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Plant-Environment Interactions. 2024;5:e10142.
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https://doi.org/10.1002/pei3.10142
wileyonlinelibrary.com/journal/pei3
1 | INTRODUCTION
Monk fruit [Siraitia grosvenorii (Swingle) C. Jef frey ex A.M. Lu & Zhi Y.
Zhang] is an herbaceous perennial vine of the Cucurbitaceae family
cultivated commerciall y mainly in the sout hern parts of Ch ina though
it is grown also in northern Thailand and has been exported to the
USA and India (Shivani et al., 2021). It is commonly grown in Yongfu,
Longsheng, and Lingui counties in northern Guangxi Province with
an annual average temperature of 16–20°C, average precipitation
of1500–2002 mm,andaveragesunshineof1237.3 ~ 1626.4 h(Zeng
et al., 2011).
The fruit of the monk fruit vine has been used as natural, calorie-
free sweeteners (Xia et al., 2008) as well as folk medicine in China
for thousands of years due to their pharmaceutical properties such
as anti- inflammation (Di et al., 2011), anti- carcinogenesis (Takasaki
et al., 2003), anti- oxidation, and anti- obesity (Sun et al., 2012).
Mogrosides are the main compounds in the fruit responsible for the
medicinal activities and sweetness.
Received:12January2024
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Revised:18M arch2024
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Accepted :19March2024
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
This is an op en access ar ticle under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provide d the original work is properly cited.
© 2024 The Aut hors. Plant-Environment Interactions published by N ew Phytologist Foundation and John Wiley & Sons Ltd.
1Institute for Sustainable Horticulture,
Kwantlen Polytechnic University, Surrey,
British Columbia, Canada
2Elmhirst Diagnostics and Research,
Abbotsford, British Columbia, Canada
Correspondence
Li Ma, Ins titute for Sustainable
Horticulture, Kwantlen Polytechnic
University,1266672ndAvenue,Surrey,
BC V3W 2M8, C anada.
Email: li.ma6@kpu.ca
Janice F. Elmhirst, Elmhirst Diagnostics
andResearch,5727River sideStreet,
Abbot sford, 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 ver y 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
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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 eco-
systems are hosts to one or more fungal endophytes, which may re-
side within roots, stems, leaves, and/or other plant parts (Petrini, 198 6;
Stone et al., 20 04). The symbiotic relationship between fungal endo-
phytes and their host s 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 trans-
mission of endophytic fungi is primarily horizontal via airborne spores;
some however can transmit vertically to new host generations via seed
infections (Aly et al., 2011; Saikkonen et al., 2002). Besides their signif-
icant impacts on the survival and fitness of plants by conferring stress
tolerance, increasing water use efficiency and plant biomass, or de-
creasing fitness by altering resource allocation (Rodriguez et al., 2009),
endophy tic fungi also have great potential as a unique source of bi-
ologically active compounds with promising applications in medicine,
pharmacy, and agriculture (Aly et al., 2010; Nisa et al., 2015; Zhang
et al., 2006).
It has been shown that both fungal and bacterial endophytes
can modify their genes by absorbing part of the host DNA into
their genome for adaptation to the specific microenvironment (Aly
et al., 2011; Germaine et al., 2004), which may help explain the ability
of some endophytes to produce the same phytochemicals as those
produced by their host plants (Stierle et al., 1993). Chen et al. (2020)
isolated 15 endophytic fungal strains from roots, stems, leaves, and
fruits of S. grosvenorii and found that two of them, Diaporthe angeli-
cae Berk. Wehm. [syn. Mazzantia angelicae (Berk.) Lar. N. Vassiljeva]
and Fusarium solani (Mart.) Sacc., could produce some of the phyto-
chemicals produced by the host plant. The other endophytic strains
isolated from monk fruit were not named in the published report
(Chen et al., 2020). There is very little information available about
the fungal endophytes in monk fruit. The present study aimed to
explore the abundance and diversity of fungal endophytes in monk
fruit grown in a Canadian research greenhouse environment, where
we can manipulate the environment to mimic the natural cultivat-
ing conditions of monk fruit and minimize their interactions with the
outdoor environment and potential contaminants. This also avoided
the introduction of novel fungal species into the environment.
2 | MATERIALS AND METHODS
2.1 | Isolating endophytic fungi from seeds
In 2020, dr y monk fruit seeds obtained via Alibaba from Guangxi
Naturix Import & Export Co., Ltd. (Nanning, Guangxi, China) and seeds
extracted from commercial fresh fruits (Figure 1) purchased from
China. Fungal endophytes were isolated from seeds following the
method used by Shearin et al. (2018) with modifications. Seeds were
surface sterilized with 10% bleach for 2 min, rinsed with sterile reverse
osmosis water three times, and then placed on two types of microbial
growth media in petri dishes: potato dextrose agar (PDA) incorporated
with 0.0 05% streptomycin, and water agar (WA) media. The rinse
water was plated as a control to ensure that the sur face sterilization
process was thorough. If fungal colonies were observed in the control
plates, the plates were discarded and new seed samples were surface-
sterilizedandplatedagain. Plates were kept in anincubator at 27°C
and monitored regularly. All fungal endophytes were recovered from
the media and each endophyte was sub- cultured up to three times
until a pure culture was obtained for identification.
