Microbial Ecology

https://doi.org/10.1007/s00248-018-1153-9

PLANT MICROBE INTERACTIONS

Community Structure of Endophytic Actinobacteria in a New Zealand

Native Medicinal Plant Pseudowintera colorata (Horopito)
and Their Influence on Plant Growth
Neeraj Purushotham 1 & Eirian Jones 1 & Jana Monk 2,3 & Hayley Ridgway 1,4

Received: 21 September 2017 / Accepted: 30 January 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
The role of plant endophytic Actinobacteria remains poorly understood with no reports of these communities in New Zealand
native plants. This first investigation of endophytic Actinobacteria in New Zealand targeted the culturally significant medicinal
shrub Pseudowintera colorata (horopito) as a model plant. Community analysis in plant tissues collected from ten geographically
distinct sites showed that tissue type had the strongest influence on diversity and richness of endophytic Actinobacteria. More
denaturing gradient gel electrophoresis (DGGE) bands were obtained from stems (n = 18) compared to roots (n = 13).
Sequencing analysis of the major bands (n = 20) identified them as uncultured bacteria, Streptomyces sp. and Angustibacter
peucedani. Using two Actinobacteria-specific media, nine isolates were recovered from surface-sterilised P. colorata tissues.
This was approximately 12% of the total taxa and correlated well with culturable numbers in international studies. In vitro
analysis of the functionality of these strains showed that Streptomyces sp. PRY2RB2 inhibited all the tested phytopathogenic
fungi (n = 4), Streptomyces sp. UKCW/B and Nocardia sp. TP1BA1B solubilised phosphate and produced siderophores. The
functionality of the phosphate solubilising strains (n = 2) in vivo was investigated by inoculation of P. colorata seedlings. After
4 months, the mean shoot height of seedlings treated with Nocardia sp. TP1BA1B was 1.65× longer, had higher shoot dry weight
(1.6×) and number of internodes (1.67×) compared to control. This study identified for the first time a key group of endophytic
Actinobacteria that are likely to be important in the ecology of New Zealand flora.

Keywords Plant-microbe interaction . Phytopathogens . Growth promotion . Microbial communities

Abbreviations DGGE denaturing gradient gel electrophoresis

PRY2RB2 isolate from Paringa Forest root sample TP1BA1B isolate from Taihape Scenic Reserve stem sample
UKCW/B isolate from Paringa Forest root sample

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s00248-018-1153-9) contains supplementary
material, which is available to authorized users.
* Neeraj Purushotham
neeraj.purushothambalraj@lincolnuni.ac.nz
1
Department of Pest-management and Conservation, Faculty of
Agriculture and Life Sciences, Lincoln University, Lincoln, New
Zealand
2
AgResearch, Christchurch, New Zealand
3
Present address: AsureQuality, Christchurch, New Zealand
4
Present address: Plant & Food Research Ltd, Christchurch, New
Zealand
Introduction
Plants are recognised as meta-organisms and normally contain
a diverse community of endophytic microorganisms that col-
lectively form the Bplant endomicrobiome^. These ancillary
genomes have become topical in plant ecology and evolution
as they are demonstrated to supplement the host genetic res-
ervoir and influence all aspects of plant metabolism [16].
Research has shown that endophytes can enhance plant
growth, improve nutrient uptake and increase adaptability to
climate change [32]. For medicinal plants, there are examples
where the endomicrobiome contributes to, or directly pro-
duces, compounds for which the plant is known. For example,
the endophytic fungus Taxomyces andreanae isolated from

