Received: 12 January 2024

| Revised: 18 March 2024 | Accepted: 19 March 2024

DOI: 10.1002/pei3.10142

RESEARCH ARTICLE

Abundance and diversity of fungal endophytes isolated from
monk fruit (Siraitia grosvenorii) grown in a Canadian research
greenhouse
Li Ma1

| Janice F. Elmhirst2 | Rojin Darvish1 | Lisa A. Wegener1 | Deborah Henderson1

1
Institute for Sustainable Horticulture,
Kwantlen Polytechnic University, Surrey,
British Columbia, Canada
2

Elmhirst Diagnostics and Research,
Abbotsford, British Columbia, Canada
Correspondence
Li Ma, Institute for Sustainable
Horticulture, Kwantlen Polytechnic
University, 12666 72nd Avenue, Surrey,
BC V3W 2M8, Canada.
Email: li.ma6@kpu.ca
Janice F. Elmhirst, Elmhirst Diagnostics
and Research, 5727 Riverside Street,
Abbotsford, BC V4X 1T6, Canada.
Email: janice.elmhirst@shaw.ca
Funding information
NutraEx Food Inc

Abstract
Monk fruit (Siraitia grosvenorii) is an herbaceous perennial vine of the Cucurbitaceae
family cultivated commercially mainly in southern China. There is very little
information available about the fungal endophytes in monk fruit. In this study, monk
fruit plants were grown from seeds in a research greenhouse at Kwantlen Polytechnic
University in British Columbia, Canada to explore the abundance and diversity of their
fungal endophytes. Fungal endophytes were isolated from seeds, seedlings, mature
monk fruit plants, and fruits, and cultured on potato dextrose agar and water agar
media. Isolates were identified by microscopic examination and BLAST comparison
of ITS sequences to published sequences in GenBank. At least 150 species of fungal
endophytes representing 60 genera and 20 orders were recovered from monk fruit
tissues. Non-­metric multidimensional scaling (NMDS) was carried out to explore the
similarity of fungal communities among roots, stems, leaves, flowers, fruits, and seeds
based on fungal orders. Our study showed that monk fruit plants are a rich source of
fungal endophytes with the greatest abundance and diversity in leaves. This work has
deepened our understanding of the intricate interactions between plants and fungi
that sustain ecosystems and underpin plant health and resilience.
KEYWORDS

abundance, diversity, fungal communities, fungal endophytes, monk fruit, Siraitia grosvenorii

1

|

I NTRO D U C TI O N

of 1500–2002 mm, and average sunshine of 1237.3 ~ 1626.4 h (Zeng
et al., 2011).

Monk fruit [Siraitia grosvenorii (Swingle) C. Jeffrey ex A.M. Lu & Zhi Y.

The fruit of the monk fruit vine has been used as natural, calorie-­

Zhang] is an herbaceous perennial vine of the Cucurbitaceae family

free sweeteners (Xia et al., 2008) as well as folk medicine in China

cultivated commercially mainly in the southern parts of China though

for thousands of years due to their pharmaceutical properties such

it is grown also in northern Thailand and has been exported to the

as anti-­inflammation (Di et al., 2011), anti-­carcinogenesis (Takasaki

USA and India (Shivani et al., 2021). It is commonly grown in Yongfu,

et al., 2003), anti-­
oxidation, and anti-­
obesity (Sun et al., 2012).

Longsheng, and Lingui counties in northern Guangxi Province with

Mogrosides are the main compounds in the fruit responsible for the

an annual average temperature of 16–20°C, average precipitation

medicinal activities and sweetness.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2024 The Authors. Plant-Environment Interactions published by New Phytologist Foundation and John Wiley & Sons Ltd.
Plant-Environment Interactions. 2024;5:e10142.
https://doi.org/10.1002/pei3.10142



wileyonlinelibrary.com/journal/pei3

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MA et al.

Endophytic fungi live symbiotically within the internal tissues of
healthy, living plants. Many are also saprophytic and some species may
become pathogenic causing external infections upon plant senescence
(Saikkonen et al., 1998; Stone et al., 2000). Most plants in natural ecosystems are hosts to one or more fungal endophytes, which may reside within roots, stems, leaves, and/or other plant parts (Petrini, 1986;
Stone et al., 2004). The symbiotic relationship between fungal endophytes and their hosts ranges from parasitism where the endophytes
benefit for growth and reproduction at the expense of the host, to
mutualism where endophytes confer positive fitness benefits to their
hosts while obtaining nutrients for their growth and reproduction (Aly
et al., 2011; Rodriguez et al., 2009; Rodriguez & Redman, 2008). Many
fungal endophytes have been shown to reduce infection by pathogens
or disease development in their hosts (Busby et al., 2016). The transmission of endophytic fungi is primarily horizontal via airborne spores;
some however can transmit vertically to new host generations via seed

F I G U R E 1 Fresh monk fruit seeds collected from fruits.

infections (Aly et al., 2011; Saikkonen et al., 2002). Besides their significant impacts on the survival and fitness of plants by conferring stress

China. Fungal endophytes were isolated from seeds following the

tolerance, increasing water use efficiency and plant biomass, or de-

method used by Shearin et al. (2018) with modifications. Seeds were

creasing fitness by altering resource allocation (Rodriguez et al., 2009),

surface sterilized with 10% bleach for 2 min, rinsed with sterile reverse

endophytic fungi also have great potential as a unique source of bi-

osmosis water three times, and then placed on two types of microbial

ologically active compounds with promising applications in medicine,

growth media in petri dishes: potato dextrose agar (PDA) incorporated

pharmacy, and agriculture (Aly et al., 2010; Nisa et al., 2015; Zhang

with 0.005% streptomycin, and water agar (WA) media. The rinse

et al., 2006).

