Corresponding author: ShuaiFei Chen (
Academic editor: D. Haelewaters
Roux J, Kamgan Nkuekam G, Marincowitz S, van der Merwe NA, Uchida J, Wingfield MJ, Chen SF (2020) Cryphonectriaceae associated with rust-infected
The
Hawaii, in the central Pacific Ocean, is comprised entirely of islands and is the northernmost island group in Polynesia (
In April 2005, a rust disease caused by
During a casual inspection of rust-infected
The dramatic death of
Surveys for
The presence on samples of fruiting structures (ascostromata, conidiomata), typical of the
DNA was extracted from all isolates using PrepMan Ultra Sample Preparation Reagent kits (Applied Biosystems, California, USA) following the manufacturer’s instructions. An Eppendorf Mastercycler (Merck, Germany) was used for PCR amplification of the nuclear rDNA region encompassing the internal transcribed spacer regions (ITS1, ITS2) and 5.8S gene of the ribosomal RNA (
A 5 µl aliquot of the PCR products was pre-stained with GelRedTM Nucleic Acid Gel stain (Biotium, Hayward, USA), separated on 1% agarose gels and visualised under UV light. PCR products were purified using Sephadex G-50 Gel (Sigma-Aldrich), following the manufacturer’s instructions. The concentrations of the purified PCR products were determined using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Rockland, USA). Sequencing reactions were performed using the Big Dye cycle sequencing kit with Amplitaq DNA polymerase FS (Perkin-Elmer, Warrington, UK), following the manufacturer’s protocols, on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Protocols for sequencing PCR amplicons were the same as those described by
A preliminary identification of the isolates was obtained by performing a similarity search (standard nucleotide BLAST) against the GenBank database (
For analyses of the
For isolates that grouped in
The sequences for each of the single gene datasets, as well as for a combined dataset consisting of two or three gene regions, were aligned using MAFFT online v. 7 (
PAUP v. 4.0 b10 (
PhyML v. 3.1 was used for the
Isolates of the
Growth in culture was examined for two isolates of each putative new species identified. The protocols used to assess growth in culture were the same as those described by
The genetic diversity of the most commonly encountered and globally important species in the
PCR products for each isolate were multiplexed for GeneScan analysis. The composition of each sample mix was the same as that described by
The allele size for each of the seven loci was scored for each isolate from the collection. These data were used to generate a multilocus haplotype profile for each isolate. Isolates that had identical alleles for each of the seven loci were treated as clones. The frequency of each allele within the collection was calculated by taking the number of times the allele was present in the population and dividing it by the population sample size. This was then used to calculate gene diversity using the formula
A total of 139
List of
Species | Island | Hosts | Number of Trees | Number of Strains |
---|---|---|---|---|
|
O’ahu |
|
18 | 19 |
˝ | ˝ |
|
3 | 3 |
˝ | ˝ | 11 | 11 | |
˝ | ˝ |
|
9 | 12 |
˝ | Hawaii |
|
28 | 38 |
˝ | ˝ | 1 | 1 | |
˝ | ˝ |
|
1 | 1 |
˝ | Maui |
|
7 | 8 |
|
O’ahu |
|
5 | 7 |
˝ | ˝ |
|
1 | 1 |
˝ | Hawaii |
|
1 | 1 |
˝ | ˝ |
|
1 | 1 |
|
Maui |
|
4 | 8 |
˝ | Hawaii |
|
2 | 4 |
˝ | ˝ |
|
1 | 2 |
|
O’ahu |
|
4 | 6 |
˝ | ˝ |
|
3 | 4 |
˝ | ˝ | 1 | 1 | |
|
O’ahu |
|
1 | 4 |
˝ | ˝ |
|
2 | 3 |
˝ | ˝ | 1 | 3 | |
˝ | Hawaii |
|
1 | 1 |
For the isolates selected for sequencing, the PCR fragments were approximately 550, 450 and 260 bp for the
List of isolates and their GenBank accession numbers used for DNA sequence comparisons.