Taxus brevifolia (yew) is capable of producing the same anti-
cancer compound, Taxol®, as the host [42].
Actinobacteria have been isolated from a variety of plants
including domesticated crop plants such as wheat and rice [38]
and medicinal plants such as Carex baccans and Dracaena
cochinchinensis (Chinese medicinal plants) [33, 39]. They
have attracted significant interest globally due to their ability
to produce secondary metabolites in addition to their unique
biological activities [24, 34]. In addition to protecting the host
plants against phytopathogens [9], endophytic Actinobacteria
can promote plant growth [5, 26] and increase host resistance
to various biotic and abiotic stresses [17, 18, 35]. For example,
Kalmia latifolia (mountain laurel) seedlings treated with en-
dophytic Streptomyces padanus strain AOK-30 remained tur-
gid in room conditions, while the untreated seedlings wilted
within 1 h under the same conditions [17]. Endophytic
Actinobacteria have been shown to influence plant growth
either directly or indirectly by solubilising nutrients, nitrogen
fixation and producing growth hormones [8, 46].
Medicinal plants have been shown to contain endophytic
Actinobacteria with novel bioactive properties [12]. For ex-
ample, three endophytic Streptomyces species isolated from
D. cochinchinensis exhibited cytotoxic effects against two hu-
man cancer cell lines, MCF-7 and Hep G2 [39]. Endophytic
Actinobacteria as a result of their long-term association with
the host plant may also be involved in the metabolic pathways
of the plant [14, 23, 37]. The presence of endophytic
Actinobacteria has not been demonstrated in any native New
Zealand plant, and there is very little information on the eco-
logical role of these microbial endophytes in general.
The community of endophytes within a host is typically
examined using molecular techniques such as 16S rRNA se-
quencing and PCR DGGE [12, 29]. For example, using
DGGE, it was revealed that the roots of a Thai medicinal plant
Aquilaria crassna growing in different locations harboured
different endophytic Actinobacteria communities [29]. The
sole s tu dy on the New Ze aland medicina l plant
Leptospermum scoparium used DGGE and showed that tissue
type was the main factor affecting the community structure of
endophytic bacteria, but Actinobacterial communities were
not investigated [51].
This study uses culture-dependent and culture-independent
approaches to describe the diversity, community structure and
functional properties of endophytic Actinobacteria in
Pseudowintera colorata, commonly known as horopito or
New Zealand pepper tree. This plant is an endemic New
Zealand medicinal shrub belonging to the Winteraceae family
and has played a key role in traditional medicine as it was used
by Māori to treat fever, skin diseases, gonorrhoea, stomach
ache, toothache and also to wean infants [4]. The leaves con-
tain a sesquiterpene dialdehyde, polygodial, which possesses
strong anti-fungal activity against filamentous fungi and
yeasts, and anti-bacterial properties [21, 22]. As the first study
Purushotham N. et al.
to depict the importance of endophytic Actinobacteria, this
study has implications for future work on the ecology of
New Zealand native plants.
Materials and Methods
Sampling Locations
Pseudowintera colorata samples were collected from 10 sites
across New Zealand (6 South Island sites and 4 North Island
sites) (Fig. 1, Table 1). The sites chosen for sampling were
national parks and forest reserves maintained by the New
Zealand Department of Conservation (DOC).
Plant Sampling
The leaves and stem tissues were cut using sterile secateurs
from approximately 1 m above the ground. Whenever possi-
ble, branches were sampled from opposite sides of the same
plant. The selected leaves were fully open, mature and free
from any visible herbivory or disease damage. For root sam-
ples, soil was excavated close to the plant, and putative lateral
roots belonging to P. colorata were traced back to the plant
being sampled. The lateral root along with root hair was col-
lected for analysis. The tissues were maintained separately in
bags, labelled, placed in an ice-bin and stored at 4 °C until
processed within 1–3 days.
Sample Processing
All plant tissues were washed with tap water to remove soil
debris, air-dried then surface sterilised using the 5-step
sterilisation method described previously [51]. The tissues
were cut into 1-mm portions using a sterile scalpel and plated
onto the Actinobacteria selective media, Bennett’s agar and
starch casein agar and incubated at 25 °C for 7–14 days in the
dark. Both the media were amended with the antifungal agents
nystatin and cycloheximide (50 μg/mL) [30]. Some portions
of each sterilised plant tissue were set aside for extraction of
DNA for DGGE. Prior to plating the tissues, an aliquot of the
final rinse water was plated onto starch casein agar and incu-
bated at 25 °C for 24–48 h. Lack of growth on medium indi-
cated that the surface sterilisation process was effective.
Diversity Analysis of Actinobacteria Communities
in P. colorata Using Culture DGGE
Surface-sterilised P. colorata tissues (leaf, stem and root) were
treated with propidium monoazide (PMA) to exclude any re-
sidual surface DNA from amplification by PCR [6, 51]. DNA
was extracted using a modified CTAB method [1]. To amplify
the 16S rRNA from Actinobacteria, the specific primer F243
Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera...