2.2 | Growing plants
Plants were grown from seeds extrac ted from the fresh fruit from
China. Af ter removing the seed coat, seeds were surface sterilized
with 10% bleach and placed on Murashige and Skoog medium in
petri dishes to germinate. Seedlings were transplanted into Sunshine
Mix #2 pot ting media in 10 cm (4-inch)p ots and kept in a g rowth
chamber at 21°C and a 16 h light period for 10–12 weeks. After
five see dlings were taken for e ndophyte isolat ion at 9–10 weeks,
the remaining seedlings were transplanted into Sunshine Mix #4 in
15 cm(one-gallon)pots,oneplantperpot,andplacedintheresearch
greenhouse located on the KPU Langley campus in January 2021.
PlantsweregrownintheresearchgreenhousewithRHaround75%,
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)andmicronutrients
(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 individualemitter in each pot. Floweringbegan in
late June to early July 2021 and pollination was conducted by hand
using a fine paintbrush early in the morning when flowers were
open. Fruits were harvested in October and November (Figure 2).
FIGURE 1 Freshmonkfruitseedscollectedfromfruits.
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MA et al.
2.3 | Isolating endophytic fungi from the fresh
tissues of monk fruit seedlings and mature plants
Samples of roots, stems, and leaves from five seedlings
(9–10 weeksold)inthegrowthchamberweretakenforendophyte
isolation following the methods described by Musa et al. (2023).
Small pie ces (about 0.5 c m × 0.5 cm in size) of plan t tissue were
surface sterilized and rinsed with sterile reverse osmosis (RO)
water using the method described above for isolation of seed en-
dophytes. Fungal hyphae emerging from the tissue were selected
and transferred repeatedly to PDA+50 ppmstreptomycintoob-
tain a pure culture. Endophytes were isolated from leaves (young
and old), stems (young and old), roots (from bulb and roots in soil),
flowers (buds and fully- open flowers), and fruit (pulp, seeds, and
ski nseparated)atdifferentmaturi tys tagesfrom17matu remonk
fruit plants grown in the greenhouse ( Table 1). The isolation and
purification procedures were the same as for seeds and seedlings
described above.
2.4 | Identifying endophytic fungi
After pure cultures of endophytes were obtained, they were iden-
tified morphologically by microscopy and genetically by DNA
sequencing. DNA was extracted using a protocol described by
Cenis (1992) and subsequently amplified by polymerase chain reac-
tion (PCR) using general internal transcribed spacer (ITS) primers,
ITS1 and ITS 4 (White et al., 1990). The PCR product s were sent
for sequencing to Psomagen Inc., Rockville, MD, USA. The internal
transcribed spacer (ITS) sequences of the endophytic fungi were
compared to sequences deposited in GenBank using the National
Centre for Biotechnology Information (NCBI) nucleotide basic local
alignment search tool (BL ASTn) (http:// www. ncbi. nlm. nih. gov/
BLAST ). Isolates were identified to genus and species based on the
highest % identity in BLASTn, and morphological characteristics
obtained by microscopy. Where more than one identification was
possible in GenBank, the genus or species was confirmed by micro-
scopic comparison of fungal morphology to published descriptions.
In a few cases where similar genera or species that could not be reli-
ably resolved by BLAST analysis or microscopic examination, both
names are shown. Subsequently, each fungal taxon was classified
using the NCBI taxonomy browser database, US National Library of
Medicine, Bethesda, MD (https:// www. ncbi. nlm. nih. gov/ taxon omy/
brows er/ wwwtax. cgi). Fif ty- 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
Cultures (DAOMC) in Ottawa, ON, Canada, under specimen num-
ber s252740 –252796.
FIGURE 2 MonkfruitplantsgrownintheresearchgreenhouseatKwantlenPolytechnicUniversity,Langley,BritishColumbia,Canada.
TABLE 1 Numberofsamplescollec tedfrom17fruitingmonkfruitplantsgrownintheresearchgreenhouseattheInstitutefor
Sustainable Horticulture, KPU in 2021.
Leaves Flowers Fruit Stems Root s Total samples
71 60 15 35 72 253
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2.5 | Analysis of endophytic fungal communities
Non- metric multidimensional scaling (NMDS) was carried out to
explore the similarit y of fungal communities among roots, stems,
leaves, flowers, fruit s, and seeds based on fungal orders (Peters
et al., 2020). NMDS was performed using Py thon (3.9.16) (Van
Rossum & Drake, 1995) with MDS implemented in the scikit- learn
(sklearn) library.
3 | RESULTS
3.1 | Overall fungal community composition
At least 150 species of fungal endophytes representing approxi-
mately 60 genera and 20 orders were recovered in culture from the
monk fruit tissues. Twenty- seven isolates of endophytic fungi were
obtained from Chinese monk fruit seeds, either dry (purchased
through Alibaba) or extracted from fresh fruit from China (Table 2).
