The first record of metacordyceps neogunnii (metacordycepss, clacicipitaceae) isolated from larva of lepidoptera in vietnam morphological, phylogenetic characterization and chem

E-mail: nguyentb@dlu.edu.vn Received: 27.8.2021 Accepted: 06.10.2021 SUMMARY An entomopathogenic íungus, specimen DL0091 parasitized on the larvae o f Lepidoptera, was collected from Lan

Vietnam Journal o f Biotechnology 20(2): 317-327, 2022

T H E F IR S T R E C O R D O F M E TA CORD YCEPS NEOGUNNII cMETACORDYCEPS,

C L A V IC IP IT A C E A E ) IS O L A T E D F R O M L A R V A O F LEPIDOPTERA IN

V IE T N A M : M O R P H O L O G IC A L , P H Y L O G E N E T IC C H A R A C T E R IZ A T IO N A N D

C H E M IC A L C O N S T IT U E N T A N A L Y S IS

Lao Duc Thuan1, Le Huyen Ai Thuy1, Le Cong H ac1, Nguyên Hoang M ai2, Nguyên Van Giang3, Truông Binh Nguyen2,M

lFaculty ofBiotechnology, Ho Chi Minh City Open Universỉty, 97 Vo Van Tan Street, Ward 6, District

3, Ho Chi Minh City, Vietnam

2Institute o f Research & Hi-Tech Application in Agriculture, Dalat University, 1 Phu Dong Thien Vuong Street, Da Lat City, Lamdong Province, Vietnam

3Facuỉty o f Biology, Dalat University, 1 Phu Dong Thien Vuong Street, Da Lat City, Lamdong Province, Vietnam

HTo whom correspondence should be addressed E-mail: nguyentb@dlu.edu.vn

Received: 27.8.2021

Accepted: 06.10.2021

SUMMARY

An entomopathogenic íungus, specimen DL0091 parasitized on the larvae o f Lepidoptera, was collected from Lang Biang Biosphere Reserve, located in Lam Dong Province, Vietnam The specimen DL0091 has been analyzed to be contained numerous Chemical constituents, especially containing adenosine o f 634 mg/Kg and cordycepin o f 35.2 mg/Kg Due to containing many bioactive compounds, DL0091 was promised to be a precious natural source that could be applied in tíelds o f medicine and tunction food for health care For classiíication, based on the morphology analysis, it was identitied as Metacordyceps neogunnii (Metacordyceps, Clavicipitaceae) sharing the similar characteristics o f M neogunniiT.c Wen & K.D Hyde Morphology o f this species differed from

Cordyceps neogunnii (Berk.) Berk., by many characteristics, such as the larger stroma o f DL0091 (15-130 mm X 2 -6 mm), o f asci (550-680 | im X 5-8 pm), etc Additionally, the combined multi-gene phylogenetic analysis, including ITS, Tef and R pbl, well supported its systematic position in the clade

o f M neogunnii, which was used as traditional herb in China and other Asian countries In summary, DL0091 was identified as M neogunnii, containing many bioactive compounds, could be used as the medicinal potential in human healthcare.

Keywords: Molecular phylogeny; morphological identiíication; entomopathogenic tungi; adenosine; cordycepin.

INTRODUCTION

The genus Cordyceps sensu lato (Cordyceps

entomopathogenic fungi, have been used as

herbal medicines for a long time (Kuo et al.,

2015; Li et aỉ., 2020) Recently, based on the

phylogenetic analysis, Cordyceps s.l was

divived again into four genera, including

Cordyceps sensu stricto (Cordyceps s.s,

belonged to the íamily of Cordycipitaceae),

Metacordyceps (belonged to the family

Clavicipitaceae), Ophỉocordyceps (belonged to

the family Ophiocordycipitacea) and

Eỉaphocordyceps (belonged to the íamily Ophiocordvcipitaceae) (Sung et al., 2007) Of

thesespecies, Metacordyceps neogunnii T c

Wen & K.D Hyde belongs to the genus

Metacordyceps The genus Metacordyceps

includes only a limit known species, remaining

one of the most poorly understood

Metacordyceps neogunnii was reported to differ

from others related Metacordyceps species

mainly in having longer asci and wider

ascospores (Wen et aỉ., 2017) Metacordyceps

neogunnii has been wrongly recognized as herbal

entomopathogenic tungi Cordyceps gunii

(Berk.) Berk in China for more than 30 years

(Wen et al., 2017; She et a i, 2019) In the study

of Wen el al (2017), the íungus named

“Cordyceps gunnii” in China has been correctly

classitied as Metacordyceps neogunnii based on

the morphology analysis and combined multi-

gene phylogenetic analysis (Wen et aỉ., 2017)

