Corresponding authors: Ishara S. Manawasinghe (
Academic editor: M. Ruszkiewicz-Michalska
Liu J-W, Manawasinghe IS, Liao X-N, Mao J, Dong Z-Y, Jayawardena RS, Wanasinghe DN, Shu Y-X, Luo M (2023) Endophytic
Species delineation of
The objectives of this study were to isolate and identify the dominant endophytic
Healthy leaves and twigs of
Endophytic fungi were isolated following the methods described by
For macro- and micro-morphological characterisation, 5 mm diameter agar plugs were cut from all the actively growing pure cultures on
Total genomic DNA was extracted from mycelium grown on
Gene regions, respective primer pairs and PCR protocols used in the study.
Gene | Primer pair | Optimised PCR protocols | References |
---|---|---|---|
ITS | ITS1 | 94 °C: 5 min (94 °C: 30 s, 53 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
ITS4 | |||
|
GDF | 94 °C: 5 min (94 °C: 30 s, 60 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
GDR | |||
|
CHS-79F | 94 °C: 5 min (94 °C: 30 s, 49 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
CHS-345R | |||
|
CYLH3F | 94 °C: 5 min (94 °C: 30 s, 53 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
CYLH3R | |||
|
ACT-512F | 94 °C: 5 min (94 °C: 30 s, 54 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
ACT-783R | |||
|
Bt2a | 94 °C: 5 min (94 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min |
|
Bt2b | |||
|
GSF1 | 94 °C: 5 min (94 °C: 30 s, 60 °C: 60 s, 72 °C: 1 min) × 35 cycles, 72 °C: 30 min |
|
GSR1 |
For the phylogenetic analysis, reference sequences for
In the
Recombination analyses were conducted to provide evidence for genetic distances for two new species identified, based on the phylogenetic analyses. The pairwise homoplasy index (
In total, 12 endophytic
In the present study, eight
The most parsimonious tree of the
China, Guangdong Province, Huazhou, isolated from healthy twigs of
The single isolate (
China, Guangdong Province, Huazhou, isolated from healthy leaf of
Three isolates obtained in this study (
China, Guangdong Province, Huazhou, isolated from healthy leaf of
A single isolate obtained in this study (
The epithet refers to the cultivar of the host plant –
Endophytic in
Colonies on
China, Guangdong Province, Huazhou, isolated from a healthy leaf of
In the phylogenetic analysis of combined six genes,
PHI analysis of combined ITS,
In the present study, a single isolate was recognised as belonging to the
The most parsimonious tree for
China, Guangdong Province, Huazhou, isolated from healthy leaf of
A single isolate (
Three of our isolates were initially recognised as belonging to the
The most parsimonious tree of the
PHI analysis of combined
The epithet refers to the Guangdong Province where the fungus was collected.
Isolated from a
Colonies on
China, Guangdong Province, Huazhou, isolated from healthy twigs of
In the phylogenetic analysis of combined
In the present study, endophytic
Species belonging to the
Endophytic fungal colonisation might vary in different tissues of the same plant (
Overall, in the present study, two novel endophytic