Another 22 isolates were obtained from seedlings grown from the
fresh seeds (Table 3). The most common genus isolated from seeds
and seedlings combined was Trichoderma(22isolates:7or8spe-
cies), followed by Diaporthe (4 isolates: 4 spp.) and Aspergillus (5
isolates: 3 spp.) from seeds, and Penicilliumspp.(9isolates:4spp.
from seedlings; 2 from seeds). In contrast, only four isolates were
obtained from seeds ex tracted from fresh fruit harvested in the
greenhouse: one each of Aspergillus fumigatus, Penicillium aethiopi-
cum, an unidentified Penicillium sp., and Pseudogymnoascus panno-
rum (Table 4).
Three hundred and twenty- five isolates of fungal endophyte
wereobtainedinculturefrom the17matureplantsgrowninthe
greenhouse: 99 from reproductive tissues (flowers, fruit, and
seeds) (Table 4) and 226 from vegetative tissues (leaves, stems,
and roots) (Table 5). Not all of these isolates could be identified to
species. Due to the large number of isolates of some genera, such
as Penicillium, not all were submitted for ITS sequencing but were
identified to genus by microscopic examination. The most com-
mon genera isolated from reproductive tissues were Arthrinium/
Apiospora spp. (22 isolates; isolated equally from flowers and
fruit), Aspergillusspp.(17isolates),Chaetomium spp. (18 isolates),
Penicillium/Talaromyces spp. (14 isolates), and Coprinellus micaceus
(six isolates). Coprinellus micaceus was isolated frequently from
leaf tissue also (six isolates), plus five isolates of Coprinellus floc-
culosus and two species of the closely related genus Coprinopsis:
Coprinopsis alnivora (two isolates) and Coprinopsis cinerea (12
isolates). Other genera frequently isolated from leaves were
Alternaria spp. (11 isolates, including three from roots), Aspergillus
spp. (13, including one Asp. ochraceus from roots), Botrytis cine-
rea (six), Chaetomium spp. [eight, including one isolate from a
stem and two Ch. aureum (teleomorph: Arcopilus aureus) from
roots], Cladosporium spp. (12), Epicoccum nigrum (seven, includ-
ing one from a root), and Hypoxylon (18: 8 H. macrocarpum and
10 H. rubiginosum). Twenty- seven isolates of Penicillium spp. were
obtained, 13 from leaves and 14 from roots. Of the 11 isolates of
Plectosphaerella obtained, nine were Pl. oligotrophica and two Pl.
cucumerinum; all were from root s except one from a stem. Genera
isolated frequently only from roots included Fusarium (13 isolates,
including 10 F. oxysp orum and one F. haematococcum/F. solani),
Paraphaeosphaeria sporulosa (five), Sarocladium kiliense/S. strictum
(11), Simplicillium spp. (five), and Trichoderma spp. (five). Only three
fungal endophytes were obtained in 35 samples from mature plant
stems: one isolate each of Chaetomium globosum, Plectosphaerella
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
fruit skin), Chaetomium globosum, Cladosporium spp., Epicoccum ni-
grum (one from fruit skin), Penicillium citrinum/P. steckii and other
Penicillium and Talaromyces spp. Many other endophytic fungi
were isolated only once from mature monk fruit plant tissues. Four
isolates produced no match to ITS sequences in GenBank at the
genus or species level and could be identified only as members of
the Lasiosphaeriaceae or Pleosporales.
3.2 | Fungal community by plant part
Fungal community composition differed among roots, stems,
leaves, flowers, fruit s, and seeds (Figures 3 and 4). The combined
isolates represented 20 taxonomic orders. The dominant orders
across all p lant part s were Eurotiales (24%), Hy pocreales (19%),
and Pleosporales (10%) (Figure 3). Leaves (12 orders) had the
greatest diversity and abundance of fungal endophytes, followed
by roots (nine orders), fruits (nine orders), flowers (eight orders),
seeds (seven orders), and stems (six orders) (Figure 3). The
dominant orders were Eurotiales (25 isolates), Agaricales (24
isolates), Pleosporales (23 isolates), and Xylariales (21 isolates)
in leaves and Hypocreales (40 isolates), Eurotiales (16 isolates),
and Glomerellales (10 isolates) in roots. The dominant orders in
flowers, fruits, and seeds were Eurotiales (40 isolates), Xylariales
(22 isolates), Sordariales (17 isolates), and Hypocreales (15
isolates), followed by Agaricales (eight isolates). The NMDS
(stress = 0.0227) analysis showed the similarity/dissimilarity
in fungal community composition among different plant parts
(Figure 4). The root and leaf fungal communities showed a strong
distinc tion from each other and those of the reproductive plant
parts (flowers, fruits, and seeds), which were more similar in their
endophyte composition. The six orders of fungal endophytes
isolated from stems were more similar to the communities found in
the reproductive tissues (flowers, fruits, and seeds) than to those
in the leaves or roots. Some of the endophy tic isolates could have
originated horizontally, that is, from the greenhouse environment,
rather than vertically from within the monk fruit plants themselves
since the greenhouse was not completely isolated from the
outdoor environment and the soilless media was not sterile.