Identity | Isolate No.1,2 | Host | Location | Collector | GenBank accession no. | Reference | ||
---|---|---|---|---|---|---|---|---|
|
|
|
||||||
|
CMW10469T |
|
New Zealand | G.J. Samuels |
|
|
N/A3 | |
CMW10470 |
|
New Zealand | G.J. Samuels |
|
|
N/A | ||
|
CMW10526 |
|
USA | S. Redlin |
|
|
N/A |
|
MES1001 | N/A | USA | W. Cullina |
|
|
N/A |
|
|
CTS1001 | N/A | USA | K. Kitka |
|
|
N/A |
|
|
|
CBS132181T |
|
Australia | B.A. Summerell & P. Summerell |
|
N/A | N/A |
|
|
CBS130826T |
|
Australia | C. Mohammed & M. Glen |
|
N/A | N/A |
|
|
CMW10030T |
|
Colombia | C.A. Rodas |
|
|
N/A | |
CMW10035 |
|
Colombia | C.A. Rodas |
|
|
N/A | ||
|
CMW28285T |
|
Cameroon | D. Begoude & J. Roux |
|
|
N/A | |
CMW28288 |
|
Cameroon | D. Begoude & J. Roux |
|
|
N/A | ||
|
CSF10757T |
|
China | S.F. Chen & W. Wang |
|
|
|
|
CSF10762 |
|
China | S.F. Chen & W. Wang |
|
|
|
|
|
|
CBL02T |
|
Brazil | M.E. Soares de Oliveira &M.A. Ferreira |
|
|
N/A |
|
CBL06 |
|
Brazil | M.E. Soares de Oliveira &M.A. Ferreira |
|
|
N/A |
|
|
|
CMW44128T |
|
La Réunion | M.J. Wingfield |
|
|
N/A |
|
CMW44139 |
|
La Réunion | M.J. Wingfield |
|
|
N/A |
|
|
|
CERC9128T | China, GuangDong | S.F. Chen |
|
|
|
|
|
CERC9125 | China, GuangDong | S.F. Chen |
|
|
|
|
||
|
CMW9976T |
|
South Africa | M. Gryzenhout |
|
|
|
|
CMW9978 |
|
South Africa | M. Gryzenhout |
|
|
|
||
|
CMW26900 | China | X.D. Zhou & S.F. Chen |
|
|
|
|
|
CMW26908T | China | X.D. Zhou & S.F. Chen |
|
|
|
|
||
|
CMW29375 |
|
Zambia | M. Vermeulen & J. Roux |
|
|
|
|
CMW29376T |
|
Zambia | M. Vermeulen & J Roux |
|
|
|
|
|
|
CMW12750T | China | T.I. Burgess |
|
|
|
|
|
|
|
|
Hawaii | J. Roux |
|
|
|
This study |
|
Hawaii | J. Roux |
|
|
|
This study | ||
|
Hawaii | J. Roux |
|
|
|
This study | ||
|
|
|
Hawaii | J. Roux |
|
|
|
This study |
|
|
Hawaii | J. Roux |
|
|
|
This study | |
|
Hawaii | J. Roux |
|
|
|
This study | ||
|
CMW10781T |
|
Indonesia | M.J. Wingfield |
|
|
|
|
|
|
Hawaii | J. Roux |
|
|
|
This study | |
|
|
Hawaii | J. Roux |
|
|
|
This study | |
|
|
Hawaii | J. Roux |
|
|
|
This study | |
|
CMW34023T |
|
China | S.F. Chen |
|
|
|
|
CMW24912 |
|
China | M.J. Wingfield & X.D. Zhou |
|
|
|
|
|
|
CMW44126T |
|
La Réunion | M.J. Wingfield |
|
|
N/A |
|
CMW44127 |
|
La Réunion | M.J. Wingfield |
|
|
N/A |
|
|
|
CMW13936T |
|
South Africa | M. Gryzenhout |
|
|
|
|
CMW13937 |
|
