INTRODUCTION {#s1}
============

*Leucaena leucocephala* is a mimosoid tree species native to southern Mexico and central America ([@R2]). In various parts of the world including Réunion island where it has been introduced, the tree is considered an invasive weed. This is largely due to its ability to easily colonize disturbed sites; outcompeting indigenous vegetation ([@R21], [@R2]). The tree also has positive attributes being used for land restoration, natural wind barriers, fodder for animals and as a source of pulp for paper production ([@R25], [@R1], [@R27], [@R9], [@R2]). Despite these beneficial properties under some conditions, countries such as South Africa utilise biological control to prevent *L. leucocephala* from spreading as an invasive ([@R25]).

*Raffaelea,* a genus in the family *Ophiostomataceae* (*Ophio-stomatales*, *Ascomycota*), accommodates obligate ambrosia beetle symbionts ([@R12], [@R5]). Most species in the genus are saprotrophic. But some, such as the causal agents of oak wilt in Japan (*R. quercivora*) ([@R18]) and Korea (*R. quercus-mogolicae*) ([@R17]), and laurel wilt in the USA (*R. lauricola*) ([@R11]) are important tree-killing pathogens. The genus is characterised as having reduced conidiogenous structures and budding secondary conidia; possibly facilitating their dispersal by ambrosia beetles ([@R7], [@R12], [@R3]). Until recently *Raffaelea* was known as an asexual genus, but the recent discovery of a sexual morph in one species, and the inclusion of two sexual morphs previously described in *Ophiostoma* have altered this view ([@R5], [@R23]).

Prior to the wide applications of DNA sequencing technology, species of *Raffaelea* were often confused with those in *Ambrosiella*. This is because species of both genera share convergent morphological traits adapted to their ambrosia beetle-associated lifestyle. Consequently, many *Raffaelea* spp. were initially treated in *Ambrosiella* and *vice versa*. This trend continued until DNA sequence data revealed that these genera reside in two separate orders of the *Sordariomycetes*; the *Ophiostomatales* (*Raffaelea*) and the *Microascales* (*Ambrosiella*) ([@R12], [@R3], [@R4]).

During a survey of fungi on the French island of Réunion in 2015, dying *L. leucocephala* trees were observed ([Fig. 1A](#F1){ref-type="fig"}). Upon closer inspection, the trees were found to have insect tunnels resembling those produced by ambrosia beetles, but insects were no longer present. The wood tissues associated with these tunnels were clearly stained ([Fig. 1B, C](#F1){ref-type="fig"}). The aim of this study was to isolate and identify the fungus responsible for the wood staining and to consider its pathogenicity.

MATERIALS AND METHODS {#s2}
=====================

Fungal Isolation {#s2a}
----------------

Pieces of wood including insect tunnels and associated stain were cut from dying *L. leucocephala* trees. Small pieces of stained wood or scrapings from the insect tunnels were transferred to 2 % malt extract agar (MEA: 20 g malt extract, 20 g Difco agar in 1 L dH~2~O) amended with the antibiotics streptomycin and cycloheximide (0.4 g streptomycin disulphate and 0.5 g cycloheximide per 1 L, Sigma-Aldrich). The plates were incubated at ambient temperature (approx. 20 °C) until visible fungal growth was observed. Colonies with growth patterns resembling those of *Raffaelea* spp. were sub-cultured on MEA plates, containing the same antibiotics as before, until pure cultures were obtained. The purified cultures were then transferred onto 2 % MEA plates and maintained for further study.

DNA extraction, amplification & sequencing {#s2b}
------------------------------------------

DNA extraction, amplification, purification and sequencing were performed following the protocols described by [@R6]. These included DNA amplification in 25 μL reaction volumes using FastStart Taq DNA Polymerase (Roche Applied Science, Mannheim, Germany) for the internal transcribed spacer region (ITS), the 28S ribosomal large subunit (LSU), and beta-tubulin (*TUB*) using the primer pairs ITS1F and ITS4 ([@R37]), LR5 and LROR ([@R36]), and T10 and Bt2b ([@R24], [@R8]). Agarose gel electrophoresis (1 % agarose) was performed on all PCR products to confirm successful amplification. PCR products were cleaned using ExoSAP-IT™ PCR Product Cleanup Reagent (ThermoFisher Scientific) following the manufacturer's protocols.