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TABLE 2 IdentityoffungalendophytesrecoveredfromdrymonkfruitseedsandseedsfromfreshfruitfromChinabasedonrDNAITSsequenceanalysesandmorphology.
Order Name # of isolates GenBank accession # % identit y Seed source
Eurotiales Aspergillus hiratsukae 3MK 841469.1;MF7736 59.1 98.39;99.82;99.38 Alibabaa, Fruitb
Aspergillus pseudoglaucus 1KX258805.1 99.61 Alibaba
Aspergillus tennesseensis 1MT 582757. 1 100 Alibaba
Botryosphaeriales Botryosphaeria dothidea 1MN6 340 11.1 100 Fr uit
Glomerellales Colletotrichum brevisporum 2KY 70505 4.1;LC 379210.1 100; 10 0 Fruit
Colletotrichum qilinense 1M Z4751 26.1 98.81 Fruit
Diaporthales Diaporthe hongkongensis 1MW202983.1 99. 25 Fruit
Diaporthe phaseolorum 1MN650843.1 100 Fruit
Diaporthe subclavata 1MT199841.1 100 Fruit
Diaporthe unshiuensis 1MW3 41 29 7.1 10 0 Fruit
Pleosporales Exserohilum mcginnisii/E. rostratum 1MT3 37556.1/MK64 0580 .1 98.8;98.8 Fruit
Eurotiales Penicillium brevicompactum 1KX42696 8.1 99. 81 Alibaba
Penicillium sumatraense 2OQ608602.1;MT529218.1 97.53;98.51 Alibaba
Hypocreales Trichoderma atroviride 7 MN63 4667.1(4);MT341775.1(2);
MT023026.1
100 Alibaba
Trichoderma viride 3MN634 490.1(2);MN634664.1 100 Alibaba, Fruit
Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown.
aDry seeds purchased via Alibaba.
bFresh seeds extrac ted from fresh fruits from China.
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4 | DISCUSSION
Monk fruit plants proved to be a rich source of fungal endophytes
with a great diversity and abundance, especially in leaves. The role
of these fungi in the monk fruit plants is likely to be as complex as
their diversity. Some may be neutral commensalists, while others,
such as the wood- decaying Xylariaeceae (Hypoxylon, Nemania),
Meruliaceae (Phlebia tremellosa), Psathyrellaceae (Coprinellus and
Coprinopsis spp.), and Polyporaceae (Trametes hirsuta), may play
a beneficial role in vegetative decay and nutrient cycling in the
natural environment, or protection against pathogens or herbivores.
Members of the Xylariales, in particular, produce a wide array of
secondary metabolites many of which are antagonists of other
fungi and bacteria (Becker & Stadler, 2021). A few of the species
isolated may be hyperparasites of other fungal endophy tes found
in the monk fruit tissues, for example, Penicillium [Eupenicillium]
cinnamopurpureum which grows on the heads of Aspergillus spp.
(Horn & Peterson, 2008).
In addition to the Xylariales, many of the other fungal species
obtained from the monk fruit plants are known to produce bioac-
tive compounds with medical or industrial applications. For example,
Talaromyces purpureogenus (Keekan et al., 2020) and Penicillium brev-
icompactum (Fonseca et al., 2022) produce pigments with commer-
cial applications in the food processing industry. Several species are
known to produce antibiotics, such as diketopiperazine, produced
by Paraphaeosphaeria sporulosa, which is effective against salmo-
nella bacteria (Carrieri et al., 2020). Panaeolus subbalteatus is one of
the most common sources of psilocybin, used in medical treatment .
The kerosene fungus, Amorphotheca resinae (anamorph: Hormoconis
resinae), which was isolated from both leaves and flower buds, dam-
ages jet fuel, diesel, petroleum and creosote- treated wood, but may
have useful environmental applications in remediation of hydrocar-
bon contaminated sites (Rafin & Veignie, 2018). Chaetomium spp. are
the source of more than 100 useful secondary metabolites (Dwibedi
et al., 2023). For example, Arcopilus aureus (anamorph: Chaetomium
aureum) produces high levels of resveratrol, a potent antioxidant,
and sclerotiorin, which has anti- cancer properties (Dwibedi &
Saxena, 2018). A. aureus has high lead tolerance and clearance, sug-
gesting a potential role in bioremediation of contaminated soils (Da
Sila et al., 2018).
Several of the endophytic species obtained in this study have
potential agricultural applications in enhancing plant growth and
tolerance to drought and other environmental stresses, or as bio-
logical control agents of disease and insect pests. The abundance
and diversity of the fungal endophytes recovered from the monk
fruit plants suggest multiple, layered means of protection against
potential pests and adaptation to environmental stresses. Many
endophy tic species with anti- fungal or plant growth- promoting
activity recovered in this study have also been isolated from
grapevines (Vitis vinifera L.) (Kulišová et al., 2021), including
species of Aspergillus, Alternaria, Chaetomium, Epicoccum, and
Penicillium. These and several other species isolated from leaves
and fruit skin, are also common epiphytes that play a role in crop
protection both on and below the leaf sur face, and are often
transmitted horizontally. In grape, the most effective antifungal
endophy tes against Botrytis cinerea, the cause of bunch rot, were
Alternaria and Epicoccum species which, along with Aspergillus
fumigatus, produce high levels of siderophores and antioxidants
TABLE 3 Identityoffungalendophytesrecoveredfromleaves,stems,androotsofmonkfruitseedlingsgrownfromseedfromChina
based on rDNA ITS sequence analyses and morphology.