Fig. 1 New Zealand map

showing the sampling sites for
Pseudowintera colorata

(5′-GGA TGA GCC CGC GGC CTA-3′) was used in the first
PCR along with the universal bacterial reverse primer 1494R
(5′-TAC GGC TAC CTT GTT ACG AC-3′) [29, 41]. The
PCR products from the primer pair F243-R1494 were then
used as templates for a second PCR with the primer pair
F341-GC (GC–CCT ACG GGA GGC AGC AG) and R534
(ATT ACC GCG GCT GG) [29]. The PCRs were carried out
in an Applied Biosystems Proflex PCR system in a total vol-
ume of 25 μL containing 1 μL of template DNA as described
[29]. DGGE was performed using a Cipher DGGE
Electrophoresis system (CBS Scientific) as described [51].
Eight microlitres of strongly amplified PCR product along
with 2 μL of loading dye was loaded onto an 8% (w/v) poly-
acrylamide gel (acrylamide/bis solution, 37.5:1) with a linear
gradient of 35–50% denaturant [29]. Analysis of the
Actinobacteria communities was carried out using Phoretix
1D Pro Gel Analysis (Totallab, UK), which generated a matrix
based on the presence or absence of bands in each sample. The
band matrix was analysed by Primer version 7 (Primer-E Ltd.,
Plymouth Marine Laboratory, UK) multivariate software
package. Using Jaccard coefficient, a resemblance matrix
based on similarity was generated. The nonmetric multidi-
mensional scaling (nMDS) ordination, main and pair-wise
PERMANOVA tests were performed to test the statistical dif-
ference between endophytic Actinobacteria communities
among samples. Diversity and richness of the endophytic
Actinobacteria communities were analysed as described pre-
viously [51].
Major bands from the DGGE gels produced were excised
using a sterile scalpel as previously described [12]. Using the
edge of a sterile scalpel, the excised gel bands were crushed
and suspended in separate tubes containing 50 μL sterile water
and incubated overnight at 4 °C. PCR was performed using
the primer pair F341-GC and R534 [29]. The amplicons were

Table 1 Details of the sampling

sites of Pseudowintera colorata Site location Latitude Longitude Region
from North and South Island
Taihape Scenic Reserve − 39.67635°S 175.80560°E Manawatu-Wanganui
Tongariro National Park − 39.02237°S 175.71810°E Manawatu-Wanganui
Kaimanawa Forest Park − 38.94721°S 175.94370°E Manawatu-Wanganui
Lake Rotopounamu Scenic Reserve − 39.02656°S 175.73502°E Manawatu-Wanganui
Kahurangi National Park − 41.07224°S 172.59166°E Nelson/Tasman
Paringa Forest − 43.69379°S 169.40724°E West Coast
Arthur’s Pass National Park − 42.94215°S 171.56414°E Canterbury
Kaituna Valley Scenic Reserve − 43.71655°S 172.7554°E Canterbury
Peel Forest − 43.91835°S 171.25934°E Canterbury
Otago Peninsula Scenic Reserve − 45.88184°S 170.58049°E Otago

