. 5 ICMP 18786 (JN225905)	100	Xylariaceae sp.
Peel forest	Leaf	Xylariaceae sp. 5 ICMP 18786 (JN225905)	100	Xylariaceae sp.

). The sequences were deposited in NCBI with the accession numbers from OK036294 to OK036333 and MH844075 to MH844084.

3.4. Activity against Phytopathogenic Fungi and Opportunistic Human Pathogens

Of the total endophytic fungi tested (n = 50), ten isolates showed activity against at least 1 test pathogen (

Table 5. Antagonistic ctivity of endophytic fungi against phytopathogenic fungi (Neofusicoccum luteum, N. parvum, Ilyonectria liriodendri, and Neonectria ditissima) and human pathogens (Candida albicans, Staphylococcus aureus, and Escherichia coli) based on the inhibition zone size (+++ high activity, ++ moderate activity, + low activity, − no activity).

Isolate	N. luteum	N. parvum	I. liriodendri	N. ditissima	C. albicans	S. aureus	E. coli
Chaetomium sp. PR1BC2	++	++	++	++	+++	+++	−
Fusarium sp. P4LC2	++	++	+	+	+++	+++	−
Metarhizium sp. PR1SB1	+++	+++	+++	+++	-	+++	−
Pezicula sp. AF2	+++	+++	+++	+++	+++	+++	−
Pezicula sp. PRY2BA2	+++	+++	+++	+++	+++	+++	−
Phoma sp. P1LA4	+++	+++	+	+	+++	+++	−
Trichoderma sp. F3	+++	+++	++	++	++	+++	−
Trichoderma sp. PRY2BA21	+++	+++	++	+	+++	+++	−
Trichoderma sp. PRY2BC1	++	+++	++	++	+++	+++	−
Xylariaceae sp. P4BB2	++	++	+	+	+++	+++	−

). Pezicula sp. PRY2BA2 and Metarhizium sp. PR1SB1 showed the highest activity and inhibited all the phytopathogenic fungi tested (Table 5). Trichoderma sp. PRY2BA21, and Xylariaceae sp. P4BB2 completely inhibited C. albicans in dual culture assays. Pezicula sp. PRY2BA2, Fusarium sp. P4LC2, Phoma sp. P1LA4, and Chaetomium sp. PR1BC2 produced clearance zones greater than 10 mm.

3.5. Influence of Endophytic Fungi on the Growth of P. colorata Seedlings

The inoculation of P. colorata seedlings with endophytic fungi significantly increased the growth for all the treatments compared to the control (p < 0.05) except for Metarhizium sp. PR1SB1 (

Table 6. Response of Pseudowintera colorata seedlings to treatment with endophytic fungi after 4 months growth. Mean of 10 replicate plants per treatment.

Treatment	Shoot Height (cm)	Shoot Dry Weight (g)	Root Dry Weight (g)	No. of Internodes
Chaetomium sp. PR1BC2	7.35 ab	0.98	0.54	6.0 b
Fusarium sp. P4LC2	6.79 ab	1.10	0.59	7.0 a
Metarhizium sp. PR1SB1	4.99 cd	1.10	0.47	4.3 d
Trichoderma sp. PRY2BA21	8.36 a 1	1.14	0.68	6.0 b
Trichoderma sp. PRY3BC1	7.46 ab	0.95	0.72	4.8 cd
Xylariaceae sp. P4BB2	6.84 ab	0.99	0.62	5.3 bc
Xylariaceae sp. P4LA3	5.97 bc	0.93	0.69	5.8 b
Untreated Control	3.72 d	1.02	0.59	4.1 d
p Value	<0.001	0.88	0.31	<0.001
LSD	1.771	NSD	NSD	0.816

¹ Means followed by the same letter are not significantly different based on least significant difference (LSD) at p = 0.05, NSD—not significantly different.

). Mean shoot height of seedlings treated with Trichoderma sp. PRY2BA21 was 2.2 × longer (8.36 cm) than the control (3.72 cm) but was not significantly different from other treatments (Table 6). Shoot and root dry weights of the treated seedlings were not significantly different from that of the control (p = 0.88 and p = 0.31, respectively) (Table 6). Treatment with Fusarium sp. P4LC2 produced significantly more internodes (mean = 7) compared with all other treatments (means = 6.0–4.1) (p < 0.005).

3.6. Colonization of P. colorata Roots by Endophytic Fungi as Shown by DGGE

Attempts to re-isolate inoculated fungi using traditional re-isolation techniques were unsuccessful as none of the recovered fungi showed any cultural similarities to the inoculated isolates. Therefore, the endophytic colonization of the roots by the isolates was confirmed using DGGE and DNA from the inoculated isolates used as reference markers. Bands corresponding to Metarhizium PR1SB1, Xylariaceae sp. P4LA3 and Fusarium sp. P4LC2 were identified in the respective treatments (Figure S1). Xylariaceae sp. P4LA3 showed a complex profile with 2 bands (Figure S1). Bands for other fungal isolates were not observed in the treatments. There was no attempt to re-isolate the endophytes onto agar from the tissues of the P. colorata seedlings.

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.

5. Conclusions

This research is the first study to show the diversity and functional potential of culturable endophytic fungi in P. colorata in a New Zealand medicinal plant. In addition to showing strong antagonistic activity against phytopathogens and opportunistic human pathogens, the application of selected endophytic fungi as soil-drench also showed a positive effect on the growth of P. colorata seedlings. These results indicate the strong potential for endophytic fungi to be used in agriculture as plant growth promoters, as potential biocontrol agents against phytopathogens, and as sources of novel bioactive compounds.