Order Name # of isolates GenBank accession # % identit y Source
Hypocreales Beauveria bassiana 1MT4 41874.1 9 9.8 Stem
Eurotiales Chromocleista sp. 1M N64476 6.1 9 9.8 3 Stem
Mortierellales Mortierella sp. 1HE605241.1 100 Stem
Eurotiales Paecilomyces tabacinus 1LT548280.1 100 Root
Eurotiales Penicillium citrinum 2MN634531.1;MT597829.1 100; 100 Stem
Penicillium meleagrinum 1MF135516.1 99. 82 Stem
Penicillium steckii 1OP615071.2 99. 82 Stem
Talaromyces islandicus 2FR670 311.1 8 9.96 ;
89.93
Root
Hypocreales Trichoderma afroharzianum 4MN6 44793.1 100;99.83
(2);
99. 66
Root; Stem
Trichoderma asperellum 2KY659 051.1;LN846 687.1 100;99.82 Root
Trichoderma atroviridae 2M T6 0 4 17 7.1 ;
MT6 26716. 1
100;99.43 Stem
Trichoderma harzianum 2MT626717.1;MF078650.1 100;99.65 Root; Leaf
Trichoderma harzianum/T. lixii 1MH339867.1/EF596951.1 10 0/100 Stem
Trichoderma sp. 1MK870660.1 100 Stem
Note: The closest match in BLASTn to sequences deposited in GenBank and percent identity are shown.
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TABLE 4 Identityoffungalendophytesrecoveredfromflowers,flowerbuds,fruits,andseedsofmaturemonkfruitplantsgrownintheKPUresearchgreenhousebasedonrDNAITS
sequence analyses and morphology.
Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Hypocreales Acremonium sclerotigenum/Scopulariopsis
gossypii
1OQ207544.1 /KU523862.1 9 9.81/9 9.8 1 Fruit pulp
Pleosporales Alternaria sp. 1Morphology only —Fruit skin
Alternaria alternata 1MK51 84 38 .1 99. 43 Flower
Heliotiales Amorphotheca resinae 1MN242723.1 97. 8 6 Flower bud
Xylariales Apiospora kogelbergensis 1OW982982.1 99.25 Fruit skin 252751
Xylariales Arthrinium spp. 20 K X378907.1;KX378907.1 99.63;96.71 Flower(7);Fruitpulp
(3); Fruit skin (10)
Arthrinium phaeospermum/Apiospora
rasikravandrae
1GU266274.1/OP237040.1 99. 65/ 99. 47 Fruit skin 252772
Eurotiales Aspergillus fumigatus 6 Morphology to leaf isolates
MT529448.1;MT529125.1;
MH793851.1
— Fruit pulp (4); Fruit
skin (1); Seed (1)
Aspergillus ochraceus 8MN533721.1;MT447480.1;
MN53 3721.1
99.62;99.81;99.63 Flower (1); Flower
bud (1)
Fruit pulp (1); Fruit
skin (5)
Aspergillus septulus 1MH861876.1 99.82 Fruit skin 252758
Aspergillus tamarii 2MH345899.1 99.12 Fruit skin
Hypocreales Beauveria bassiana 4MT111139.1 99.6 2 Fruit skin
Helotiales Botrytis cinerea 1OP794013.1 99. 8 Fruit skin
Sordariales Chaetomium spp. 9Morphology only — Flower (1); Fruit skin
(8)
Chaetomium cochliodes 1MT520580.1 9 9.0 3 Fruit skin 252796
Chaetomium globosum 5KY132166.1;KP067224.1 98.43;99.81 Flower (1) ;
Fruit skin (4)
252 743
Chaetomium novozelandicum 3M Z7248 83.1 96.7 Fruit skin
Cladosporiales Cladosporium spp. 4Morphology only — Flower bud (3); Fruit
pulp (1)
Agaricales Coprinellus micaceus 6LR961895.1;MF156262.1;
MH855975.1;LR961895.1
99.69;99.08;99.7;
98.06
Flower (3); Flower
bud (3)
Agaricales Crustomyces sp./C. subabruptus 1MN905889.1/MK454922.1 9 9.67/99. 50 Fruit skin 252789
Pleosporales Epicoccum nigrum 1FM20 0455.1 99.4 Fruit skin
Hypocreales Fusarium graminearum 1KJ017740.1 99.59 Flower 252744
(Continues)
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Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Saccharomycetales Hyphopichia burtonii 1MG554248.1 99.75 Fruit skin
Eurotiales Paecilomyces variotii 1OW98830 0.1 99.47 Fruit skin
Eurotiales Penicillium spp. 5Morphology only —Fl ower (1);
Fruit pulp (1); Fruit
skin (2); Seed (1)
Penicillium aethiopicum 1ON428665.1 98.89 Seed 2 52742
Penicillium citrinum /P. steckii 1MG55 4368.1/K X610136.1 9 9.8 2/ 99. 64 Fruit skin 252748
Penicillium glabrum /P. corylophilum 1MT797199.1/MT441635.1 99. 44 /99.4 4 Flower bud 252 741
Polyporales Phlebia tremellosa 1OL436998.1 99. 7 Flower bud
Incertae sedis Pseudogymnoascus pannorum 1KF156305.1 99.61 Seed 252762
Chaetothyriales Rhinocladiella similis 1MH063252.1 100 Flower
Eurotiales Talaro myc es sp. 5aMK 450749. 1 99.29/97.7 Fruit skin
Talaromyces pupureogenus 1MT635321.1 99.8 1 Fruit skin 252747
Polyporales Trametes hirsuta 1MF 161 297.1 9 9.6 6 Flower 252776
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).