Cordyceps gunnỉỉ (Berk.) Berk is known only

from Australia (Berkeley, 1848) “Cordyceps

g u m ii” is morphologically and combined

multigene phylogentically different to

Cordyceps gunnii of Tasmania (an island State of

Australia) Cordyceps gunnii of Tasmania also

shows them to differ and different genus

belonged to the family Ophiocordycỉpitaceae

(Wen et aỉ., 2017) “Cordyceps gunnir1 has been

reported to have Chemical position and medical

value similar to those of traditional Cordyceps

sinensis in China (Zhu et aỉ., 2013, 2016)

Additionally, it has various medical effects, such

as anti-tumor, anti-aging, promoting sleep and

enhancing memory (Menget al., 2019; She et al.,

2019; Zhu et al., 2013, 2016) Thereíbre,

“Cordyceps gunnii” has been used as a medicinal

mushroom by local people in China (Zhu et aỉ.,

2013, 2016; She et al., 2019) Recently, several

important secondary metabolites have been

found in “Cordyceps gunnii”, including

polysaccharide, isoílavone, cordycepin,

adenosine, anti- ultraviolet radiation components

(Kuo et al., 2015; Zhu et al., 2016; She et al.,

2019) These secondary metabolites have been

shown to have pharmacological potential, and

could be used as herbal medicines to enhance

human health (She et al., 2019) For this reason,

the search for entomopathogenic íungi diversity,

including “Cordyceps gunnii”, may provide an

insight into the preventive and therapeutic potentials of these íimgi for the biotechnological research as well as development of potential product Vietnam is located in a tropical region with terrestrial ecosystems The íòrests íeature a rich biodiversity of both flora and fauna due to the tropical monsoon climate with high temperature and rainfall This is a favorable environment for the development of entomopathogenic íungi Lang Biang Biosphere Reserve is located in Lam Dong Province and comprises a vast primitive jungle with the Lang Biang Mountain at its core, one of VietnanTs four biodiversity centers During our expedition

to discover the diversity of entomopathogenic íungi, we have collected the sample DL0091

In this study, species DL0091 was morphologically and phylogenetically described

as Metacordyceps neogunnii, containing numerous bioactive constituents, especially a high amount of adenosine and cordycepin, thereíore, it was considered a valuable resource

in medicine

MATERIALS AND METHODS

Fungal sample collection

The specimen, DL0091, used for this study was collected from Lang Biang Biosphere Reserve (elevation 1640 - 1750m) from May to October 2018 The specimen, including the host, was extracted careíully, noted, and photographed

in the field using a digital camera The specimen was immediately wrapped in wax paper, placed

in a collection bag, and taken to the laboratory

M orphology analysis

Morphological observations were carried out and recorded according to the guidelines of

Kobayashi (1941; 1982) and Sung et al (2007) (Kobayashi, 1941, 1982; Sung et al., 2007) The

macroscopic characteristics of the fresh fruit body were careM ly observed, including the stipe, stroma, etc Additionally, the host insect was identified based on morphological characteristics, such as mandibulate mouth parts, antennae, shape of head and thorax For the

Vỉetnam Journal o f Biotechnology 20(2): 317-327, 2022

micromorphological analysis, one or two

perithecia were removed from the stroma and

placed on a microscope slide in lactophenol-

cotton blue to measure the sizes and shapes of the

perithecia, asci and ascospores

DNA extraction, PCR amplitìcation, target

gene sequencing

Genomic DNA was isolated by using the

phenol/chloroform method (pH = 8)