sequenced directly at the Lincoln University Sequencing
Facility. The sequences obtained were trimmed using
DNAMAN v4 (Lynnon Biosoft, Canada) to remove ambigu-
ous regions. The sequences were then compared against those
of known origin using NCBI BLAST (basic local search
alignment tool) and the GenBank database (www.ncbi.nlm.
nih.gov). All the sequences were aligned using MUSCLE
and the distance matrices and phylogenetic trees were
calculated by maximum likelihood algorithms with 1000
bootstrap replication using MEGA 6 software (Molecular
Evolutionary Genetic Analysis).
Isolation and Identification of Culturable Endophytic
Actinobacteria
Emerging colonies with morphology typical of Actinobacteria,
being powdery or elevated with margins pulling the agar, were
sub-cultured individually onto Bennett’s agar plates [43]. The
DNA for each isolate was extracted using the PureGene kit
(Qiagen) as per the manufacturer’s instructions. Cultured
Actinobacteria were identified by sequencing the 16S rRNA
gene using the primer pair F243 and R1494.
Functional Properties of Endophytic
Actinobacteria Isolated from P. colorata
Dual Culture Assay Against Phytopathogenic Fungi
The endophytic Actinobacteria isolated from P. colorata were
tested against phytopathogenic fungi Neofusicoccum luteum
ICMP 16678, N. parvum MM562, Ilyonectria liriodendri
WPA1C and Neonectria ditissima ICMP 14417. All the
strains of phytopathogenic fungi were obtained from the
Lincoln University Plant Microbiology culture collection, ex-
cept for N. ditissima ICMP 14417 which was obtained from
the International Collection of Microorganisms from Plants
(ICMP, Landcare Research, Auckland, New Zealand). The
assay was carried out on Waksman agar (WA) plates. A 6-
mm disc of each Actinobacteria strain was taken from 7 to
10-day-old culture plates and each placed 1 cm from the edge
on a fresh WA plates and incubated at 25 °C for 7–10 days.
For isolates of Actinobacteria that did not produce confluent
growth, the cultures were streaked 1 cm from the edge of the
plate. A 6-mm disc of 7-day-old cultures of N. parvum
MM562, N. luteum ICMP 16678 and I. liriodendri WPA1C
and 14-day-old culture of N. ditissima ICMP 14417 were
placed 5 cm from the Actinobacteria colony in their respective
plates and incubated again at 25 °C in a 12-h light/12 h dark
cycle for 5–10 days. For control plates, a 6-mm disc of fungal
pathogen without the Actinobacteria was set up. Antagonistic
activity was classified on the basis of the zone of inhibition
against the test pathogen, high activity (inhibition zone >
Purushotham N. et al.
5 mm), moderate activity (inhibition zone < 5 mm but >/=
2 mm) and low activity (inhibition zone < 2 mm but > 1 mm).
Dual-Culture Assay Against Phytopathogenic Bacteria
and Opportunistic Human Pathogenic Bacteria/Yeasts
The phytopathogenic bacteria (Pectobacterium atrosepticum
and P. brasiliensis) were obtained from Dr. Abigail Durant at
Bioprotection Research Centre, Lincoln University. The op-
portunistic human pathogens (Staphylococcus aureus 297,
Escherichia coli 916 and Candida albicans 3395) were ob-
tained from the Institute of Environmental Science and
Research (ESR), Porirua, New Zealand. Using a sterile cork
borer, a 6-mm disc of each Actinobacteria strain was inocu-
lated in the centre of WA plates and incubated as described
previously. After 7–10 days, using a sterile loop, a single
colony of the test pathogen was streaked away from the
Actinobacteria colony towards the edge of the plate. The
plates were sealed and incubated at 25 °C for 24 h in the dark.
Uninoculated control plates containing only the test pathogens
were also set up. A clearance zone ≥ 1 mm around the
Actinobacteria colony was recorded as positive.
Secretion of Siderophores
The ability of the Actinobacteria to secrete siderophores was
tested on Chrom-azurol S agar as previously described [40].
Using a cork borer, 6-mm plugs of the Actinobacteria strains
were inoculated face down in the centre of the CAS agar and
the plates were incubated at 25 °C in the dark. A Pseudomonas
sp. (Lincoln University Plant Microbiology Collection) known to
produce siderophores was used as a positive control. The plates
were prepared in triplicate. The plates were observed daily for 7–
10 days. Positive results were indicated by the presence of an
orange halo around the Actinobacteria colony.
Phosphate Solubilisation
The ability of endophytic Actinobacteria to solubilise phosphate
was tested on tricalcium phosphate (TCP) agar as described pre-
viously [13]. Using a sterile cork borer, a 6-mm plug of
Actinobacteria culture was transferred and placed face down onto
the centre of TCP agar plates, and the plates were sealed and
incubated at 25 °C in the dark. The plates were prepared in
triplicate. Three positive control plates were inoculated with a
known phosphate solubilising Bacillus sp. (Lincoln University
Plant Microbiology Collection). Positive results were indicated
by the presence of a clear zone ≥ 0.5 mm around the colony.
Strains which solubilised phosphate were further tested for their
influence on the growth of P. colorata.
Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera...
Influence of Endophytic Actinobacteria on the Growth
of P. colorata Seedlings in the Glasshouse
Six-week-old seedlings were purchased from Southern Woods
Plant Nursery (Christchurch, New Zealand). The seedlings
were acclimatised in the shade house for 3–4 weeks in
February 2017. Immediately prior to inoculation with the en-
dophyte treatments, the length of the shoots and the stem girth
were measured using a digital callipers.
Two 6-mm plugs of 7–10-day-old Actinobacteria cul-
tures were inoculated into flasks containing 150 mL of ster-
ile Waksman broth (WB) and incubated in a shaking incu-
bator (Labnet 211DS) set at 25 °C for 150 rpm in the dark for
5–7 days. The cultures were harvested by centrifuging at
20,000×g at 4 °C for 15 min to pellet the cells and the pellet
was resuspended in sterile distilled water (SDW). The spore
concentration of the suspension was determined using a
haemocytometer and adjusted to 105 to 106 spores/mL using
SDW [51]. Prior to inoculation with the Actinobacteria, the
P. colorata seedlings were not watered for 24 h. Each seed-
ling was transferred to a 1-L pot containing the potting mix
medium on the day of inoculation. The potting mix was
composed of 20% pumice, 80% composted bark, 2 kg/m3
Osmocote® standard 3–4 months gradual release fertiliser
(NPK 16-3.5-10 plus trace elements), 1 kg/m3 agricultural
lime, 500 g/m3 Hydraflo® 2 (granular wetting agent, Scott
Australia Pty Ltd., Auckland, New Zealand). Using a sterile
pipette, the root region of P. colorata seedlings was
drenched with 50 mL of the appropriate cell suspension
[36]. For control plants, SDW without any cell suspension
was added. Pots were arranged in a randomised complete
block design. The plants were watered once daily and ob-
served regularly for any dead or diseased plants following
the treatments. The shoot height of the seedlings was mea-
sured after 3 months (March 2017 to May 2017) and the
difference in heights pre-treatment (X) and post-treatment
(Y) was calculated (Y-X). After 3-month growth, the potting
mix around the root zone of each treatment of P. colorata
seedlings was reinoculated by drenching with 50 mL of
freshly prepared spore suspension (105 to 106 spores/mL)
of each of the respective treatments. The seedlings were
destructively harvested 4 weeks after the second inoculation
(June 2017).
At harvest, the shoot height was measured from the stem
base (at the soil level) to the top leaf using a digital callipers.
The number of internodes was measured for each plant stop-
ping before the top 2 leaves. The shoot and root portions were
weighed after drying in an oven at 60 °C for 2 days. The data
was analysed using a general analysis of variance (ANOVA)
using Minitab 17 (Lead Technologies, Australia). Fisher’s
protected least significant difference (LSD) was used to test
the mean difference between shoot lengths, shoot weights and
root weights of treated plants with untreated controls.
Influence of the Endophytic Inoculants
on the Microbial Communities in the Roots
of P. colorata
A small portion of the roots from the harvested
P. colorata seedlings were surface sterilised, then treated
with PMA [51] and the DNA extracted using a modified
CTAB method as described previously [1]. DNA was am-
plified by nested PCR using group specific primers for
Alphaproteobacteria (F203-L1401 and 341FGC-518R),
Betaproteobacteria (Beta359F-Beta682R and 518FGC-
Beta682R), Gammaproteobacteria (Gamma395F-
Gamma871R and 518FGC-785R) and total fungi (AU2-
AU4 and FF390-FR1GC) as previously described [51]
(Supplementary Data S1). DGGE was performed as de-
scribed [51], and 8 μL of strongly amplified PCR product
along with 2 μL of loading dye was loaded onto an 8%
(w/v) polyacrylamide gel (acrylamide/bis solution, 37.5:1)
with a linear gradient of 35–50% denaturant for
Actinobacteria [29], 40 to 60% for Alphaproteobacteria
and Gammaproteobacteria [51], 40 to 55% for
Betaproteobacteria [51] and 25 to 55% for total fungi
[51]. Diversity and richness of endophytic microbial com-
munities were analysed as described previously [51].
Results
Community Structure of Endophytic Actinobacteria
Based on DGGE
Plant tissue, location (n = 10) and the interactions between
location and plant tissue influenced the Actinobacteria com-
munities (PERMANOVA P ≤ 0.05) (Table 2). The DGGE pat-
terns grouped the microbial communities in roots and stems
separately according to the tissue, whereas the communities in
leaves were more diverse (Fig. 2). Actinobacterial taxa were
richer in stems (n = 18) and leaves (n = 16) compared to roots
(n = 13) (LSD; P ≤ 0.05) (Supplementary Data S2). Plant lo-
cation influenced the richness in stems (PERMANOVA; P ≤
0.05) and leaves (PERMANOVA; P ≤ 0.005), but not roots
(PERMANOVA; P = 0.255) (Supplementary Data S3).
Effect of Plant Maturity on the Endophytic
Actinobacteria Community Structure and Richness
in P. colorata
Collection of both mature and immature plants at each site
was not always possible. Thus, for analysing the influence
of plant maturity on the community structure of endophytes,
a subset of three sites was selected where plants of different
maturities were present. These sites were Kaituna Valley
Forest Park, Paringa Forest and Peel Forest. The plants
 Purushotham N. et al.