We would like to thank Dr Shaun Pennycook, Nomenclature Editor of Mycotaxon, for his guidance on the species names. M. Luo would like to thank for the grant from the Guangdong Rural Science and Technology Commissioner project (KTP20210313) and the Research Project of Innovative Institute for Plant Health (KA21031H101). Z.Y. Dong would like to thank the Key Realm R & D Program of Guangdong Province (2018B020205003). R.S. Jayawardena would like to thank Thailand Science Research and Innovation, grant number 652A01003 entitled ‘Biodiversity, taxonomy, phylogeny and evolution of
Fungal isolates and sequences of molecular marker used in
Species | Culture | Type | Genbank accession number | ||||||
---|---|---|---|---|---|---|---|---|---|
ITS |
|
|
|
|
|
|
|||
|
ICMP 18608 | Holotype |
|
|
|
- |
|
|
|
ICMP 18686 |
|
|
|
- |
|
|
|
||
|
ICMP 17673 | Holotype |
|
|
|
- |
|
|
|
|
ICMP 17919 | Holotype |
|
|
|
- |
|
|
|
|
ICMP 12071 | Holotype |
|
|
|
- |
|
|
|
ICMP 18621 |
|
|
|
- |
|
|
|
||
|
ICMP 18537 | Holotype |
|
|
|
- |
|
|
|
|
Holotype |
|
- | - | - | - | - | - | |
|
MFLUCC 181167 | Holotype |
|
- | - | - | - | - | - |
|
ICMP 18580 | Holotype |
|
|
|
- |
|
|
|
ICMP 18696 |
|
|
|
- |
|
|
|
||
|
|
|
- |
|
|
- | |||
|
ICMP 17904 |
|
|
|
- |
|
- | - | |
|
CGMCC3 14925 | Holotype |
|
|
|
- |
|
|
|
|
MFLUCC 150022 | Holotype |
|
|
|
- |
|
|
- |
|
CMM 4268 | Holotype |
|
|
|
- |
|
|
|
|
ICMP 18539 | Holotype |
|
|
- | - |
|
- | - |
|
ICMP 18658 | Holotype |
|
|
|
- |
|
|
|
|
BRIP 66219 | Holotype |
|
|
|
- |
|
|
- |
|
CAUG17 | Holotype |
|
|
|
- |
|
|
- |
|
ICMP 18579 | Holotype |
|
|
|
- |
|
|
|
LC0856 |
|
|
- | - |
|
|
|
||
|
CAUG28 |
|
|
|
- |
|
|
- | |
MFLUCC 13-0418 | Holotype |
|
|
- | - |
|
- | - | |
|
LF130 |
|
|
- | - |
|
|
- | |
CPC:28644 |
|
|
|
- |
|
|
- | ||
CPC:28645 |
|
|
|
- |
|
|
- | ||
CPC:30253 |
|
|
|
- |
|
|
- | ||
UOM 1138 |
|
|
|
- |
|
|
- | ||
|
ICMP 18581 | Holotype |
|
|
|
- |
|
|
|
|
CBS 133125 | Holotype |
|
|
|
- |
|
|
- |
|
IMI 356878 |
|
|
|
- |
|
|
|
|
|
CBS 132879 | Holotype |
|
|
|
- |
|
|
|
|
CAUG7 | Holotype |
|
|
|
- |
|
|
- |
|
MFLUCC 13-0726 | Holotype |
|
|
|
- |
|
|
- |
|
MFLU 150689 | Holotype |
|
- |
|
- |
|
- | - |
|
CPC:26844 |
|
|
|
- |
|
|
- | |
|
Holotype |
|
|
|
- |
|
|
|
|
|
ICMP 17920 |
|
|
|
- |
|
- | - | |
|
NBRC 7478 | Holotype |
|
|
|
- |
|
|
|
|
CBS 142411 | Holotype |
|
|
|
- |
|
|
- |
|
Holotype |
|
|
- | - |
|
|
|
|
|
ICMP 17816 |
|
|
|
- |
|
|
|
|
|
CPC:28612 | Holotype |
|
|
|
- |
|
|
|
CPC:28555 |
|
|
|
- |
|
|
|
||
CPC:28556 |
|
|
|
- |
|
|
|
||
|
CBS:116870 | Holotype |
|
|
|
- |
|
|
|
CMM 4458 |
|
|
|
- |
|
|
|
||
|
CBS 470.96 | Holotype |
|
|
|
- |
|
|
- |
CBS 469.96 |
|
|
|
- |
|
|
- | ||
|
MFLUCC 170571 | Holotype |
|
|
|
- |
|
|
- |
|
GA100 | Holotype |
|
|
- | - |
|
|
|
|
CBS 132882 | Holotype |
|
|
|
- |
|
|
|
|
MFLUCC 181602 | Holotype |
|
|
|
- |
|
|
- |
|
CBS 145.29 | Holotype |
|
|
|
- |
|
|
|
|
ICMP 1778 | Holotype |
|
|
|
- |
|
|
|
ICMP 18705 |
|
|
|
- |
|
|
|
||
|
CBS 133134 | Holotype |
|
|
|
- |
|
- | - |
|
ICMP 19051 | Holotype |
|
|
|
- |
|
|
|
CBS 119296 |
|
|
|
- |
|
- | - | ||
|