South Africa | M. Gryzenhout |
|
|
|
|
|
|
CERC8780 |
|
China | J. Roux & S.F. Chen |
|
|
N/A |
|
CERC8810T |
|
China | S.F. Chen |
|
|
N/A |
|
|
|
CMW62 |
|
South Africa | M.J. Wingfield |
|
|
N/A | |
CMW9327 |
|
South Africa | J. Roux |
|
|
N/A |
|
|
CMW2113T |
|
South Africa | M.J. Wingfield |
|
|
N/A | ||
|
CMW10453 |
|
Democratic Republic of the Congo | N/A |
|
|
N/A | |
CMW8758 | Venezuela | M.J. Wingfield |
|
|
N/A | |||
CMW10669 | Republic of the Congo | J. Roux |
|
|
N/A |
|
||
CMW10639 |
|
Colombia | C.A. Rodas |
|
|
N/A |
|
|
|
CMW11290 | Indonesia | M.J. Wingfield |
|
|
N/A |
|
|
CMW8651 |
|
Indonesia | M.J. Wingfield |
|
|
N/A |
|
|
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
CMW11287T |
|
Ecuador | M.J. Wingfield |
|
|
N/A |
|
CMW11286 |
|
Ecuador | M.J. Wingfield |
|
|
N/A |
|
|
|
CMW10625 |
|
Colombia | C.A. Rodas |
|
|
N/A |
|
CMW9995 |
|
Colombia | R. Arbelaez |
|
|
N/A |
|
|
CMW10641T= CBS115854 |
|
Colombia | R. Arbelaez |
|
|
N/A |
|
|
|
CMW12727T |
|
Colombia | R. Arbelaez |
|
|
N/A |
|
CMW12729 |
|
Colombia | R. Arbelaez |
|
|
N/A |
|
|
|
CMW29940T= CBS124488 |
|
Zambia | D. Chungu & J. Roux |
|
|
N/A |
|
CMW29942= CBS124490 |
|
Zambia | D. Chungu & J. Roux |
|
|
N/A |
|
|
|
CMW29928T= CBS124503 |
|
Zambia | D. Chungu & J. Roux |
|
|
N/A |
|
CMW29930= CBS124502 |
|
Zambia | D. Chungu & J. Roux |
|
|
N/A |
|
|
|
CERC3629T |
|
China | S.F. Chen & G.Q. Li |
|
|
N/A |
|
CERC3631 |
|
China | S.F. Chen & G.Q. Li |
|
|
N/A |
|
|
|
CMW7048 |
|
USA | R.J. Stipes |
|
|
N/A | |
CMW13749 |
|
Japan | N/A |
|
|
N/A |
|
|
|
CFCC52138T |
|
China, ShaanXi | N. Jiang |
|
|
N/A |
|
CFCC52139 |
|
China, ShaanXi | N. Jiang |
|
|
N/A |
|
|
|
CMW10455 |
|
Italy | A. Biraghi |
|
|
N/A |
|
CMW10477 |
|
Italy | A. Biraghi |
|
|
N/A | ||
|
CMW18790 |
|
Indonesia | M.J. Wingfield |
|
|
N/A | |
CMW18793 |
|
Indonesia | M.J. Wingfield |
|
|
N/A | ||
CMW28535T= CBS124009 |
|
North Sumatra, Indonesia | M.J. Wingfield |
|
|
N/A |
|
|
|
CMW37321 |
|
South Africa | J. Roux |
|
|
N/A |
|
CMW37322T |
|
South Africa | J. Roux |
|
|
N/A |
|
|
|
CMW2091 |
|
USA | R.J. Stipes |
|
|
N/A | |
CMW10442 |
|
USA | R.J. Stipes |
|
|
N/A | ||
|
CMW37887T |
|
South Africa | J. Roux, S.F. Chen & F. Roets |
|
|
|
|
CMW37329 |
|
South Africa | J. Roux & S.F. Chen |
|
|
|
|
|
|
CMW7033T |
|
South Africa | M. Venter |
|
|
|
|
CMW7035 |
|