water was plated as a control to ensure that the surface sterilization

It has been shown that both fungal and bacterial endophytes

process was thorough. If fungal colonies were observed in the control

can modify their genes by absorbing part of the host DNA into

plates, the plates were discarded and new seed samples were surface-­

their genome for adaptation to the specific microenvironment (Aly

sterilized and plated again. Plates were kept in an incubator at 27°C

et al., 2011; Germaine et al., 2004), which may help explain the ability

and monitored regularly. All fungal endophytes were recovered from

of some endophytes to produce the same phytochemicals as those

the media and each endophyte was sub-­cultured up to three times

produced by their host plants (Stierle et al., 1993). Chen et al. (2020)

until a pure culture was obtained for identification.

isolated 15 endophytic fungal strains from roots, stems, leaves, and
fruits of S. grosvenorii and found that two of them, Diaporthe angelicae Berk. Wehm. [syn. Mazzantia angelicae (Berk.) Lar. N. Vassiljeva]

2.2 | Growing plants

and Fusarium solani (Mart.) Sacc., could produce some of the phytochemicals produced by the host plant. The other endophytic strains

Plants were grown from seeds extracted from the fresh fruit from

isolated from monk fruit were not named in the published report

China. After removing the seed coat, seeds were surface sterilized

(Chen et al., 2020). There is very little information available about

with 10% bleach and placed on Murashige and Skoog medium in

the fungal endophytes in monk fruit. The present study aimed to

petri dishes to germinate. Seedlings were transplanted into Sunshine

explore the abundance and diversity of fungal endophytes in monk

Mix #2 potting media in 10 cm (4-­inch) pots and kept in a growth

fruit grown in a Canadian research greenhouse environment, where

chamber at 21°C and a 16 h light period for 10–12 weeks. After

we can manipulate the environment to mimic the natural cultivat-

five seedlings were taken for endophyte isolation at 9–10 weeks,

ing conditions of monk fruit and minimize their interactions with the

the remaining seedlings were transplanted into Sunshine Mix #4 in

outdoor environment and potential contaminants. This also avoided

15 cm (one-­gallon) pots, one plant per pot, and placed in the research

the introduction of novel fungal species into the environment.

greenhouse located on the KPU Langley campus in January 2021.
Plants were grown in the research greenhouse with RH around 75%,

2

|

M ATE R I A L S A N D M E TH O DS

2.1 | Isolating endophytic fungi from seeds

temperature at 18–32C in soilless media with drip irrigation. All
plants were fertigated daily with a solution containing macro-­(N,
162; P, 30; K, 222; Ca, 136; Mg, 62; S, 100 ppm) and micronutrients
(Fe, 1.0; Mn, 0.45; B, 0.1; Zn, 0.33; Cu, 0.035; Mo, 0.01; and NH4,
8.2 ppm), via an individual emitter in each pot. Flowering began in

In 2020, dry monk fruit seeds obtained via Alibaba from Guangxi

late June to early July 2021 and pollination was conducted by hand

Naturix Import & Export Co., Ltd. (Nanning, Guangxi, China) and seeds

using a fine paintbrush early in the morning when flowers were

extracted from commercial fresh fruits (Figure 1) purchased from

open. Fruits were harvested in October and November (Figure 2).

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MA et al.

F I G U R E 2 Monk fruit plants grown in the research greenhouse at Kwantlen Polytechnic University, Langley, British Columbia, Canada.

TA B L E 1 Number of samples collected from 17 fruiting monk fruit plants grown in the research greenhouse at the Institute for
Sustainable Horticulture, KPU in 2021.
Leaves

Flowers

Fruit

Stems

Roots

Total samples

71

60

15

35

72

253

2.3 | Isolating endophytic fungi from the fresh
tissues of monk fruit seedlings and mature plants

sequencing. DNA was extracted using a protocol described by
Cenis (1992) and subsequently amplified by polymerase chain reaction (PCR) using general internal transcribed spacer (ITS) primers,

Samples of roots, stems, and leaves from five seedlings

ITS1 and ITS 4 (White et al., 1990). The PCR products were sent

(9–10 weeks old) in the growth chamber were taken for endophyte

for sequencing to Psomagen Inc., Rockville, MD, USA. The internal

isolation following the methods described by Musa et al. (2023).

transcribed spacer (ITS) sequences of the endophytic fungi were

Small pieces (about 0.5 cm × 0.5 cm in size) of plant tissue were

compared to sequences deposited in GenBank using the National

surface sterilized and rinsed with sterile reverse osmosis (RO)

Centre for Biotechnology Information (NCBI) nucleotide basic local

water using the method described above for isolation of seed en-

alignment search tool (BLASTn) (http://​w ww.​ncbi.​nlm.​nih.​gov/​

dophytes. Fungal hyphae emerging from the tissue were selected

BLAST​). Isolates were identified to genus and species based on the

and transferred repeatedly to PDA+ 50 ppm streptomycin to ob-

highest % identity in BLASTn, and morphological characteristics

tain a pure culture. Endophytes were isolated from leaves (young

obtained by microscopy. Where more than one identification was

and old), stems (young and old), roots (from bulb and roots in soil),

possible in GenBank, the genus or species was confirmed by micro-

flowers (buds and fully-­o pen flowers), and fruit (pulp, seeds, and

scopic comparison of fungal morphology to published descriptions.

skin separated) at different maturity stages from 17 mature monk

In a few cases where similar genera or species that could not be reli-

fruit plants grown in the greenhouse (Table 1). The isolation and

ably resolved by BLAST analysis or microscopic examination, both

purification procedures were the same as for seeds and seedlings

names are shown. Subsequently, each fungal taxon was classified

described above.

using the NCBI taxonomy browser database, US National Library of
Medicine, Bethesda, MD (https://​w ww.​ncbi.​nlm.​nih.​gov/​t axon​omy/​

2.4 | Identifying endophytic fungi

brows​er/​w wwtax.​cgi). Fifty-­seven isolates from the mature plants
that were less common, or had potential agronomic or other useful
applications, have been stored in the Canadian Collection of Fungal

After pure cultures of endophytes were obtained, they were iden-

Cultures (DAOMC) in Ottawa, ON, Canada, under specimen num-

tified morphologically by microscopy and genetically by DNA

bers 252740–252796.