Table 2 Effect of location and plant tissue on the similarity and richness Table 3 Effect of plant location, plant tissue and plant maturity on the
of endophytic Actinobacteria communities in Pseudowintera colorata similarity and richness of endophytic Actinobacteria communities of
Pseudowintera colorata
Treatment †Microbial Microbial
community richnessa Treatment Microbial Microbial
similarity community richnessa
similaritya
Location 0.007* 0.177
Plant tissue 0.001** 0.045* Location 0.001** 0.13
Location vs plant tissue 0.002** 0.298 Plant tissue 0.001** 0.013*
Maturity 0.010* 0.096
a
Asterisk denotes levels of statistical significance of microbial commu- Location vs plant tissue 0.001** < 0.001**
nity similarity based on PERMANOVA
Location vs maturity 0.011* 0.178
*Significantly different (P ≤ 0.05)
Plant tissue vs maturity 0.001** 0.407
**High significant difference (P ≤ 0.005)
Plant tissue vs location vs 0.001** 0.029*
maturity
collected at these sites were classified based on their height as
a
mature plants (> 3 m) and immature plants (≤ 1 m). Asterisk denotes levels of statistical significance of microbial commu-
nity similarity based on PERMANOVA
Plant tissue, location, maturity and the interaction between
*Significantly different (P ≤ 0.05)
these factors influenced Actinobacteria communities
(PERMANOVA; P ≤ 0.05) (Table 3). The Actinobacteria **High significant difference (P ≤ 0.005)
communities were not distinguished in mature and immature
plants (Fig. 3a), nor in the leaves and stems of immature/ 1500 bp long. Sequencing of these PCR products revealed
mature plants (PERMANOVA; P = 0.246 and P = 0.044, re- that the strains belonged to the genera Streptomyces (n = 3),
spectively) (Fig. 3b, c), while only the communities in stems Nocardia (n = 1), Micromonospora (n = 3), Microlunatus
and roots of immature plants formed discrete clusters (n = 1) and Nakamurella (n = 1) (Fig. 4). PCR of excised
(PERMANOVA; P = 0.001) (Supplementary Data S4) (Fig. bands from DGGE gels using F341GC and R534 yielded
3d). PCR products approximately 150–180 bp long. Of the 20
bands selected for PCR and sequenced from the DGGE gels,
only 10 bands were successfully sequenced. Sequencing of
Identity of the Culturable and Unculturable the PCR product from the DGGE bands identified that bands
Endophytic Actinobacteria from the Tissues 1L12B, 1L1B, 1L2A as uncultured bacteria. Bands L19A and
of P. colorata 1L4B were identified as a strain of Angustibacter peucedani
and a Streptomyces sp., respectively (Fig. 4). Three bands
A total of nine endophytic Actinobacteria were cultured from could not be resolved due to the small size, while the other
the surface-sterilised tissues (5 from stems and 4 from roots) two bands were identified as chloroplasts.
of P. colorata. PCR of the 16S rRNA subunit using the
primers F243 and R1494 yielded products approximately
Activity Against Phytopathogenic Fungi