aAll five isolates were the same Tal ar omy ces species; no specific ID in GenBank.
TABLE 4 (Continued)
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TABLE 5 Identityoffungalendophytesrecoveredfromleaves,stems,androotsofmaturemonkfruitplant sgrownintheKPUresearchgreenhousebasedonrDNAITSsequenceanalyses
and morphology.
Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Hypocreales Acremonium roseolum 1MH858153.1 98.66 Leaf
Acremonium hyalinulum 1KP131521.1 98.68 Leaf
Pleosporales Alternaria alternata 2OP696965.1;OL711657.1 99.61;99. 62 Leaf (1); Root
bulb (1)
Alternaria infectoria 1MK801346.1 95.15 Leaf 252756
Alternaria spp. 8OK274326.1;OK274326.1;
KX139150.1;MK6 40587.1;
MW53 4563.1;HQ6 49962.1
100;99.46;84.0;99.5;
98.67;99.38
Leaf (6); Root
bulb (2)
Heliotiales Amorphotheca resinae 2MN242723.1;KJ207403.1 98.34;95.96 Leaf (2) 252753
Xylariales Apiospora kogelbergensis 1OW982982.1 99. 25 Leaf
Xylariales Arthrinium spp. 4KX378907.1;KX148691.1 100;99.24 Leaf (4)
Orbiliales Arthrobotr ys amerospora 1KU702707.1 99.8 3 Root hair
Eurotiales Aspergillus flavipes 1MN956655.1 99.6 2 Leaf 252764
Aspergillus fumigatus 7 MT529448.1;MT529125.1;
MH793851.1
98.92;99.2895.01 Leaf (6)
Aspergillus ochraceus 4 Morphology to flower/fruit isolates
MN533721.1;MT447480.1;
MN53 3721.1
— Leaf (3); Root
hair (1)
Aspergillus tamarii 1MK332591.1 9 7.9 9 Leaf 252759
Dothidiales Aureobasidium pullulans 1MT6 45 93 0.1 93.0 Leaf 252754
Hypocreales Beauveria bassiana 3OK331343.1 98.46 Leaf
Hypocreales Bionectria sp. (anamorph: Clonostachys
sp.)
2MH729023.1;KU951245.1 99. 61; 99.67 Root bulb (1);
Root hair (1)
Saccharomycetales Blastobotrys sp. 1MK 2 4 61 87. 1 99. 81 Root bulb
Helotiales Botrytis cinerea 6OM349592.1;MT150132.1;
MH992148.1;AB693927.1;
MK513827.1;MF661902.1
100;100;99.39;100;
100; 88. 25
Leaf
Cephalothecales Cephalotheca sulfurea 1OM262341 .1 99. 61 Leaf 252787
Microascales Cephalotrichum purpureofuscum/
Doratomyces sp.
OP0 38661.1/KU95 4345.1 99. 29/9 9.4 7 Leaf 252788
Sordariales Chaetomidium leptoderma 4N R_164219.1;JN573175.1 97.61;97.68;97.86 Root bulb (3);
Root hair (1)
252 763
(Continues)
10 of 16
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MA e t al.
Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Sordariales Chaetomium aureum (teliomorph:
Arcopilus aureus)
2KP278194.1;MW533023.1 10 0; 100 Root bulb 252779
Chaetomium globosum 3KP067223.1;MF476072.1 100;98.21 Leaf (2);
Stem (1)
252 766;
252775
Chaetomium novozelandicum 2MZ724883.1;MZ724884.1 98.82;99.81 Leaf 2 52765
Chaetomium spinosum 1M H861746 .1 99. 05 Leaf
Cladosporiales Cladosporium herbarum 1O N71 2476.1 99.4 Leaf
Cladosporium ramotenellum 1OP006753.1 9 9.8 Leaf
Cladosporium tenuissimum 1MK9 05 459.1 99. 39 Leaf
Cladosporium spp. 9 ON208763.1;KT826671.1;
MH137774.1
93.16;98.99;98.39 Leaf
Agaricales Coprinellus flocculosus 5MK656240.1 96.88;96.88;97.31 Leaf
Coprinellus micaceus 6MF156262.1;MF156262.1;LR961895.1;
LR961895.1;MF156262.1;
LR9 61895.1
100;99.84;99.84;
99. 23 ;99.84;9 8. 06
Leaf
Agaricales Coprinopsis alnivora 2MZ407758 .1 98.02 Leaf
Coprinopsis cinerea 12 MN8 41919.1;MF351861.1;
MN8 41919.1;M N841919.1
96.91;99.68;99.69;
99. 85
Leaf
Pleosporales Curvularia canadensis /C. inaequalis 1NR_1700 04.1;OK117928.1 9 9.6 2; 99. 62 Leaf
Curvularia coatesiae 1LC605635.1 96 .53 Leaf 252755
Diaporthales Diaporthe eres 1MK335735.1 99. 63 Root bulb 252770
Pleosporales Didymella anserina 1M N6127 79.1 99.14 Leaf 252740
Pleosporales Epicoccum nigrum 7 OP315769.1;OP315769.1;OP315769.1;
MH861752.1
100;100;99.59;99.79 Leaf (6); Root
bulb (1)
Polyporales Fomitopsis mounceae 1MH086786.1 98.03 Leaf
Hypocreales Fusarium haematococcum/F. solani 1MH729 023.1/KU951245.1 9 9.61/9 9.67 Root hair 252778
Fusarium lichenicola 1KM921661.1 9 9.4 2 Root bulb
Fusarium oxysporum 10 K R906700.1;KC30 4797.1;FJ824032.1;
MT52 9814.1
95.63;99.8;99.8;
98.29
Root bulb (6);
Root hair (4)
Fusarium tricinctum 1MN833356.1 99.81 Root bulb 252752
TABLE 5 (Continued)
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MA et al.
Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Xylariales Hypoxylon macrocarpum 8HM192912.1 96.38;99.46;98.6;
98.43;99.3;98.78;
97.69;99.47
Leaf
Hypoxylon rubiginosum 10 AY787708.2;MT214998.1 99.80;99.80;99.80;
99.41;99.80;100;
91.2;92.88
Leaf 252769;252773
Sordariales Lasiosphaeriaceae 3KX343155.1;MN5410 90.1 99. 8; 99.59 Root hair 252774
Mortierellales Linnemannia zychae 1MH857054.1 9 9.8 3 Root bulb
Pleosporales Lophiostoma corticola /
Angustimassarina coryli
1MK907710.1/M F167431.1 100/100 Leaf 252794
Mortierellales Mortierella hyalina 1MT003063.1 99.8 3 Root bulb
Xylariales Nemania sp. 1MT 153669.1 9 9.2 2 Leaf 252749
Xylariales Nigrospora oryzae 1KC131293 .1 99. 41 Leaf 252757
Agaricales Panaeolus subbalteatus 1MH855553.1 98.73 Leaf 252791
Pleosporales Paraconiothyrium fuckelii 1M K05270 0.1 99. 29 Leaf 252792
Pleosporales Paraphaeosphaeria sporulosa 5KX302013.1;MH859903.1 99.82; 9 9.8 2 Root bulb (4);
Root hair (1)
252761;2527 71
Eurotiales Penicillium canescens 1MH865756.1 99. 62 Leaf
Penicillium cataractarum /P.
simplicissimum
2MK534497.1/KM613146.1;
MK534497.1/MT303132.1
99. 44 /10 0;
99. 63 /99.45
Root bulb (1);
Root hair (1)
252 767
Penicillium cinnamopurpureum 1MH655003.1 99.65 Leaf 252746
Penicillium citrinum/P. steckii 1KX610174.1/MT582790.1 95.16/94.72 Root bulb
Penicillium spp. 22 OP 035353.1;OP647345.1;
KY401082.1;MH512953.1;
MH512953.1;MH512953.1;
ON182131.1; ON182131.1;
ON182131.1; ON182131.1
99.62;99.06;99.44;
99.26;99.45;
99.26;100;100;
99. 81;9 9.8 1
Leaf (11); Root
bulb (11)
Pleosporales Periconia byssoides 1MK907734.1 99.63 Leaf 252795
Pleosporales Phaeosphaeria sp. 1ON 52 076 7.1 9 9.6 Leaf 252785
Cephalothecales Phialemonium inflatum 1NR_165996.1;MH857776.1 99.61;96.58 Stem 252783
Glomerellales Plectosphaerella cucumerinum 2ON9 27102.1;MW850542.1 9 9.4;9 9.8 Root bulb 2527 77;252790
Plectosphaerella oligotrophica 9MT4 474 99.1 99.6 ;99. 8 Root bulb (3);
Root hair (5);
Stem (1)
252782;252784
Pleosporales Unidentified 1MG916998 .1 99. 22 Leaf 2 5276 8
TABLE 5 (Continued)
(Continues)
12 of 16
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MA e t al.