DNA sequencing was performed in both the forward and reverse direction using the same primers as for PCR. Sequencing PCR products were precipitated using the NaOAc/Ethanol precipitation method and analysed on an Applied Biosystems^®^ 3130 (4-capillary) Genetic Analyzer at the sequencing facility of the University of Pretoria (Pretoria, South Africa). Contig sequences were assembled and those produced for the ITS region were used for preliminary identification of isolates by performing BLAST searches against the non-redundant sequence database NCBI GenBank (<https://blast.ncbi.nlm.nih.gov/Blast.cgi>). All sequence data generated in this study were submitted to NCBI GenBank, and those for ex-type isolates will be included in the RefSeq Targeted Loci (RTL) database ([@R31]) ([Table 1](#T1){ref-type="table"}). For some species, ITS and LSU sequence data were extracted from whole genomes and these sequences have been deposited in NCBI GenBank.

Phylogenetic analyses {#s2c}
---------------------

Based on the putative identification of the isolates, datasets were constructed using reference sequences available in NCBI GenBank (<https://www.ncbi.nlm.nih.gov/genbank/>). Datasets were aligned using an online version of MAFFT v. 7 ([@R16]) and the default parameters. Maximum Likelihood (ML) analyses were performed for each individual gene region using raxmlGUI v. 1.3 ([@R32], [@R34]) with 1 000 bootstrap replicates and the substitution model GTR+I+G. Bayesian Inference analyses were conducted using MrBayes v. 3.2 ([@R29]) and run until the standard deviation of frequencies reached a value \< 0.01, sampling every 10 000 generations. Run times differed for each gene region: *TUB* (795 000 generations), ITS (6.64 million generations), LSU (6.02 M generations).

Morphological characterisation {#s2d}
------------------------------

### Growth in culture

A growth study was conducted to determine the optimal growth temperature for the isolated fungus. Three isolates, representing three cultural variants (CMW 51553, CMW 51724, CMW 51555) were used. Isolates were grown on 2 % MEA and the study was performed following the method described by [@R23].

### Microscopy

Fungal structures were initially mounted in water on microscope slides. The water was later replaced with 85 % clear lactic acid in which visualization and measurements were made. The structures were studied and photographed using differential interference contrast (DIC) microscopy. Where possible, fifty measurements were made for each morphologically characteristic structure. The dimensions were described as minimum-maximum (mean ± standard deviation).

Pathogenicity {#s2e}
-------------

A pathogenicity trial was conducted using two isolates of the fungus isolated from infected *L. leucocephala* trees. Seeds were collected from pods on a tree in nature, and after germination these were planted in bags (25 L) containing a soil mixture composed of 1:2:4 mixture of river sand, red top soil and pine bark potting medium. Seedlings were maintained at 25 °C in a phytotron with natural day/night light cycle for approximately 2 yr. Inoculations were made on 2-yr-old saplings.

Eleven saplings were each inoculated with isolates CMW 51553 (M13) and CMW 51724 (M15), and 10 were inoculated with sterile agar plugs to serve as controls. A 3 mm diam. drill bit was used to make a wound of approximately 2 mm deep into the stem of each sapling with the drill bit being sterilized in 80 % ethanol between each sapling. To prepare the inoculum, a 3 mm agar plug was cut from actively growing cultures of the test isolates and placed inside the wound.

Wounds were sealed using parafilm to prevent drying of the inoculum or contamination. The inoculated saplings were monitored for approximately 6 wk. The bark of the area around the inoculation points was peeled back to expose the lesions. Lesions were measured above and below the inoculation points. To fulfil Koch's postulates, isolations were made from tissue associated with the inoculations, including the controls. Re-isolated cultures from two trees of each treatment and the controls were subjected to DNA sequencing to determine whether the fungus arising from the lesions was the same as that inoculated.

Statistical analyses were performed on lesions using the program R ([@R26]; <https://www.R-project.org/>). The data were first analysed using Grubbs test to detect outliers with "outliers" package ([@R30]). Normal distribution and homogeneity of variance were tested by performing a Shapiro-Wilk test and a Bartlett test. The data were then subjected to analysis of variance (ANOVA) and Tukey's honestly significance difference (Tukey's HSD).

RESULTS {#s3}
=======

Isolates and phylogenetic analyses {#s3a}
----------------------------------

Primary isolations from the wood samples yielded 33 isolates resembling *Raffaelea sensu stricto* as defined by [@R3]. These isolates had three different colony morphologies ([Fig. 2](#F2){ref-type="fig"}).