Of the endophytic Actinobacteria strains tested, Streptomyces

sp. PRY2RB2 showed highest activity against the four phyto-
pathogenic fungi tested. Micromonospora sp. KVYPSA1
showed high activity against I. liriodendri and N. ditissima
(Table 4).

Activity Against Phytopathogenic Bacteria

and Opportunistic Human Pathogens

Only Streptomyces sp. PRY2RB2 showed activity against

Fig. 2 Nonmetric multidimensional scaling (MDS) plot showing
actinobacterial communities from different plant tissues of
Pseudowintera colorata. Root (∇); stem (×); leaf (●)
P. atrosepticum. None of the strains were active against
P. brasiliensis, E. coli 916 and C. albicans 3395. Streptomyces
sp. PRY2RB2 and Streptomyces sp. KVP1RA1 showed low
activity against S. aureus (Table 4).
Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera...
Fig. 3 Nonmetric multidimensional scaling (MDS) plots showing Actinobacteria communities from mature and immature plants. a All tissues, b leaves,
c stems and d roots of Pseudowintera colorata. Immature plant ∇; mature plant ●
Production of Siderophores and Solubilisation of TCP
Of the strains tested (n = 9), only Streptomyces sp. PRY2RB2,
Streptomyces sp. UKCW/B and Nocardia sp.TP1BA1B pro-
duced faint halo zones (< 1 mm) on CAS agar. Other test
isolates failed to grow on CAS agar.
On TCP agar, Streptomyces sp. UKCW/B produced a
clearance zone greater than 4 mm. Nocardia sp.
TP1BA1B showed low activity by producing a faint clear-
ance zone (< 1 mm) (Table 4).
Effect of Endophytic Actinobacteria on the Growth
of P. colorata Seedlings
Inoculation with Actinobacteria increased the growth of
P. colorata seedlings for both the treatments compared to the
control (P < 0.05) (Table 5). Mean shoot height of seedlings
treated with Nocardia sp. TP1BA1B was 1.7× longer
(5.17 cm) than the control (3.12 cm). Shoot dry weight of
seedlings treated with Nocardia sp. TP1BA1B was 1.6×
heavier (1.22 g) than that of the control (0.76 g), but was not
significantly different from seedlings treated with
Streptomyces sp. UKCW/B (1.07 g) (Table 5). Root dry
weight of seedlings treated with Nocardia sp. TP1BA1B
was 1.6× heavier (0.73 g) than the control (0.46 g). Number
of internodes produced by the treated plants were significantly
higher than the control, with Nocardia sp. TP1BA1B and
Streptomyces sp. UKCW/B producing 1.7× and 1.3× more
internodes than the control (3.7) (Table 5).
Influence of the Endophytic Inoculants
on the Microbial Communities in the Roots
of P. colorata
The influence of inoculating endophytic Actinobacteria on the
microbial communities in the root tissues of the P. colorata
seedlings was analysed using DGGE. Treatment of seedlings
with Nocardia sp. TP1BA1B and Streptomyces sp. UKCW/B
did not influence the endophytic communities in the roots of
P. colorata seedlings in comparison to the control
(Supplementary Data S5).
The endophytic colonisation of the roots by the inoc-
ulant strains was confirmed using DNA from the pure
inoculated isolates as reference markers on DGGE gels.
Bands corresponding to Nocardia sp. TP1BA1B and
Streptomyces sp. UKCW/B were identified in the treat-
ment lanes and thus confirmed colonisation (data not
shown).
Discussion
This is the first study to analyse the diversity of culturable and
unculturable endophytic Actinobacteria in a New Zealand