Order Name # of isolates GenBank accession # % identity Source DAOMC ID #
Hypocreales Purpureocillium lilacinum 1KJ862077.1 9 9.6 4 Leaf
Hypocreales Sarocladium kiliense/S. strictum 11 KX384658.1/MF077236.1;
KX384658.1/MF077236.1;
KF293986.1/MF077237.1;
KF293986.1/ON500613.1
99. 44 /99.2 5;
99. 81 /99. 62 ;
99. 62 /99.43;
10 0/9 9.81
Rootbulb(9);
Root hair (2)
Agaricales Schizophyllum commune 1ON500589.1 99. 83 Leaf
Microascales Scopulariopsis brevicaulis 1OW987158.1 99.8 3 Leaf
Hypocreales Simplicillium aogashimaense 3AB604 004.1;MK579181.1;
MK579181.1
99.63;99.28;99.27 Root bulb 252780;252781
Simplicillium obclavatum 1KC40 3970.1 91 .9 Root bulb 252786
Simplicillium subtropicum 1MW26 0103.1 9 9.47 Root bulb 252750
Sordariales Sordaria fimicola 1JX273473.1 99.26 Leaf
Hypocreales Trichoderma ghanense 1MT52 0628.1 96 .0 Root bulb 252745
Trichoderma spp. 4Morphology only — Root bulb (1);
Root hair (3)
Ustilaginales Ustanciosporium appendiculatum 1GQ888733.1 91.12 Leaf 252760
Helotiales Varicosporium delicatum 1JQ 4128 64.1 93.75 Leaf 252793
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).
TABLE 5 (Continued)
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13 of 16
MA et al.
(Kulišová et al., 2021). Endophytic strains of E. nigrum have
been shown to reduce the incidence and severity of a range
of plant diseases (Taguiam et al., 2021). In British Columbia,
an isolate of E. nigrum from mummy berry- infected blueberries
suppressed spring apothecia production of Monilinia vaccinii-
corymbosi when applied to soil after infected berries dropped
(Kitura et al., 2023). Hypoxylon rubiginosum has shown promise
as a biocontrol for dieback of European ash (Fraxinus excelsior
L.), associated with its production of the anti- fungal metabolite,
phomopsidin (Halecker et al., 2020). Simplicillium aogashimaense
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 effec tive biocontrol
agent of gummy stem blight and powdery mildew of cucumber,
and has been shown to inhibit other plant pathogens including
nematodes (Moreno- Gavíra et al., 2021). Purpureocillium lilaci-
num [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 para-
sitic nematodes in crops. Arthrobotrys amerispora, isolated from
a root hair of the monk fruit, may be playing a role in root pro-
tection; Arthrobotrys spp. are well- known nematode- trapping
fungi as well as mycoparasites (Gams et al., 2004). Eight endo-
phytic 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
Trichoderma spp., which are well- known protectors of plants
from pathogen and insect attack, as well as plant growth pro-
moters (Sharma & Gothalwal, 2017).
For some plant pathogenic fungi, existence as an endophy te
may be a latent stage in pathogenesis. Disease develops as the
host plant reaches a certain life stage or begins to senesce, or
as the plant experiences environmental stress or other damage.
Botrytis cinerea, for example, is a common pathogen causing gray
mold disease of many crops but is often found as an endophyte
in healthy plant tissues. The two Colletotrichum spp. isolated
FIGURE 3 Numberoffungalisolatesindifferenttaxonomicordersisolatedfromroots,stems,leaves,flowers,fruits,andseedsofmonk
fruit.
FIGURE 4 Measureofdissimilarityintheendophyticfungi
composition among the root, stem, leaf, flower, fruit, and seed of
monk fruit using non- metric multidimensional scaling.
14 of 16
|
MA e t al.
from the internal tissues of monk fruit seeds in this study are
known plant pathogens and may be a quiescent stage in the de-
velopment of anthracnose disease. Plectosphaerella cucumerinum
(syn. Plectosporium tabacinum) causes wilt and root rot of several
crops including cucurbits, tomato, potato, and basil (Raimondo &
Carlucci, 2018) and may be a quiescent pathogen in the monk fruit
plants, while Pl. oligotrophica is a low- carbon feeding, soil sapro-
phyte (Liu et al., 2013) that may be neutral, or play a beneficial role
in the presence of biotic or abiotic stresses. As an example of the
multiple potential roles of a single endophytic species, Pl. cucumer-
inum is also nematophagous and has been tested for biocontrol
of potato cyst nematode (Atkins et al., 2003), although, more re-
cently, 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 patho-
genic strains in tomato and other crops (de Lamo & Takken, 2020).
The endophytic strains of F. ox ys porum 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 summar y, monk fruit plants can be easily grown in the
greenhouse and are a prolific source of endophytic fungi and sec-
ondary 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- friendly 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 po-
tential source of these endophytes may reveal even more useful
strains and advance our understanding of how endophytes inter-
act with their hosts.
ACKNOWLEDGMENTS
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.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
DATA AVAIL ABILI TY STATEMENT
The data that support the findings of this study are openly available
in figshare: 10.6084/m9.figshare.24530542.
ORCID
Li Ma https://orcid.org/0000-0003-4739-1670
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How to cite this article: Ma, L ., Elmhirst, J. F., Darvish, R.,
Wegener, L. A., & Henderson, D. (2024). Abundance and
diversit y of fungal endophytes isolated from monk fruit
(Siraitia grosvenorii) grown in a Canadian research
greenhouse. Plant- Environment Interactions, 5, e10142.
htt ps://doi.org/10.1002/pei3.10142