(Chomczynski, 1993) The íruiting body was

incubated in a lysis buffer (2.0% SDS, Tris-HCl

pH 8.0, 150 mM NaCl, lOmM EDTA, 0.1

mg/mL Proteinase K) at 65°c ovemight The

supematant was collected by centriíiigation, and

phenol/chloroform/isoamyl alcohol (25:24:1)

Table 1 The primers’ sequences used in current study.

was supplemented and centriíliged The supematant was collected and precipitated with absolute isopropanol Finalỉy, the isolated genomic DNA was stored in Tris-EDTA buffer

at -20°c for íurther studies

The primer pairs used to ampliíy ITS, Tef,

rpbl gene were shown in Table 1 The fínal

volume of PCR was done in a total of 15 pL with the thermal program: 1 cycle at 95°c for 5 min,

40 cycles at 95°c for 30 s, 55°c for 30 s, 72°c

for 2 min, 1 cycle at 72°c for 5 min Five pL aliquots of ampliíĩcation product were electrophoresed on a 2.0% agarose gel and

visualized in a u v trans illuminator The

ampliííed product was sequenced by Sanger method

Rpb1

Taxa and ITS, T ef,R p b l sequences collection,

and phylogenetic analysis

The data set of ITS, T ef and Rpbl sequences

were established by sequences downloaded from

Genbank (NCBI) and based on the previous data

published by Sung et al (2007) and Wen et al

(2017) The ITS, Tef and Rpbl were noted with

accession number and name of taxon The

ampliííed DNA sequences were proofread to

remove ambiguous signals at both ends by

different software, including Seaview 4.2.12 and

Chromas Lite 2.1.1 The phylogenetic tree was

constructed based on neighbor-joining (NJ),

maximum parsimony (MP), maximum

likelihood (ML), and UPGMA (UP) using

Molecular Evolutionary Genetics Analysis

(MEGA) version 5

Chemical constituents and bioactive compound analysỉs

For determination of Chemical positions of specimen DL0091, powder of specimen DL0091 was sent for analysis at Center of Analytical Services and Experimentation HCMC, Vietnam (www.case.com.vn)

RESULTS

Taxonomy

Metacordyceps neogunnii (Figs 1, 2)

Typiíication VIETNAM Lam Dong Province, Lang Biang Biosphere Reserve, Lang Biang

mountain Elevation 1640 - 1750 m; humidity: over

85%; temperature: day 20°c - 22°c, night: 14°c -

16°C; collected in May - October 2018, from the

larvae of Lepidoptera in moist soil surrounded by

dried leaves (Figs 1A and B)

Host: On the larva of Lepidoptera, 40 - 60 mm

X 4 - 7 mm, buried in the soil (Figs 1C and 2A)

H abitat: Individuals of associated species

appeared at the type locality, including pioneer

species such as Acer laurinum (Aceraceae),

Castanopsis chinensis (Fagaceae), Erỉobotrya

poỉỉaneỉ (Rosaceae), Jasminum longisepalum

('Oỉeaceae), Phoebe petelotiỉ (Lauraceae) and

Tetrastigma lanceolarium ( Vitaceae).

Stromata: arose from head of host, íleshy,

rather tough, rarely branched (solitary or in

group of two stromata), white to grey (Fig 1), 15

- 130 mm X 2 - 6 mm (Figs 1C and 2A, E)

Stipe: cylindrical, 15 - 100 mm X 2 - 4 mm,

white (the part in underground) to grey (the

above part), tleshy, enlarging abruptly at fertile

part (Figs 1C and 2A) Fertile part: cylindrical

or obtuse, round head shape, white (in young) to

grey (in mature), 1 0 - 3 0 mm X 4 - 6 mm (Figs

2A and B); Surface: grey with several irregular

striate, black dots (Fig 2C); cortex: white (Fig

2D) Perithecicr immersed, elongated or

ampuliíòrm, even distribution, dark grey at the ostiole, 700 - 800 pm X 250 - 270 pm (Figs 2F

and G) Asci: cylindrical, hyaline, thick apical

cap, 550 - 680 pm X 5 - 8 pm (Fig 2H)

Ascospores: 3.0 - 4.0 pm X 1.8 - 2.1 pm, hyaline, filiform, multi-septate, disarticulating into secondary ascospores after released from the asci (Fig 21)

Phylogenetic analysis

The dataset of taxa in current study assembled from previously published studies

(Sung et al., 2007; Wen et al., 2017), and were

downloaded from GenBank (NCBI) for the construction of phylogenetic tree We obtained

25 sequences of each ITS, T ef and Rpbl gene

from 18 different species (Table 2) The

combined dataset if three gene, ITS, T ef and

Rpbỉ gene, consisted of 1127 bp and 24 taxa

were analyzed, representing the genus

Metacordyceps (Clavicipitaceae), Cordyceps,

(Ophiocordycipitaceae), the outgroup taxon

Glomereỉỉales).