Fig. 4 Phylogenetic tree based on
alignment of partial 16S rRNA
sequences of endophytic
Actinobacteria associated with
Pseudowintera colorata
recovered as culturable isolates
(●) and from DGGE gels (○)
native plant. This work identified the community structure of
endophytic Actinobacteria that inhabit P. colorata and dem-
onstrated their functional role in planta.
The application of molecular techniques for the detection
and identification of endophytes using markers such as 16S
rRNA is frequently employed to study the diversity and struc-
ture of endophytic communities [27]. This study used DGGE
as a molecular tool to analyse the Actinobacteria communities
in P. colorata tissues. The band distribution pattern in DGGE
gels showed that tissue type was the main factor influencing
the composition and richness of endophytic Actinobacteria,
suggesting that it is an overriding factor in the formation of
these microbial communities in planta.
Pseudowintera colorata maturity and its interaction with
tissue type and location influenced the diversity of endophytic
A c t i n o b a c t e r i a . T h e com m u ni t i e s o f en do ph yt i c
Actinobacteria grouped together in the immature roots, while
those in mature roots were more diverse, indicating that there
could be a community shift as the plants mature. Similar re-
sults have been reported in the perennial plant Boechera
stricta, where the plant age affected the abundance of
leaf-associated Actinobacteria [48]. In this study, the
richness of endophytic Actinobacteria was not
Purushotham N. et al.
influenced by tissue maturity for the sites analysed.
Chemotype variations in P. colorata have been shown
in previous studies [31, 50], and thus, variability in
polygodial levels could contribute to the differences in
endophytic Actinobacteria communities across different
locations, or vice versa. Actinobacteria taxa were richer
in stems and leaves compared to that in roots. Greater
richness in stems was also shown for endophytes recov-
ered from Jatropha curcas [36].
Although the DGGE profiles showed diverse endophytic
Actinobacteria in the tissues of P. colorata, the number of
culturable endophytic Actinobacteria isolated was relative-
ly low (n = 9; 12% of total Actinobacteria taxa). No endo-
phytic Actinobacteria were recovered from the leaves.
Similar results were reported in a study of a Thai medicinal
plant, A. crassna, where a total of 10 culturable endophytic
Actinobacteria were isolated from the roots and stems and
none from the leaves [28]. A preference for the recovery
media and the natural antimicrobial nature of sequestered
compounds in the tissues may have affected the number of
endophytic Actinobacteria recovered [25, 49]. In order to
increase the frequency of Actinobacteria isolated in similar
studies, researchers use 5–10 types of specific media [20,
Table 4 Activity of endophytic Actinobacteria against a range of fungal and bacterial phytopathogens and human pathogens, siderophore production on Chrom-azurol S agar (CAS) and phosphate
solubilisation on tricalcium phosphate agar (TCP) and identification based on 16S rRNA sequencing

Isolate Tissue N. luteum N. parvum I. liriodendri N. ditissima P. atrosepticum P. brasiliensis S. aureus E. coli C. albicans CAS TCP

Micromonospora sp. KVYPRB2 Root – – – – – – – – – NG –

Micromonospora sp. KVYPSA1 Stem – – +++ +++ – – – – – NG –
Micromonospora sp. KVYPSA11 Stem – – – – – – – – – NG –
Microlunatus sp. KVYPSC1B Stem – – – – – – – – – NG –
Streptomyces sp. PRY2RB2 Root +++ +++ +++ +++ ++ – + – – +
Streptomyces sp. UKCW/B Root – – – – – – – – – + ++
Streptomyces sp. KVYPRA1 Root – – – – – – + – – NG –
Nocardia sp. TP1BA1B Stem – – – – – – – – – + +
Nakamurella sp. KVP1BC1 Stem – – – – – – – – – NG –

+++ High activity, ++ moderate activity, + low activity, – no activity

NG no growth
Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera...