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MA et al.

2.5 | Analysis of endophytic fungal communities

obtained, 13 from leaves and 14 from roots. Of the 11 isolates of

Non-­
metric multidimensional scaling (NMDS) was carried out to

cucumerinum; all were from roots except one from a stem. Genera

explore the similarity of fungal communities among roots, stems,

isolated frequently only from roots included Fusarium (13 isolates,

leaves, flowers, fruits, and seeds based on fungal orders (Peters

including 10 F. oxysporum and one F. haematococcum/F. solani),

Plectosphaerella obtained, nine were Pl. oligotrophica and two Pl.

et al., 2020). NMDS was performed using Python (3.9.16) (Van

Paraphaeosphaeria sporulosa (five), Sarocladium kiliense/S. strictum

Rossum & Drake, 1995) with MDS implemented in the scikit-­learn

(11), Simplicillium spp. (five), and Trichoderma spp. (five). Only three

(sklearn) library.

fungal endophytes were obtained in 35 samples from mature plant
stems: one isolate each of Chaetomium globosum, Plectosphaerella

3

|

R E S U LT S

3.1 | Overall fungal community composition

oligotrophica, and Phialemonium inflatum. In addition to Coprinellus
micaceus, species isolated from both reproductive and vegetative
tissues were Acremonium spp., Amorphotheca resinae, Arthrinium
spp. and Apiospora kogelbergensis, Aspergillus fumigatus and Asp.
ochraceus, Beauveria bassiana (three from leaves and four from

At least 150 species of fungal endophytes representing approxi-

fruit skin), Chaetomium globosum, Cladosporium spp., Epicoccum ni-

mately 60 genera and 20 orders were recovered in culture from the

grum (one from fruit skin), Penicillium citrinum/P. steckii and other

monk fruit tissues. Twenty-­seven isolates of endophytic fungi were

Penicillium and Talaromyces spp. Many other endophytic fungi

obtained from Chinese monk fruit seeds, either dry (purchased

were isolated only once from mature monk fruit plant tissues. Four

through Alibaba) or extracted from fresh fruit from China (Table 2).

isolates produced no match to ITS sequences in GenBank at the

Another 22 isolates were obtained from seedlings grown from the

genus or species level and could be identified only as members of

fresh seeds (Table 3). The most common genus isolated from seeds

the Lasiosphaeriaceae or Pleosporales.

and seedlings combined was Trichoderma (22 isolates: 7 or 8 species), followed by Diaporthe (4 isolates: 4 spp.) and Aspergillus (5
isolates: 3 spp.) from seeds, and Penicillium spp. (9 isolates: 4 spp.

3.2 | Fungal community by plant part

from seedlings; 2 from seeds). In contrast, only four isolates were
obtained from seeds extracted from fresh fruit harvested in the

Fungal community composition differed among roots, stems,

greenhouse: one each of Aspergillus fumigatus, Penicillium aethiopi-

leaves, flowers, fruits, and seeds (Figures 3 and 4). The combined

cum, an unidentified Penicillium sp., and Pseudogymnoascus panno-

isolates represented 20 taxonomic orders. The dominant orders

rum (Table 4).

across all plant parts were Eurotiales (24%), Hypocreales (19%),

Three hundred and twenty-­five isolates of fungal endophyte

and Pleosporales (10%) (Figure 3). Leaves (12 orders) had the

were obtained in culture from the 17 mature plants grown in the

greatest diversity and abundance of fungal endophytes, followed

greenhouse: 99 from reproductive tissues (flowers, fruit, and

by roots (nine orders), fruits (nine orders), flowers (eight orders),

seeds) (Table 4) and 226 from vegetative tissues (leaves, stems,

seeds (seven orders), and stems (six orders) (Figure 3). The

and roots) (Table 5). Not all of these isolates could be identified to

dominant orders were Eurotiales (25 isolates), Agaricales (24

species. Due to the large number of isolates of some genera, such

isolates), Pleosporales (23 isolates), and Xylariales (21 isolates)

as Penicillium, not all were submitted for ITS sequencing but were

in leaves and Hypocreales (40 isolates), Eurotiales (16 isolates),

identified to genus by microscopic examination. The most com-

and Glomerellales (10 isolates) in roots. The dominant orders in

mon genera isolated from reproductive tissues were Arthrinium/

flowers, fruits, and seeds were Eurotiales (40 isolates), Xylariales

Apiospora spp. (22 isolates; isolated equally from flowers and

(22 isolates), Sordariales (17 isolates), and Hypocreales (15

fruit), Aspergillus spp. (17 isolates), Chaetomium spp. (18 isolates),

isolates), followed by Agaricales (eight isolates). The NMDS

Penicillium/Talaromyces spp. (14 isolates), and Coprinellus micaceus

(stress = 0.0227) analysis showed the similarity/dissimilarity

(six isolates). Coprinellus micaceus was isolated frequently from

in fungal community composition among different plant parts

leaf tissue also (six isolates), plus five isolates of Coprinellus floc-

(Figure 4). The root and leaf fungal communities showed a strong

culosus and two species of the closely related genus Coprinopsis:

distinction from each other and those of the reproductive plant

Coprinopsis alnivora (two isolates) and Coprinopsis cinerea (12

parts (flowers, fruits, and seeds), which were more similar in their

isolates). Other genera frequently isolated from leaves were

endophyte composition. The six orders of fungal endophytes

Alternaria spp. (11 isolates, including three from roots), Aspergillus

isolated from stems were more similar to the communities found in

spp. (13, including one Asp. ochraceus from roots), Botrytis cine-

the reproductive tissues (flowers, fruits, and seeds) than to those

rea (six), Chaetomium spp. [eight, including one isolate from a

in the leaves or roots. Some of the endophytic isolates could have

stem and two Ch. aureum (teleomorph: Arcopilus aureus) from

originated horizontally, that is, from the greenhouse environment,

roots], Cladosporium spp. (12), Epicoccum nigrum (seven, includ-

rather than vertically from within the monk fruit plants themselves

ing one from a root), and Hypoxylon (18: 8 H. macrocarpum and

since the greenhouse was not completely isolated from the

10 H. rubiginosum). Twenty-­s even isolates of Penicillium spp. were

outdoor environment and the soilless media was not sterile.