Figure 1 The DL0091 sample “Metacordyceps neogunnii” A: stroma appeared in moist soil surrounded by

dried leaves; B: Immature stroma; C: Variation size of stroma stromata arose from head of host.

Vietnam Journaỉ o f Biotechnoỉogy 20(2): 317-327, 2022

Table 2 List of species and taxa used in this phylogenetic analysis.

Metacordyceps

In the phylogenetic analysis, the best model HM149362, and R p b l: H M 149367) the was TN93+G, -lnL = 6473.37, G=0.25 The

parameters used included base ữequcncies -

iìeqA = 0.24, freqT = 0.20, freqC = 0.30, and

freqG = 0.26 The NJ, MP, ML and UP analyses

showed the similar topologies resolving the

taxonomic relationship between species DL0091

and others The NJ, MP, ML and UP phylogenetic

trees could be broadly separated into different

aenera: Metacordyceps, Cordyceps,

Tolypocladium, and Ophiocordyceps (Fig 3).

The species DL0091 and taxon Cordyceps

gunniiừơm China (ITS: HM149352, Tef.

other species of Metacordyceps neogunnii with

credible bootstrap support (NJ: 96, MP: 100, ML: 100, UPMA: 100) (Fig 4) The clade of

Metacordyceps neogunnii from the well-

supported separate clade of Metacordyceps genus with other species o f Metacordyceps in the family o f Clavicipitaceae, including

Metacordyceps ỉndigotica, Metacordyceps kusanagỉensis, Metacordvceps martialis, Metacordyceps shibinensis, Metacordyceps

taii, Metacordyceps yongmunensis (NJ: 99,

MP: 100, ML: 100, UPMA: 99) (Fig 4) Two

specimens o f Cordyceps gunniỉ from Tasmania

(Australia) formed a separate clade of

Cordyceps genus with well-supported value

(NJ: 100, MP: 100, ML: 100, UPMA: 100),

closely to the genus of Metacordyceps and

Tolipocladium (Fig 4).

Figure 2 Morphology analysis of DL0091 “Metacordyceps neogunnií’ A: stroma; B: Fertile part; C: Suríace of tertile part; D: White cortex; E: Host: larva of Lepidoptera', F, G: Perithecia; H: Asci, thick apical cap; I:

Ascospores.

- Metacordyceps genus

1ỌỌr- Cordyceps genus

- T ữlypocladium genus

- ũphiocordycepỉ genus

- ũutgroup

Metacordyceps genus

— 125- Cordyceps genus

- T olypocíadiurn genus

- Ophiocordyceps genus -ũutgroup

• Metacordyceps genus

— 152 Cordyceps genus - — T olypocladium genus

- Ophiocordyceps genus -ũiigroup

• Melacordyceps genus

- Cordyceps genus

- T olypocladium genus

- Ophiocordyceps genus

- ũutgroup

Figure 3 Schematic diagrams of phylogenetic relationships A: Neighbor joining; B: Maximum parsimony; C: Maximum likelihood (ML), UPGMA in genus sampling Bootstrap: 1000 replicates The bootstrap value was

indicated above nodes The tree is rooted to Glomerella cingulate (Outgroup).