South Africa | M. Venter |
|
|
|
|
|
|
CMW37334T |
|
South Africa | J. Roux & S.F. Chen |
|
|
|
|
CMW37335 |
|
South Africa | J. Roux & S.F. Chen |
|
|
|
|
|
|
CMW37337T |
|
South Africa | J. Roux & S.F. Chen |
|
|
|
|
CMW37338 |
|
South Africa | J. Roux & S.F. Chen |
|
|
|
|
|
CMW6246 |
|
Australia | M.J. Wingfield |
|
|
|
|
|
CMW10015 |
|
New Zealand | R.J. van Boven |
|
|
|
|
|
|
CMW37314T |
|
South Africa | M.J. Wingfield & J. Roux |
|
|
N/A |
|
CMW37315 |
|
South Africa | M.J. Wingfield & J. Roux |
|
|
N/A |
|
|
|
CMW28274 |
|
Swaziland | J. Roux |
|
|
N/A |
|
CMW28276T |
|
Swaziland | J. Roux |
|
|
N/A | ||
CMW28275 |
|
Swaziland | J. Roux |
|
|
N/A |
|
|
|
CBS130775 |
|
Australia | C. Crane |
|
|
N/A |
|
CBS130776T |
|
Australia | C. Crane |
|
|
N/A |
|
|
|
CMW11301 |
|
Azores | C.S. Hodges & D.E. Gardner |
|
|
N/A |
|
|
CMW14550 |
|
Mexico | C.S. Hodges |
|
|
N/A |
|
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
|
Hawaii | J. Roux |
|
|
N/A | This study | |
|
CMW46433T |
|
South Africa | D.B. Ali & J. Roux |
|
|
N/A |
|
CMW46435 |
|
South Africa | D.B. Ali & J. Roux |
|
|
N/A |
|
|
|
CSF2061T | China | S.F. Chen & G.Q. Li |
|
|
|
|
|
CSF8777 | China | J.Roux & S.F. Chen |
|
|
|
|
||
|
CSF10460T | China | S.F. Chen & W. Wang |
|
|
|
|
|
CSF10738 | China | S.F. Chen & W. Wang |
|
|
|
|
||
|
CMW9972 |
|
Ecuador | M.J. Wingfield |
|
|
N/A | |
CMW10796T |
|
Ecuador | M.J. Wingfield |
|
|
N/A |
|
|
CMW9971 |
|
Ecuador | M.J. Wingfield |
|
|
N/A |
|
|
|
CMW18119T |
|
USA | C.S. Hodges |
|
|
N/A | |
CMW18115 |
|
USA | C.S. Hodges |
|
|
N/A |
|
|
|
CMW5587 |
|
South Africa | W.A. Smit |
|
|
N/A |
|
CMW5288 |
|
South Africa | W.A. Smit |
|
|
N/A |
|
1 Designation of isolates and culture collections:
For the
Phylogenetic trees based on Maximum Likelihood (
In the
Phylogenetic trees, based on Maximum Likelihood (
Fruiting bodies developed for all six isolates grown on
Colonies on 2% MEA were fluffy and white when young, turning yellow or greenish-grey to greenish when old. The optimal growth temperatures for novel species was 30 °C, at which colonies reached 59–80 mm within 4 days.
Based on phylogenetic analyses of sequence data for the three gene regions, three previously unknown
The species name refers to the Hawaiian word for happy, “Hau’oli”, describing the collector’s joy in visiting and discovering
Micrographs of
Not observed.