Table 5 Response of Pseudowintera colorata seedlings following treatment with endophytic Actinobacteria after 4-month growth. Mean of 10
replicate plants per treatment
Treatment Shoot
height (cm)
Nocardia sp. TP1BA1B 5.17 aa
Streptomyces sp. UKCW/B 4.93 a
Control 3.12 b
P value 0.030
LSD (5%) 1.628
a
Means within a column followed by the same letter are not significantly different based on least significant difference (LSD) at P = 0.05
30]. In a similar study, using ten different media, a total of
576 endophytic Actinobacteria were isolated from four dif-
ferent Australian native trees [20]. In addition to using dif-
ferent media, crushing the plant sample prior to plating has
been reported to increase the number of endophytic
Actinobacteria isolated [20]. This is unlikely to have im-
proved recovery in P. colorata due to the release of
polygodial. The effect of polygodial on endophytes was
not assessed in this study, but previous studies have demon-
strated that polygodial has a broad range of activity against
bacteria, filamentous fungi and yeasts [21, 22].
Sequencing the 16S rRNA gene identified the culturable
endophytic Actinobacteria into the common genera
Streptomyces and Micromonospora along with the less com-
mon genera Microlunatus and Nakamurella. Several studies
have reported Streptomyces spp. and Micromonospora spp. as
common endophytes in plants [19, 45]. The results of this
study are consistent with the findings in another study for
investigating endophytes in 10 different medicinal plants of
the Compositae family where 93% of the total cultured endo-
phytic Actinobacteria (n = 131) belonged to the genera
Streptomyces, Nocardiopsis and Micromonospora [10].
Sequencing some of the dominant bands from DGGE gels
identified them as uncultured bacteria, uncultured
Streptomyces and Angustibacter peucedani. The uncultured
bacteria may represent novel Actinobacteria present in
P. colorata that could not be resolved due to the small size
of the obtained sequences.
Streptomyces sp. PRY2RB2 was active against all the phy-
topathogenic fungi tested. The other strains which showed
activity against phytopathogenic bacteria/fungi and opportu-
nistic human pathogens were members of genera
Streptomyces and Micromonospora. Streptomyces spp. are
ubiquitous and are known to produce several bioactive metab-
olites and antibiotics [3]. For example, commercial products
such as Mycostop® and Actinovate® are produced by
Streptomyces and are used in controlling damping-off caused
by Rhizoctonia solani in tomato seedlings [15]. The faint or-
ange halos on CAS agar suggested that production of
siderophores is not a major mechanism against
Purushotham N. et al.
Shoot dry Root dry Number
weight (g) weight (g) of internodes
1.22 a 0.73 a 6.2 a
1.07 a 0.61 ab 4.7 b
0.76 b 0.46 b 3.7 c
0.002 0.012 < 0.001
0.244 0.172 0.490
phytopathogenic fungi and is consistent with other studies
[11]. In addition to producing siderophores, endophytic
Actinobacteria can promote plant growth by solubilising
phosphate as shown previously [47]. In this study,
Streptomyces sp. UKCW/B and Nocardia sp. TP1BA1B
solubilised TCP. These phosphate solubilising strains (n = 2)
were selected for further study on their influence in planta on
P. colorata seedlings.
Phosphate-solubilising microbes are able to convert insol-
uble phosphorus to a soluble form thereby presenting a possi-
ble mechanism of directly increasing plant growth [47].
Although Streptomyces sp. UKCW/B formed bigger clear-
ance zones compared to Nocardia sp. TP1BA1B, in the in
planta study, Nocardia sp. TP1BA1B produced greater in-
creases in root and shoot biomass. The results from inoculat-
ing plants with endophytes that can solubilise phosphate
in vitro are often highly variable [2]. A similar study in peanut
showed that of the 110 bacteria that solubilised TCP in vitro,
only one increased growth of plant in vivo [44]. In our present
study, mean shoot length was increased by both treatments.
These results were similar to another study where the resulting
shoots of seedlings from tomato seeds treated with spore sus-
pensions of endophytic Streptomyces sp. strains were signifi-
cantly longer than the untreated control [48]. Both Nocardia
sp. TP1BA1B and Streptomyces sp. UKCW/B were detected
in roots following reinoculation and absent in the control lanes
indicating that the strains were able to recolonise the roots of
P. colorata seedlings. These Actinobacteria did not influence
the endophytic bacteria, Actinobacteria and fungal communi-
ties in the roots of P. colorata, suggesting that the effects are
due to these strains and not alteration in microbial community.
Similar findings were reported in an earlier study where treat-
ment of wheat with endophytic Streptomyces sp. strain EN27,
Microbiospora sp. strain EN2 and Nocardiodes albus EN46
did not influence the indigenous endophytic Actinobacteria in
wheat seedlings [7].
In conclusion, the results of this study showed that endo-
phytic Actinobacteria are present in the tissues of a New
Zealand native medicinal plant and have a potential role in
plant ecology. This Bproof of concept^ showed that they can
Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera...
influence plant growth and the in vitro tests demonstrated
antimicrobial activity on plates. These findings support the
presence of a functional relationship between Actinobacteria
and their host, the complete dynamics of which has yet to be
fully explored.
Acknowledgements The authors thank Lincoln University for funding
this research and New Zealand Department of Conservation (DOC) for
permission to collect samples. The first author (NP) was funded through
NZAID Commonwealth scholarship administered by the New Zealand
Ministry of Foreign Affairs and Trade (MFAT).
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