MN634490.1 (2); MN634664.1

3

Trichoderma viride

Fresh seeds extracted from fresh fruits from China.

Dry seeds purchased via Alibaba.

b

a

Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown.

MN634667.1 (4); MT341775.1 (2);
MT023026.1

7

Trichoderma atroviride

OQ608602.1; MT529218.1

2

Penicillium sumatraense

Hypocreales

KX426968.1

1

Penicillium brevicompactum

Eurotiales

MT337556.1/MK640580.1

MW341297.1

1

Diaporthe unshiuensis
1

MT199841.1

1

Diaporthe subclavata

Exserohilum mcginnisii/E. rostratum

MN650843.1

1

Diaporthe phaseolorum

Pleosporales

MW202983.1

MZ475126.1

1
1

Diaporthe hongkongensis

Colletotrichum qilinense

Diaporthales

KY705054.1; LC379210.1

MN634011.1

2

Colletotrichum brevisporum

1

Botryosphaeria dothidea

MT582757.1

1

Aspergillus tennesseensis

Glomerellales

KX258805.1

1

Aspergillus pseudoglaucus

Botryosphaeriales

MK841469.1; MF773659.1

3

Aspergillus hiratsukae

GenBank accession #

Eurotiales

# of isolates

Name

Order

100

100

97.53; 98.51

99.81

98.8; 98.8

100

100

100

99.25

98.81

100; 100

100

100

99.61

98.39; 99.82; 99.38

% identity

TA B L E 2 Identity of fungal endophytes recovered from dry monk fruit seeds and seeds from fresh fruit from China based on rDNA ITS sequence analyses and morphology.

Alibaba, Fruit

Alibaba

Alibaba

Alibaba

Fruit

Fruit

Fruit

Fruit

Fruit

Fruit

Fruit

Fruit

Alibaba

Alibaba

Alibabaa, Fruitb

Seed source

MA et al.

| 5 of 16

6 of 16

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MA et al.

TA B L E 3 Identity of fungal endophytes recovered from leaves, stems, and roots of monk fruit seedlings grown from seed from China
based on rDNA ITS sequence analyses and morphology.
Order

Name

# of isolates

GenBank accession #

% identity

Source

Hypocreales

Beauveria bassiana

1

MT441874.1

99.8

Stem

Eurotiales

Chromocleista sp.

1

MN644766.1

99.83

Stem

Mortierellales

Mortierella sp.

1

HE605241.1

100

Stem

Eurotiales

Paecilomyces tabacinus

1

LT548280.1

100

Root

Eurotiales

Penicillium citrinum

2

MN634531.1; MT597829.1

100; 100

Stem

Hypocreales

Penicillium meleagrinum

1

MF135516.1

99.82

Stem

Penicillium steckii

1

OP615071.2

99.82

Stem

Talaromyces islandicus

2

FR670311.1

89.96;
89.93

Root

Trichoderma afroharzianum

4

MN644793.1

100; 99.83
(2);
99.66

Root; Stem

Trichoderma asperellum

2

KY659051.1; LN846687.1

100; 99.82

Root

Trichoderma atroviridae

2

MT604177.1;
MT626716.1

100; 99.43

Stem

Trichoderma harzianum

2

MT626717.1; MF078650.1

100; 99.65

Root; Leaf

Trichoderma harzianum/T. lixii

1

MH339867.1/EF596951.1

100/100

Stem

Trichoderma sp.

1

MK870660.1

100

Stem

Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown.

4

|

DISCUSSION

resinae), which was isolated from both leaves and flower buds, damages jet fuel, diesel, petroleum and creosote-­treated wood, but may

Monk fruit plants proved to be a rich source of fungal endophytes

have useful environmental applications in remediation of hydrocar-

with a great diversity and abundance, especially in leaves. The role

bon contaminated sites (Rafin & Veignie, 2018). Chaetomium spp. are

of these fungi in the monk fruit plants is likely to be as complex as

the source of more than 100 useful secondary metabolites (Dwibedi

their diversity. Some may be neutral commensalists, while others,

et al., 2023). For example, Arcopilus aureus (anamorph: Chaetomium

such as the wood-­
decaying Xylariaeceae (Hypoxylon, Nemania),

aureum) produces high levels of resveratrol, a potent antioxidant,

Meruliaceae (Phlebia tremellosa), Psathyrellaceae (Coprinellus and

and sclerotiorin, which has anti-­
cancer properties (Dwibedi &

Coprinopsis spp.), and Polyporaceae (Trametes hirsuta), may play

Saxena, 2018). A. aureus has high lead tolerance and clearance, sug-

a beneficial role in vegetative decay and nutrient cycling in the

gesting a potential role in bioremediation of contaminated soils (Da

natural environment, or protection against pathogens or herbivores.

Sila et al., 2018).