ICMP 18578 | Holotype |
|
|
|
- |
|
|
|
CPC:30210 |
|
|
|
- |
|
|
|
||
|
CPC:30211 |
|
|
|
- |
|
|
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|
CPC:30212 |
|
|
|
- |
|
|
|
||
CPC:30221 |
|
|
|
- |
|
|
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||
CPC:30209 |
|
|
|
- |
|
|
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||
|
|
|
- |
|
|
- | |||
|
|
|
- |
|
|
- | |||
|
|
|
- |
|
|
- | |||
|
DNCL021 | Holotype |
|
|
- | - |
|
|
|
DNCL028 |
|
|
- | - |
|
|
|
||
DNCL018 |
|
|
- | - |
|
|
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||
|
CBS 143666 | Holotype |
|
|
|
- |
|
|
|
UOM 1119 |
|
|
|
- |
|
|
- | ||
|
|
|
- |
|
|
- | |||
|
CBS 133122 | Holotype |
|
|
|
- |
|
- | - |
|
CBS 124945 | Holotype |
|
|
|
- |
|
|
|
|
ICMP 4832 | Holotype |
|
|
|
- |
|
|
|
ICMP 5285 |
|
|
|
- |
|
|
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||
|
Holotype |
|
|
|
- |
|
|
|
|
|
|
|
- |
|
|
|
|||
|
|
|
- |
|
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|
|||
|
CBS 124946 |
|
|
|
- |
|
|
- | |
CBS 124943 |
|
|
|
- |
|
- | - | ||
CBS 124949 | Holotype |
|
|
|
- |
|
|
|
|
|
GZAAS 5.08601 | Holotype |
|
|
- | - |
|
|
- |
|
Holotype |
|
|
|
- |
|
|
|
|
|
BRIP 45094 | Holotype |
|
|
|
- |
|
|
|
|
CFCC 50818 | Holotype |
|
|
|
- |
|
|
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BCC 38876 | Holotype |
|
|
|
|
|
|
- |
CBS 129958 |
|
|
|
|
|
|
- | ||
|
CBS 119297 | Holotype |
|
|
|
|
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|
- |
|
CBS 125375 |
|
|
|
|
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|
- | |
|
Holotype |
|
|
|
|
|
|
- | |
|
|
|
|
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|
- | |||
|
|
|
|
|
|
- | |||
|
|
Holotype |
|
|
|
|
|
|
- |
|
CBS519.97 | Holotype |
|
|
|
|
|
|
- |
|
|
|
|
|
|
|
- | ||
CAUOS2 |
|
|
|
|
|
|
- | ||
|
|
Holotype |
|
|
|
|
|
|
- |
|
MAFF240517 | Holotype |
|
|
- | - |
|
|
- |
SB 08 |
|
|
- |
|
|
- | - | ||
|
|
|
|
|
|
|
|
- | |
|
|
Holotype |
|
|
|
|
|
|
- |
MH0413 |
|
|
|
|
|
|
- | ||
|
BCC 38876 |
|
|
|
|
|
|
- | |
|
CBS 170.49 | Holotype |
|
|
|
|
|
|
- |
MAFF 238321 |
|
- | - | - | - |
|
- | ||
|
CBS 133705 |
|
|
|
|
|
|
- | |
CBS 125375 | Holotype |
|
|
|
|
|
|
- | |
|
CBS519.97 |
|
|
|
|
|
|
- | |
|
|
Holotype |
|
|
|
|
|
|
- |
|
|
Holotype |
|
|
|
|
|
|
- |
|
|
|
|
|
|
|
- | ||
|
|
Holotype |
|
|
|
|
|
|
- |
|
|
|
|
|
|
|
- | ||
|
|
Holotype |
|
|
|
|
|
|
- |
|
|
Holotype |
|
|
|
|
|
|
- |
|
|
|
|
|
|
|
- | ||
LC8240 |
|
|
|
|
|
|
- | ||
LC8244 |
|
|
|
|
|
|
- | ||
LC8322 |
|
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|
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|
- | ||
LC8337 |
|
|
|
|
|
|
- | ||
|
|
|
- |
|
|
- | |||
|
LC8230 | Holotype |
|
|
|
|
|
|
- |
LC8248 |
|
|
|
|
|
|
- | ||
|
ATCC62257 | Holotype |
|
|
|
|
|
|
- |
CBS128510 |
|
|
|
|
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|
- | ||
LC8335 |
|
|
|
|
|
|
- | ||
LC8492 |
|
|
|
|
|
|
- | ||
|
LC8894 | Holotype |
|
|
|
|
|
|
- |
LC8895 |
|
|
|
|
|
|
- | ||
LC8896 |
|
|
|
|
|
|
- | ||
|
CBS126.25 |
|
|
|
|
|
|
- | |
CBS181.82 | Holotype |
|
|
|
|
|
|
- |