Vietnam Journal o f Biotechnology 20(2): 317-327, 2022

-M ôlac& rd yee ps_ yon gm un en sisJTS _JN O 4 98 $6 _TE F_ E F4 -$ 87 70 _R P 8 1_ E F4 6§ 87 6

- M eta cofdyce psJcu san ag ie nsisJT S _ JN 04 93 73_ T E F _ JF4 16 01 4_ R P B f_JN 04 98 9Ũ -M e ta cofdyce ps_ sbibin en sis_ frS _ K R 15 35 85 _T E F _ K R 15 35 89 _R P B 1_ K R 15 35 90 -M e ta cữ rd yce p s_ ch la m yd o sp o ria JT S _ A J2 9 2 3 9 8 _ T E F _ E F 4 6 9 0 6 9 _ R P B 1 _ £ F 4 6 9 0 9 8

H—

Ỉũl— M et M e t3 to fđ y c e ps_ chla rrtyd osp Q na JT S _JN G 4 93 21 _T E F_ D Q 52 23 27 _R P ei_D Q 5 22 37 2

M e ta co rđ ỵce p sJ a iiJ T S J N 0 4 9 8 2 9 _ T E F _ A F 5 4 3 ? 7 5 _ R P B 1 _ D Q 5 2 2 3 8 3

M e ta c & rd y c e p s jn c fig o tic a J T S _ J N 0 4 9 8 7 5 _ T E F _ J =416011_RPB1_JNQ49887

9 8 1 M e ta c o rdyce psJn dig otica JT S _JN 0 49 87 4_ T E F _JF 4 16 01 Q _R P B t_JN 0 49 88 &

-M e ta c o fd ycep s_m aríia? isJT S _JN 0 49 88 1_ T E F _JF 4 16 01 6_ R P B 1 _JN 04 98 92

- D L0 09 1JT S _ T E F _ R P B 1

M e ta cordyce ps_ ne og un nji_IT S _K U 72 971 6_ ĨE F_ K U 7 29 72 7_R P B 1_ K U 7 29 73 2

M et3 C ordyce ps_ ne og un m i_ íT S _ K U ?2 & ? 17J'E F _K U 72 97 28 _R P 6 1_ K U 72 97 33

C ordyceps _gurtn iiJT S _H M 14 935 2_ T E F _ H M 14 93 62 _R P B 1_ H M 1 49 36 7

M eta cD fđyce ps_ ne og un niiJTS _ K U 72 9? 15 _TE F_ K U 72 97 26 _R P B 1_ K U 72 97 31

— M et3C 0rđyceps_necgunm f ÍTS_M H143811_TEF_M H143861_RPB 1_MH143876

Metacordyceps neogunnii clade

Metacordyceps genus

r uo

iõl C ordyce ps_gunm i_rrS _H M 14063Ũ _TE F_H M 140€36_R P B 1_H M 140639

c59 I—

- C ordyce ps_ gu nn ii_ ITS _ JN 04 98 22 _TE F_ A Y 4 89 61 6_ R P B 1 _A Y 48 96 50

- T o ty p o c la d iu m ja p o n tc a _ !T S _ JN 04 9& 24_TEF_DQ 522330_R P81_D Q 5223?5 - Totypocla dtum _o ph io glo ssaide sJTS _ JN 9 43 32 G _ TE F_A Y 48 96 18 _R P B 1_ A Y 48 96 52

Corc^iiceps genuỉ

T olypocladium genus

- T otypocla dium _subsessitós_ITS _JN 049844_TE F_EF469061_RP B1_EF46909Q 100Ị-O p h io co rd ycep s_sin en sis_ IT S _ JN S 4 98 54 _T E F EF46876 7_ RP B 1 _E F4 68 â7 4

O p h io co rđ yce ps_ sin en sisJTS _H M 53 59 81 _T E F _ H M 5 95 91 8_ R P B 1 _H M 59 595 2 -O p hto cord ycep s_stylo ph ora_ ITS _ JN0 49 82 8_ TE F_D Q 5 22 33 7_ R P B 1 _D Q 5 22 38 2 -O ph ioco rd yce p s_ rtiizo id e a JT S _ JN G 4 9 8 5 ? _ T E F _ £ F 4 $ 8 7 6 4 _ R P 8 1 _ E F 4 6 8 8 ? 3

Opíiiocordyceps genus

- G lom e fella_ cin g u la ta JT S _ D Q 2 8 6 2 0 2 _ T E F_ A F 5 4 3 ? 7 3 _ R P B -L A Y 4 8 9 6 5 9 ]Outgroup

(A)