Members of the Xylariales, in particular, produce a wide array of

Several of the endophytic species obtained in this study have

secondary metabolites many of which are antagonists of other

potential agricultural applications in enhancing plant growth and

fungi and bacteria (Becker & Stadler, 2021). A few of the species

tolerance to drought and other environmental stresses, or as bio-

isolated may be hyperparasites of other fungal endophytes found

logical control agents of disease and insect pests. The abundance

in the monk fruit tissues, for example, Penicillium [Eupenicillium]

and diversity of the fungal endophytes recovered from the monk

cinnamopurpureum which grows on the heads of Aspergillus spp.

fruit plants suggest multiple, layered means of protection against

(Horn & Peterson, 2008).

potential pests and adaptation to environmental stresses. Many

In addition to the Xylariales, many of the other fungal species

endophytic species with anti-­f ungal or plant growth-­p romoting

obtained from the monk fruit plants are known to produce bioac-

activity recovered in this study have also been isolated from

tive compounds with medical or industrial applications. For example,

grapevines (Vitis vinifera L.) (Kulišová et al., 2021), including

Talaromyces purpureogenus (Keekan et al., 2020) and Penicillium brev-

species of Aspergillus, Alternaria, Chaetomium, Epicoccum, and

icompactum (Fonseca et al., 2022) produce pigments with commer-

Penicillium. These and several other species isolated from leaves

cial applications in the food processing industry. Several species are

and fruit skin, are also common epiphytes that play a role in crop

known to produce antibiotics, such as diketopiperazine, produced

protection both on and below the leaf surface, and are often

by Paraphaeosphaeria sporulosa, which is effective against salmo-

transmitted horizontally. In grape, the most effective antifungal

nella bacteria (Carrieri et al., 2020). Panaeolus subbalteatus is one of

endophytes against Botrytis cinerea, the cause of bunch rot, were

the most common sources of psilocybin, used in medical treatment.

Alternaria and Epicoccum species which, along with Aspergillus

The kerosene fungus, Amorphotheca resinae (anamorph: Hormoconis

fumigatus, produce high levels of siderophores and antioxidants

Morphology only
MK518438.1

1
1

Alternaria sp.

Alternaria alternata

GU266274.1/OP237040.1

1

Arthrinium phaeospermum/Apiospora
rasikravandrae

Morphology only

9

Chaetomium spp.

Cladosporium spp.

Coprinellus micaceus

Crustomyces sp./C. subabruptus

Epicoccum nigrum

Fusarium graminearum

Agaricales

Agaricales

Pleosporales

Hypocreales

1

1

1

6

4

KJ017740.1

FM200455.1

MN905889.1/MK454922.1

LR961895.1; MF156262.1;
MH855975.1; LR961895.1

Morphology only

MZ724883.1

3

Chaetomium novozelandicum

Cladosporiales

MT520580.1
KY132166.1; KP067224.1

1
5

Chaetomium cochliodes

Chaetomium globosum

OP794013.1

MT111139.1

Sordariales

1

4

Botrytis cinerea

MH861876.1
MH345899.1

1
2

Aspergillus septulus

Aspergillus tamarii

Beauveria bassiana

MN533721.1; MT447480.1;
MN533721.1

8

Aspergillus ochraceus

Helotiales

Morphology to leaf isolates
MT529448.1; MT529125.1;
MH793851.1

6

Aspergillus fumigatus

Hypocreales

Eurotiales

KX378907.1; KX378907.1

OW982982.1

20

Arthrinium spp.

1

Apiospora kogelbergensis

Xylariales

MN242723.1

Xylariales

1

Amorphotheca resinae

Heliotiales

OQ207544.1/KU523862.1

Pleosporales

1

Acremonium sclerotigenum/Scopulariopsis
gossypii

GenBank accession #

Hypocreales

# of isolates

Name

Order

99.59

99.4

99.67/99.50

99.69; 99.08; 99.7;
98.06

—

96.7

98.43; 99.81

99.03

—

99.8

99.62

99.12

99.82

99.62; 99.81; 99.63

—

99.65/99.47

99.63; 96.71

99.25

97.86

99.43

—

99.81/99.81

% identity

Flower

Fruit skin

Fruit skin

Flower (3); Flower
bud (3)

Flower bud (3); Fruit
pulp (1)

Fruit skin

Flower (1);
Fruit skin (4)

Fruit skin

Flower (1); Fruit skin
(8)

Fruit skin

Fruit skin

Fruit skin

Fruit skin

Flower (1); Flower
bud (1)
Fruit pulp (1); Fruit
skin (5)

Fruit pulp (4); Fruit
skin (1); Seed (1)

Fruit skin

Flower (7); Fruit pulp
(3); Fruit skin (10)

Fruit skin

Flower bud

Flower

Fruit skin

Fruit pulp

Source

(Continues)

252744

252789

252743

252796

252758

252772

252751

DAOMC ID #

TA B L E 4 Identity of fungal endophytes recovered from flowers, flower buds, fruits, and seeds of mature monk fruit plants grown in the KPU research greenhouse based on rDNA ITS
sequence analyses and morphology.

MA et al.

| 7 of 16

Morphology only

ON428665.1
MG554368.1/KX610136.1
MT797199.1/MT441635.1

5

1
1
1

Paecilomyces variotii

Penicillium spp.

Penicillium aethiopicum

Penicillium citrinum /P. steckii

Penicillium glabrum /P. corylophilum

Eurotiales

99.66

99.81

99.29/97.7

100

99.61

99.7

99.44/99.44

99.82/99.64

98.89

—

99.47

99.75

% identity

Flower

Fruit skin

Fruit skin

Flower

Seed

Flower bud

Flower bud

Fruit skin

Seed

Flower (1);
Fruit pulp (1); Fruit
skin (2); Seed (1)

Fruit skin

Fruit skin

Source

252776

252747

252762

252741

252748

252742

DAOMC ID #

a

All five isolates were the same Talaromyces species; no specific ID in GenBank.

Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown, and the specimen ID # of isolates deposited in the Canadian Collection of Fungal Cultures
(DAOMC).

MF161297.1

MT635321.1

1

1

Talaromyces pupureogenus

Trametes hirsuta

MK450749.1

5a

Talaromyces sp.

Polyporales

MH063252.1

1

Rhinocladiella similis

Eurotiales

KF156305.1

Chaetothyriales

OL436998.1

1

Pseudogymnoascus pannorum

1

Phlebia tremellosa

Incertae sedis

OW988300.1

Polyporales

1

MG554248.1

Eurotiales

1

Hyphopichia burtonii

GenBank accession #

Saccharomycetales

# of isolates

Name

Order

TA B L E 4 (Continued)

8 of 16

|
MA et al.

Aureobasidium pullulans

Beauveria bassiana

Bionectria sp. (anamorph: Clonostachys
sp.)

Blastobotrys sp.

Botrytis cinerea

Cephalotheca sulfurea

Cephalotrichum purpureofuscum/
Doratomyces sp.

Chaetomidium leptoderma

Hypocreales

Saccharomycetales

Helotiales

Cephalothecales

Microascales

Sordariales

4

1

6

1

2

3

1

NR_164219.1; JN573175.1

OP038661.1/KU954345.1

OM262341.1

OM349592.1; MT150132.1;
MH992148.1; AB693927.1;
MK513827.1; MF661902.1

MK246187.1

MH729023.1; KU951245.1

OK331343.1

MT645930.1

MK332591.1

1

Aspergillus tamarii

Hypocreales

Morphology to flower/fruit isolates
MN533721.1; MT447480.1;
MN533721.1

4

Aspergillus ochraceus

Dothidiales

MN956655.1
MT529448.1; MT529125.1;
MH793851.1

1
7

KU702707.1

KX378907.1; KX148691.1

OW982982.1

MN242723.1; KJ207403.1

Aspergillus flavipes

Eurotiales

1

4

1

2

Aspergillus fumigatus

Arthrinium spp.

Arthrobotrys amerospora

Xylariales

Apiospora kogelbergensis

Xylariales

Orbiliales

Amorphotheca resinae

Heliotiales

MK801346.1

1
8

Alternaria infectoria

Alternaria spp.

OK274326.1; OK274326.1;
KX139150.1; MK640587.1;
MW534563.1; HQ649962.1

OP696965.1; OL711657.1

2

Alternaria alternata

KP131521.1

1

Acremonium hyalinulum

Pleosporales

MH858153.1

1

Acremonium roseolum

GenBank accession #

Hypocreales

# of isolates

Name

Order

97.61; 97.68; 97.86

99.29/99.47

99.61

100; 100; 99.39; 100;
100; 88.25

99.81

99.61; 99.67

98.46

93.0

Root bulb (3);
Root hair (1)

Leaf

Leaf

Leaf

Root bulb

Root bulb (1);
Root hair (1)

Leaf

Leaf

Leaf

Leaf (3); Root
hair (1)

—

97.99

Leaf (6)

Leaf

Root hair

Leaf (4)

Leaf

Leaf (2)

Leaf (6); Root
bulb (2)

Leaf

Leaf (1); Root
bulb (1)

Leaf

Leaf

Source

98.92; 99.28 95.01

99.62

99.83

100; 99.24

99.25

98.34; 95.96

100; 99.46; 84.0; 99.5;
98.67; 99.38

95.15

99.61; 99.62

98.68

98.66

% identity

252763

252788

252787

252754

252759

252764

252753

252756

(Continues)

DAOMC ID #

TA B L E 5 Identity of fungal endophytes recovered from leaves, stems, and roots of mature monk fruit plants grown in the KPU research greenhouse based on rDNA ITS sequence analyses
and morphology.

MA et al.

| 9 of 16

MK905459.1

1
9

Cladosporium tenuissimum

Cladosporium spp.

MH729023.1/KU951245.1
KM921661.1
KR906700.1; KC304797.1; FJ824032.1;
MT529814.1
MN833356.1

10
1

Fusarium oxysporum

Fusarium tricinctum

MH086786.1

1

Hypocreales

1

OP315769.1; OP315769.1; OP315769.1;
MH861752.1

MN612779.1

1

Fomitopsis mounceae

Polyporales

7

1

Fusarium haematococcum/F. solani

Epicoccum nigrum

Pleosporales

Fusarium lichenicola

Didymella anserina

Pleosporales

MK335735.1

LC605635.1

1

1

Curvularia coatesiae

Diaporthe eres

NR_170004.1; OK117928.1

1

MN841919.1; MF351861.1;
MN841919.1; MN841919.1

12

Curvularia canadensis /C. inaequalis

MZ407758.1

2

Coprinopsis cinerea

MF156262.1; MF156262.1; LR961895.1;
LR961895.1; MF156262.1;
LR961895.1

6

Coprinellus micaceus

Coprinopsis alnivora

MK656240.1

5

Coprinellus flocculosus

Diaporthales

Pleosporales

Agaricales

Agaricales

OP006753.1

1
ON208763.1; KT826671.1;
MH137774.1

ON712476.1

1

Cladosporium ramotenellum

MZ724883.1; MZ724884.1
MH861746.1

2
1

Chaetomium novozelandicum

Chaetomium spinosum

Cladosporium herbarum

KP067223.1; MF476072.1

3

Chaetomium globosum

Cladosporiales

KP278194.1; MW533023.1

2

Chaetomium aureum (teliomorph:
Arcopilus aureus)

GenBank accession #

Sordariales

# of isolates

Name

Order

TA B L E 5 (Continued)

99.81

95.63; 99.8; 99.8;
98.29

99.42

99.61/99.67

98.03

100; 100; 99.59; 99.79

99.14

99.63

96.53

99.62; 99.62

96.91; 99.68; 99.69;
99.85

98.02

100; 99.84; 99.84;
99.23; 99.84; 98.06

96.88; 96.88; 97.31

93.16; 98.99; 98.39

99.39

99.8

99.4

99.05

98.82; 99.81

100; 98.21

100; 100

% identity

Root bulb

Root bulb (6);
Root hair (4)

Root bulb

Root hair

Leaf

Leaf (6); Root
bulb (1)

Leaf

Root bulb

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf

Leaf (2);
Stem (1)

Root bulb

Source

252752

252778

252740

252770

252755

252765

252766;
252775

252779

DAOMC ID #

10 of 16

|
MA et al.