Formed after two months on
Colonies on 2% MEA, when young showing circular growth with smooth margins, above white with tint of yellow (30 °C) or orange (25 °C) towards the edge of Petri dish, reverse yellow, except for at 30 °C becoming brown towards the edge; with age above becoming brown, except for 30 °C at which each colony showing variable yellow with white mycelial clumps, reverse dark brown at all temperatures; optimal growth at 30 °C (9.4 mm/d), followed by 25 °C (7.9 mm/d) and 20 °C (4.8 mm/d), minimal growth at 35 °C (0.2 mm/d), no growth at 5 °C; mycelia fluffy, density sparce in centre becoming thicker towards the edge.
On/in bark of
Hawaii, USA
Nucleotide differences observed in the
Species/Isolate No. |
|
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
82 | 61 | 75 | 76 | 80 | 112 | 161 | 162 | 186 | 187 | 193 | 194 | 467 | |
T3 | A |
|
|
|
– | – |
|
T | A | – |
|
– | |
|
T | A |
|
|
|
– | – |
|
T | A | – |
|
– |
T | A |
|
|
|
– | – |
|
T | A | – |
|
– | |
– | A | – | T | G | G |
|
A | T | A | – | A | – | |
|
– | A | – | T | G | G |
|
A | T | A | – | A | – |
T |
|
– | T | G | G | – | A |
|
|
|
A |
|
|
|
T |
|
– | T | G | G | – | A |
|
|
|
A |
|
1 Polymorphic nucleotides occurring only in all isolates are shown, not alleles that partially occur in individuals per phylogenetic group. 2 Numerical positions of the nucleotides in the DNA sequence alignments are indicated. 3 Fixed polymorphisms for each group are in bold. 4 Ex-type isolates are indicated in italic.
Nucleotide differences observed in the
Species/Isolate No. |
|
|||||||||
---|---|---|---|---|---|---|---|---|---|---|
1052 | 127 | 130 | 131 | 132 | 182 | 183 | 188 | 191 | 201 | |
G3 | C | – | – | – | – | – | – | T | C | |
|
G | C | – | – | – | – | – | – | T | C |
G | C | – | – | – | – | – | – | T | C | |
|
C | C | T | C | – | – | – | T | C | |
|
|
C | C | T | C | – | – | – | T | C |
G |
|
C | T | C |
|
|
|
|
|
|
|
G |
|
C | T | C |
|
|
|
|
|
1 Polymorphic nucleotides occurring only in all isolates are shown, not alleles that partially occur in individuals per phylogenetic group. 2 Numerical positions of the nucleotides in the DNA sequence alignments are indicated. 3 Fixed polymorphisms for each group are in bold. 4 Ex-type isolates are indicated in italic.
Nucleotide differences observed in the
Species/Isolate No. |
|
||||||
---|---|---|---|---|---|---|---|
232 | 43 | 44 | 112 | 113 | 114 | 127 | |
C3 | G | C | – | – | – | T | |
C | G | C | – | – | – | T | |
C | G | C | – | – | – | T | |
|
G | C | T | T | T | T | |
|
G | C | T | T | T | T | |
C |
|
|
T | T | T |
|
|
C |
|
|
T | T | T |
|
1 Polymorphic nucleotides occurring only in all isolates are shown, not alleles that partially occur in individuals per phylogenetic group. 2 Numerical positions of the nucleotides in the DNA sequence alignments are indicated. 3 Fixed polymorphisms for each group are in bold. 4 Ex-type isolates are indicated in italic.
The species name refers to the Hawaiian Islands where the holotype was collected.
Micrographs of
Not observed.
Formed after two months on
Colonies on 2% MEA, when young showing circular growth with smooth margins, above white with yellow tint towards edge (25 °C), reverse pale brown, becoming darker in centre at 25 °C and 30 °C; with age above becoming darker yellow to brown, reverse dark brown, except at 20 °C, 25 °C having yellow with dark brown patches; optimal growth at 30 °C (6.6 mm/d), followed by 25 °C (6.0 mm/d) and 20 °C (4.1 mm/d), minimal growth at 35 °C (0.1 mm/d), growth at 5 °C restricted to mycelial plug; mycelia fluffy, density sparse in centre becoming thicker towards the edge.