- Metacordyceps_yongrĩHjnensisJTS_JN049856_TEF_EF468770_RPB1_EF468876

100 I -Metacordyceps_chlamydosponaJTS_AJ292398_TEF_EF4690€9_RPB1_EF469038

I— M e

&

- Metacordyceps_chlamydospofiaJTS_JN049821_TEF_OQ522327_RPB1_DQ522372

- Metacordyceps_shihinensis_rTS_KR153S85_TEF_KR153£89_RPB1_KR153590

— Metacordyceps_kusanagief\sisJTS_JN049S73_TEF_JF416Q14_RPB1_JN049890

|-Metacordyceps_taiiJTS_JNa49829_TEF_AF543775_RPB1_DQ522383 Metacordyceps JndigoticaJTS_JN0498?5_TEF_JF416011_RPB1_JN049887 Gol Metacordyceps_indigotica_ITS_JN049874_TEF_JF41601 Ũ_RPB1_JN049886

Cofdyc eps_gunn ĨĨ_ITS_H M 149352_7EF_HM 149362_RPB1 _ H M 149367

; Metacordyceps_neogunniiJTS_KU729716_TEF_KU729727_RPB1_KU729732 Metacordyceps_neogunnii_ITS_KU729715_TEF_KU729726_RPB1_KU729731 Metacordyceps_neogunnii_ITSJ<U729717_TEF_KU729728_RPB1_KU729733 -DL0091_fTS_TEF_RPB1

-Metacordyceps_neogunnii_rrS_MH143811_TEF_MH143861_RPB1_MH143876 -Metacordyceps_maftialisJTS_JN049881_TEF_JF416016_RPB1_JN049892

(B)

M elacordyceps neogunnii clade

M etacordyceps genưs

-Metacordyceps_yongnwnensisJTS_JN049856_TEF_EF488770_RPB1_EF468876 -MetacofdycepsJíusanagtensisJTS_JN049873_TEF_JF416014_RPB1_JN049890

— Metacofdyceps_shibinensis_ITS_KR153585_IEF_KR153589_Rrei_KR153S9B

- Metacordyceps_chlamydosporiaJTS_AJ292398_TEF_EF469069_RPB1_EF469098

- r “ *

ũl— Msl

1 0 0 1— Metacofdyceps_dilamydosporiaJTS_JN049821_TEF_OQ522327_RPB1_DQ522372 Metac<wdycepsJaiiJTSiN049829_TEF_AF54377S_RPB1_ĐQS22383

M etacordycepsjndigaticaJTS_JN049875 TEF_JF416011_RPB1_JN049887

9 8 1 MetacordycepsJnểigoticaJTS_JN049874_TEFJF416<m_RPB1_JN049886

- Metacofdyceps_martialisJTS_JN049881_TEF_JF416016_RPB1_JN049892

- ĐL0091JTS_TEF_RPB1

Metacordyeeps_neogunniiJTS_KU729716_TEF_KU729727_RPB1_KU729732

Metacordyceps_neogunmiJTS_KU729717_TEF_KU729728_RPB1_KU729733

Cofdyceps_gunnii_ITS_HM149352JEF_HM149362_RPB1_HM149367

Metacordyceps_neogunfiiiJTS_KU729715_TEF_KU729726_RPB1_KU729731

— Metacofdyceps_neogunniiJTS_MH143811_TEF_IWH14386t_RP81_MH14387i

M etacordyceps neogunnii clade

Metacordyceps genưs

(C)

69 100

Metacordyceps_neogunniiJTS_KU729715_TEF_KU729726_RPB1 _KU723731 Met3cordyceps_neogurmịi_ITS_KU729717_TEF_KU729728_RPB1_KU729733

^Metacordyceps_neogunnii_!TS_KU729716_TEF_KU729727_RPB1_KU729732 Cordyceps_gunnii_ỈTS_HMl49352_TEF_HM149362_RPB1_HM149367 Met3CQrdyceps_neogunnii_ITS_MH143811_ĨEF_MH143861_RPB1_MH143876 DL0091 rrS_TEF_RPB1