MH865756.1
MK534497.1/KM613146.1;
MK534497.1/MT303132.1
MH655003.1
KX610174.1/MT582790.1

1
2
1
1
22

Lophiostoma corticola /
Angustimassarina coryli

Mortierella hyalina

Nemania sp.

Nigrospora oryzae

Panaeolus subbalteatus

Paraconiothyrium fuckelii

Paraphaeosphaeria sporulosa

Penicillium canescens

Penicillium cataractarum /P.
simplicissimum

Penicillium cinnamopurpureum

Penicillium citrinum/P. steckii

Penicillium spp.

Mortierellales

Xylariales

Xylariales

Agaricales

Pleosporales

Pleosporales

Eurotiales

Pleosporales

Unidentified

ON927102.1; MW850542.1
MT447499.1

2
9

Plectosphaerella cucumerinum

Plectosphaerella oligotrophica

Glomerellales

1

1

MG916998.1

NR_165996.1; MH857776.1

ON520767.1

Phialemonium inflatum

1

Phaeosphaeria sp.

Cephalothecales

MK907734.1

Pleosporales

1

Periconia byssoides

OP035353.1; OP647345.1;
KY401082.1; MH512953.1;
MH512953.1; MH512953.1;
ON182131.1; ON182131.1;
ON182131.1; ON182131.1

KX302013.1; MH859903.1

MK052700.1

MH855553.1

KC131293.1

MT153669.1

MT003063.1

MK907710.1/MF167431.1

Pleosporales

5

1

1

1

1

1

1

MH857054.1

KX343155.1; MN541090.1

Pleosporales

1

Linnemannia zychae

3

Lasiosphaeriaceae

Mortierellales

AY787708.2; MT214998.1

10

Hypoxylon rubiginosum

Sordariales

HM192912.1

8

Hypoxylon macrocarpum

GenBank accession #

Xylariales

# of isolates

Name

Order

TA B L E 5 (Continued)

99.22

99.6; 99.8

99.4; 99.8

99.61; 96.58

99.6

Leaf

Root bulb (3);
Root hair (5);
Stem (1)

Root bulb

Stem

Leaf

Leaf

Leaf (11); Root
bulb (11)

99.63

Root bulb
99.62; 99.06; 99.44;
99.26; 99.45;
99.26; 100; 100;
99.81; 99.81

Leaf

Root bulb (1);
Root hair (1)

Leaf

Root bulb (4);
Root hair (1)

Leaf

Leaf

Leaf

Leaf

Root bulb

Leaf

Root bulb

Root hair

Leaf

Leaf

Source

95.16/94.72

99.65

99.44/100;
99.63/99.45

99.62

99.82; 99.82

99.29

98.73

99.41

99.22

99.83

100/100

99.83

99.8; 99.59

99.80; 99.80; 99.80;
99.41; 99.80; 100;
91.2; 92.88

96.38; 99.46; 98.6;
98.43; 99.3; 98.78;
97.69; 99.47

% identity

252768

(Continues)

252782; 252784

252777; 252790

252783

252785

252795

252746

252767

252761; 252771

252792

252791

252757

252749

252794

252774

252769; 252773

DAOMC ID #

MA et al.

| 11 of 16

Varicosporium delicatum

Helotiales

1

1

JQ412864.1

GQ888733.1

Morphology only

93.75

91.12

—

96.0

99.26

99.47

91.9

99.63; 99.28; 99.27

99.83

99.83

99.44/99.25;
99.81/99.62;
99.62/99.43;
100/99.81

99.64

% identity

Leaf

Leaf

Root bulb (1);
Root hair (3)

Root bulb

Leaf

Root bulb

Root bulb

Root bulb

Leaf

Leaf

Root bulb (9);
Root hair (2)

Leaf

Source

252793

252760

252745

252750

252786

252780; 252781

DAOMC ID #

Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown, and the specimen ID # of isolates deposited in the Canadian Collection of Fungal Cultures
(DAOMC).

Ustanciosporium appendiculatum

4

Trichoderma spp.

Ustilaginales

JX273473.1

1
1

Sordaria fimicola

Trichoderma ghanense

Sordariales

Hypocreales

MT520628.1

KC403970.1
MW260103.1

1
1

Simplicillium obclavatum

AB604004.1; MK579181.1;
MK579181.1

OW987158.1

3

1

Simplicillium subtropicum

Simplicillium aogashimaense

Hypocreales

ON500589.1

1

Schizophyllum commune

Scopulariopsis brevicaulis

Agaricales

Microascales

KJ862077.1
KX384658.1/MF077236.1;
KX384658.1/MF077236.1;
KF293986.1/MF077237.1;
KF293986.1/ON500613.1

1
11

Purpureocillium lilacinum

Sarocladium kiliense/S. strictum

Hypocreales

GenBank accession #

Hypocreales

# of isolates

Name

Order

TA B L E 5 (Continued)

12 of 16

|
MA et al.

| 13 of 16

MA et al.