On/in bark of
Hawaii, USA
Nucleotide differences observed in the
Species/Isolate No. |
|
|||||||
---|---|---|---|---|---|---|---|---|
562 | 57 | 59 | 98 | 160 | 161 | 193 | 467 | |
|
|
|
|
– | – | A |
|
– |
|
|
|
– | – | A |
|
– | |
|
|
|
– | – | A |
|
– | |
– | – | G | – | – | – | – | T | |
– | – | G | – | – | – | – | T | |
|
– | – | G | – | – | – | – | T |
|
– | – | G |
|
|
A | – | T |
1 Polymorphic nucleotides occurring only in all isolates are shown, not alleles that partially occur in individuals per phylogenetic group. 2 Numerical positions of the nucleotides in the DNA sequence alignments are indicated. 3 Ex-type isolates are indicated in italic. 4 Fixed polymorphisms for each group are in bold.
Nucleotide differences observed in the
Species/Isolate No. |
|
|
||||||
---|---|---|---|---|---|---|---|---|
572 | 131 | 139 | 175 | 272 | 77 | 220 | 222 | |
|
Cd | T |
|
C |
|
C | – |
|
C | T |
|
C |
|
C | – |
|
|
C | T |
|
C |
|
C | – |
|
|
C |
|
G | C | G |
|
A | C | |
C |
|
G | C | G |
|
A | C | |
|
C |
|
G | C | G |
|
A | C |
|
|
T | G | – | G | C | A | C |
1 Polymorphic nucleotides occurring only in all isolates are shown, not alleles that partially occur in individuals per phylogenetic group. 2 Numerical positions of the nucleotides in the DNA sequence alignments are indicated. 3 Ex-type isolates are indicated in italic. 4 Fixed polymorphisms for each group are in bold.
The species name refers to the fact that Hawaii, where the holotype of this fungus was collected, is regarded as a paradise by travellers.
Micrographs of
Not observed.
Produced after two months on
Colonies on 2% MEA, when young, showing circular growth with smooth edges, above white, reverse pale to dark brown (30 °C) and yellow (25 °C); with age, above becoming brown and reverse dark yellow; optimal growth at 30 °C (7.7 mm/d), followed by 25 °C (7.0 mm/d) and 20 °C (4.6 mm/d), minimal growth at 35 °C (0.1 mm/d), no growth at 5 °C; mycelia fluffy, density-sparse in centre, becoming thicker towards the edge, aerial hyphae more abundant at 25 °C than at 30 °C when young.
On/in bark of
Hawaii, USA
Inoculation with two isolates each of
Vertical bar chart showing results of inoculation trial (xylem lesion) with
Five species of
The occurrence of
The occurrence of a single clone of
Three new species of
Preliminary pathogenicity trials on
In the surveys conducted in this study, samples with symptoms of the
We thank the National Research Foundation of South Africa (NRF), The Centre of Excellence in Tree Health Biotechnology (CoE-CTHB), the National Key R&D Program of China (China–South Africa Forestry Joint Research Centre Project; project No. 2018YFE0120900) for providing funding and the facility to conduct this study. We are also most grateful to Chris Kadooka and JB Friday, University of Hawaii and Lloyd Loope of the US Geological Survey, Makawao, Hawaii for invaluable advice and local support of the survey carried out in Hawaii. This work is based on the research supported by the National Research Foundation of South Africa, Grant specific unique reference number (UID83924). The grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by NRF supported research are that of the authors and that the NRF accepts no liability whatsoever in this regard.
Table S1
PCR-based microsatellite markers
List of PCR-based microsatellite markers used in this study.
Table S2
datasets and statistics
Datasets used and the statistics resulting from the phylogenetic analyses.