M etacordyceps neogunnii clade

Met3cordyceps_chl3rnydospoFí3jTS_AJ292393_TEF_EF469Ũ69_RPB1_EF469Ũ&8

-Metacofđyceps_yongmunensis_rrS_JN049856_TEF_EF468770_RPB1_EF468876 -Metacordyceps_kusanagiensis_ITS_JN049873JEF_JF416014_RPB1_JN(M9890 -Metacordyceps_shibinensìsJTS_KR153585_7EF_KR153589_RPB1_KR153590 - Metacordyceps_martialis_rĩS_JN049881_TEFJF416016_RPB1_JN049B92 rMetacordỵcepsJaii_ITS_JN049829_TEF_AF543775_RPB1_DQ522383 _ ^MetacordycepsJndigoticaJTS_JN()49874_TEF_JF41601D_RPB1_JN049886

991 MetacordycepsJndigoticaJTS_JN049875_TEF_JF416011_RPB1_JN043887

(D )

M elacordyceps genus

Figure 4 Schematic diagram of phylogenetic analysis A: Phylogenetic relationships among DL0091 and related

species based on combined analysis of ITS, Tef, Rpb1 data from ML analysis; B: Enlargement of Metacordyceps

genus of Neighbor joining; C: Maximum likelihood; D: UPGMA tree Bootstrap: 1000 replicates The bootstrap

value was indicated above nodes The tree is rooted to Glomerella cingulate (Outgroup).

Chemical and bioactỉve compounds analysỉs

Numerous bioactive constituents, such as

cordycepin, adenosine, polysaccharides,

phytosterol, as well as other Chemical positions,

such as protein, amino acid, ash, fat, carbohydrate, have been extracted from specimen DL0091 The bioactive constituents and other Chemical positions were shown in Table 4

Table 3 Synopsis of the characteristics of DL0091 and related species.

S p e cie s H o s t s tro m a ta A s c o m a ta A s c i A s c o s p o re s R e te ren ce

DL0091 Larvae of

L e p id o p te ra

Fleshy, white to grey, rarely branched, 1 5 -

130 mm X 2-6 mm

Embeded

Cylindrical, hyalíne, 5 5 0 -

680 ụm X 5 -

8 ụm, thick apical cap

3 0 -4 0 pm X 1.8-2.1 pm, hyaline, íiíiíorm,

m ulti-septate, This study disarticulating

into secondary ascospores

M e ta c o rd y c e p s n e o g u n n iiT c

W en & K.D Hyde

Larvae o f

L e p id o p te ra

R eshy, w hite to gray, rarely branched, 4 0 -

80 mm X 2-6 mm

Embedded

Cylindrical, hyaline, 250-

480 X 3 -5 , possessing a prom inent apical cap

330-460 ụm X

2 - 3 pm, hyaline, tilitorm , m ulti- W en T c e t

septate, .Tòn 171

breaking into ^ ' secondăry

ascospores

C o rd y c e p s g u n n ii (Berk.) Berk

Larvae of

E n d o c lita

e x c re s c e n s

Singularia, calvata, ecaptehospite;

s tip e 3 6 6 -5 2 3

mm X 4 8 -8 6

mm, head

18 5 -1 9 3 mm

X 4 -9 4 mm

Embedded

Cylindrical,

3 4 5 -5 3 0 X

4 4 -6 9

Hyaline, tilitorm ,

m ulti-septate, breaking into 2 - Li, z e t a l

4.3 ụ m x 1 -1 8 (1999)

ụm secondary ascospores

Vietnam Journaỉ o f Biotechnology 20(2): 317-327, 2022

Table 4 Chemical positions of DL0091.

7 Phytosterol

DISCUSSION

In the fíeld of pharmaceutical industry and

traditional medicine fíelds, the search for the

natural resources, especially entomopathogenic

fungi, has been considered to be important to

develop biological product Lang Biang

Biosphere Reserve, located in Lam Dong

Province, is classiíied as Vietnam’s biodiversity

center and considered a hotspot of ủingal

biodiversity, including entomopathogenic íìingi

During our expedition to validate the diversity of

entomopathogenic íungi in Lang Biang Biosphere Reserve, the species DL0091 was collected Morphologically, the species DL0091