F I G U R E 3 Number of fungal isolates in different taxonomic orders isolated from roots, stems, leaves, flowers, fruits, and seeds of monk
fruit.

and S. obclavatum, isolated here from monk fruit root bulbs, are
mycoparasites that have shown efficacy against, respectively,
powdery mildew and stripe rust of wheat (Wang et al., 2020;
Zhu et al., 2022). Paecilomyces variotii is an effective biocontrol
agent of gummy stem blight and powdery mildew of cucumber,
and has been shown to inhibit other plant pathogens including
nematodes (Moreno-­G avíra et al., 2021). Purpureocillium lilacinum [syn. Paecilomyces lilacinus (Thom) Samson] is a parasite of
nematode eggs (Kiewnick & Sikora, 2004), an entomopathogen,
and has been shown to promote the growth of tomato under
heavy metal stress (Musa et al., 2023). Strains of P. lilacinum
have been registered in the USA and Europe for control of parasitic nematodes in crops. Arthrobotrys amerispora, isolated from
a root hair of the monk fruit, may be playing a role in root proF I G U R E 4 Measure of dissimilarity in the endophytic fungi
composition among the root, stem, leaf, flower, fruit, and seed of
monk fruit using non-­metric multidimensional scaling.

tection; Arthrobotrys spp. are well-­
k nown nematode-­
t rapping
fungi as well as mycoparasites (Gams et al., 2004). Eight endophytic strains of the entomopathogen Beauveria bassiana were
recovered from the monk fruit tissues, in addition to a Bionectria
sp. (anamorph: Clonostachys; syn. Gliocladium) and several

(Kulišová et al., 2021). Endophytic strains of E. nigrum have

Trichoderma spp., which are well-­
k nown protectors of plants

been shown to reduce the incidence and severity of a range

from pathogen and insect attack, as well as plant growth pro-

of plant diseases (Taguiam et al., 2021). In British Columbia,

moters (Sharma & Gothalwal, 2017).

an isolate of E. nigrum from mummy berry-­infected blueberries

For some plant pathogenic fungi, existence as an endophyte

suppressed spring apothecia production of Monilinia vaccinii-­

may be a latent stage in pathogenesis. Disease develops as the

corymbosi when applied to soil after infected berries dropped

host plant reaches a certain life stage or begins to senesce, or

(Kitura et al., 2023). Hypoxylon rubiginosum has shown promise

as the plant experiences environmental stress or other damage.

as a biocontrol for dieback of European ash (Fraxinus excelsior

Botrytis cinerea, for example, is a common pathogen causing gray

L.), associated with its production of the anti-­f ungal metabolite,

mold disease of many crops but is often found as an endophyte

phomopsidin (Halecker et al., 2020). Simplicillium aogashimaense

in healthy plant tissues. The two Colletotrichum spp. isolated

14 of 16

|

MA et al.

from the internal tissues of monk fruit seeds in this study are

DATA AVA I L A B I L I T Y S TAT E M E N T

known plant pathogens and may be a quiescent stage in the de-

The data that support the findings of this study are openly available

velopment of anthracnose disease. Plectosphaerella cucumerinum

in figshare: 10.6084/m9.figshare.24530542.

(syn. Plectosporium tabacinum) causes wilt and root rot of several
crops including cucurbits, tomato, potato, and basil (Raimondo &

ORCID

Carlucci, 2018) and may be a quiescent pathogen in the monk fruit

Li Ma

https://orcid.org/0000-0003-4739-1670

plants, while Pl. oligotrophica is a low-­c arbon feeding, soil saprophyte (Liu et al., 2013) that may be neutral, or play a beneficial role

REFERENCES

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multiple potential roles of a single endophytic species, Pl. cucumerinum is also nematophagous and has been tested for biocontrol
of potato cyst nematode (Atkins et al., 2003), although, more recently, it has also been shown to cause potato wilt disease in China
(Gao et al., 2016) and Pakistan (Alam et al., 2021). Paraconiothyrium
fuckelii (syn. Leptosphaeria coniothyrium, basionym: Coniothyrium
fuckelii) is a wound pathogen causing cane blight of raspberry,
rose, and other woody hosts worldwide (Guarnaccia et al., 2022).
It is also known as a saprobe, but its potential role as an endophyte
in these hosts has not been explored.
Among some species of plant pathogens, endophytic and
pathogenic strains have quite different relationships and effects
on their hosts. Endophytic strains of Fusarium oxysporum have
been shown to reduce root rot and wilt diseases caused by pathogenic strains in tomato and other crops (de Lamo & Takken, 2020).
The endophytic strains of F. oxysporum have fewer effectors and
exhibit different patterns of tissue colonization and triggering of
host defenses than pathogenic strains. Further understanding
of the role of endophytes in plant protection and pathogenesis
may reveal additional new, sustainable methods of plant disease
control.
In summary, monk fruit plants can be easily grown in the
greenhouse and are a prolific source of endophytic fungi and secondary metabolites for potential research and development. This
work has deepened our understanding of the intricate interactions
between plants and fungi that sustain ecosystems and underpin
plant health and resilience. These findings can inform strategies
for developing climate-­resilient crops and restoring ecosystems in
the face of climate challenges and developing more sustainable
and eco-­
f riendly strategies for plant health management. Our
analysis did not include bacterial or viral endophytes, or fungi that
did not grow on PDA. Further investigation of monk fruit as a potential source of these endophytes may reveal even more useful
strains and advance our understanding of how endophytes interact with their hosts.
AC K N OW L E D G M E N T S
We thank NutraEx Food Inc. for financial support. We also thank
Erwin Yamzon for his advice on statistical analyses and Yasaman
Morshedikermani for preparing the isolates for DAOMC.
C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

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MA et al.

How to cite this article: Ma, L., Elmhirst, J. F., Darvish, R.,
Wegener, L. A., & Henderson, D. (2024). Abundance and
diversity of fungal endophytes isolated from monk fruit
(Siraitia grosvenorii) grown in a Canadian research
greenhouse. Plant-­Environment Interactions, 5, e10142.
https://doi.org/10.1002/pei3.10142