was identiííed as Metacordyceps neogunnii,

belonged to Metacordyceps genus,

Clavicipitaceae family The species DL0091

shared the common characteristics of the genus

of Metacordyceps The genus of Metacordyceps G.H Sung, et al was íĩrst introducedby Sung et

al (2007) which was characterized by (1)

stromata: solitary, simple or branched; (2) stipe: íleshly or touch, cylindrical to enlarging in fertile

part; (3) perithecia: partially or completely

immersed in stromata, ordinal or oblique in

aưangement; (4) asci: cylindrical, thickened

ascus apex; (5) ascospores: cylindrical,

multiseptate, disarticulating into part-spores; (6)

host: almost always buried in soil (Sung et ai.,

2007) Compared to species belonged to the

genus Metacordyceps, DL0091 was similar to

Metacordyceps neogunnỉi T c Wen & K.D

Hyde, and different from the species Cordyceps

gunnii (Berk.) Berk (Table 3) Here, the

phylogenetic trees were conducted based on the

combined sequence data from multi-gene loci,

including ITS, T ef and Rpbl Phylogenetically,

the species DL0091 clustered with

Metacordyceps neogunnii, and Cordyceps gunnii

from China (ÍTS: HM149352, Tef HM149362,

and Rpbl: HM 1493 67) to form the separate

Metacordyceps neogunnii clade (belonged to the

genus Metacordyceps) with well-supported

bootstrap samplings The taxon Cordyceps

gunnỉỉ from China was clustered within this

group, it could be explained that it was wrongly

classiíied as Cordyceps gunnii for more than 30

years, and has been coưectly classiíied as

morphology analysis and combined multi-gene

phylogenetic analysis (Wen et al., 2017) The

correct Cordyceps gunnii from Tasmania

(Australia) fonned a clade that split a separate

clade from Metacordyceps neogunnii clade with

well-supported nodal value

This study conclusively demonstrates that

species DL0091, collected from Lang Biang

Biosphere Reserve, Vietnam, was

morphology and phylogenetic analysis

Additionally, this is the íĩrst record of

Metacordyceps neogunnii in Vietnam Due to

the bioactivities o f Metacordyceps neogunnỉi,

the record of DL0091 as Metacordyceps

neogunniỉ may provide an insight into the

preventive and therapeutic potentials of this

fungi for the biotechnological research as well

as development o f potential product

Moreover, the strategies used for identifying

morphology analysis and phylogenetic analysis based on multi-gene loci could have a wide application in entomopathogenic fungi Generally, many Chemical constituents of specimen DL0091 have been identiíled including protein, fat, carbohydrate, amino acid, cordycepin and adenosine, therefore, the understandings o f Chemical compositions in this study could apply in the development of new drugs and therapeutics Amount of protein, fat, ash, carbohydrate, polysaccharide were 27.6%, 3.75%, 5.06%, 2.51% and 3.71 Essential and non-essential amino acid shown

in Table 4 revealed the presence of 16 amino acids with the amount of 13.30 f/100 g Nine essential amino acids were detected, including Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Valine, of which Arginine and Valine were the highest (0.94 g/100 g and 0.91 g/100 g, respectively) Seven non-essential amino acids were Alanine, Aspartic acid, Glutamic acid, Glycin, Proline, Serine, and Tyrosine, with the highest amount for Glutamic acid (1.88 g/100 g) Free phytosterol, including Campesterol and Beta-sistosterol, were identiíìed in DL0091 It has been evidenced that Beta-sistosterol, a major phytosterol, possessed many biological activities, such as anti-tumorigenesis, anti- inílammatory, hepato-protective, antioxidant

as well as anti-diabetic ủmctions (Yang et al.,

2009) Cordycepin and Adenosine concentration in DL0091 were presented in the amount of 634 mg/Kg, and 35.2 mg/Kg Cordycepin and Adenosine were the categories

o f compounds that exhibited signiíĩcant therapeutic potential, such as anti- inílammatory, analgesic, and regulation of immune response, anti-tumorigenesis, anti- metastatic, and anti-proliferative effects, as

well as inducing apoptosis (Shin et ai., 2009; Liu et ai., 2015; Jin et al., 2018) Therefore,

the nutritional and bioactive values of DL0091 detected indicated its potential use in medical application as well as source o f development

o f íunctional food for healthcare