Preliminary identification of the isolates based on the BLAST results of the ITS region confirmed that they were species of *Raffaelea*. Maximum likelihood phylogenetic analyses of the ITS, LSU and *TUB* regions revealed that all isolates were of a single species, regardless of their different colony morphology. They resided in a well-supported lineage sister to *Raffaelea crossotarsa* and were distinct from described species ([Fig. 3](#F3){ref-type="fig"}). Phylogenies produced for the different gene regions using Bayesian inference analyses were highly congruent with those produced by the ML analyses and supported the grouping of the isolates obtained in this study as their own distinct, well supported species.

Pathogenicity {#s3b}
-------------

The two isolates used in the pathogenicity trial produced distinct lesions following the 6-wk observation period. Most control seedlings showed small lesions ([Fig. 4A](#F4){ref-type="fig"}), while larger lesion lengths were observed for both isolates ([Fig. 4B, C](#F4){ref-type="fig"}). Lesion lengths above the inoculation point were approximately 10 mm longer on average than those below this point. Exceptionally long lesions were found on two saplings: one inoculated with isolate CMW 51553 and the other with isolate CMW 51724, with one sapling showing fungal spread into the vascular system ([Fig. 4D, F](#F4){ref-type="fig"}).

Most of the control saplings displayed small lesions but four had somewhat longer lesions. One of the control saplings showed discolouration of the vascular system ([Fig. 4E, G](#F4){ref-type="fig"}). These data were removed as outliers. The result of the Shapiro Wilk test and Bartlett test showed that the data were normally distributed and had equal variance. Results of the ANOVA indicated that there was a significant within the data difference (P-value \< 0.01, at the 95 % confidence level). Based on the results of Tukey's HSD, there was a significant difference between the lesions associated with the inoculated versus the control plants but no significant difference in the lesion lengths produced by either of the two fungal isolates ([Fig. 5](#F5){ref-type="fig"}). Mortality was not observed in any of the inoculated saplings for the duration of the trial.

Re-isolations were performed from the lesion margins on both the control and inoculated saplings. Isolates resembling the *Raffaelea* species were obtained from all 11 saplings inoculated with CMW 51724, six of the saplings inoculated with CMW 51553 and one of the control saplings showing larger lesions. DNA sequence analyses confirmed the identity of the isolates as the same as those used in the inoculations.

Taxonomy {#s3c}
--------

***Raffaelea borbonica*** M. Procter, M.J. Wingf & Marinc., ***sp. nov.*** MycoBank MB836438. [Fig. 6](#F6){ref-type="fig"}.

*Etymology*: Name refers to the island of Réunion, previously known as *Île Bourbon* where this fungus was collected.

*Conidiophores* mononematous, micro- or macronematous, arising from vegetative hyphae, mostly simple, occasionally branched once, upright, straight, curved or undulate, tapering towards apex, 27--118 (69±23) μm long, 2--5.5 (4.0±0.7) μm wide near base, occasionally reduced to conidiogenous cell. *Conidiogenous cells* integrated, hyaline, cylindrical or peg-like when micronematous, tapering towards apex, blastic, showing percurrent growth, 3--31 × 1.5--2.5 (17±8 × 2±0.3) μm. *Conidia* hyaline, aseptate, majority oblong with the upper part swollen, apex round, tapering toward base, base truncate, 5--8.5 × 2--4 (6.8±0.7 × 2.9±0.4) μm, yeast-like budding from primary conidia observed in fresh culture.

*Typus*: **Réunion**, on *Leucaena leucocephala*, Jun. 2015, *M.J. Wingfield* & *P.W. Crous* (**holotype** PREM 62884, living culture ex-holotype CMW 51553 = PPRI 27953).

*Additional material examined*: **Réunion**, on *Leucaena leucocephala*, Jun. 2015, *M.J. Wingfield* & *P.W. Crous,* PREM 62885, living culture CMW 51724 = PPRI 27954.

*Culture characteristics*: Cultures on 2 % MEA in dark showing optimum growth at 25 °C reaching 80 mm in 12 d, circular growth with smooth margin, mycelia mostly submerged (aerial hyphae sparse), flat, initially whitish to creamy becoming darker with age, sporodochium-like mycelial mass formed in 4-mo-old culture.

*Notes*: *Raffaelea borbonica* can be distinguished from its closest relative *R. crossotarsa* by conidial shape and in having longer but narrower dimensions. Conidia of *R. borbonica* are predominantly oblong with an enlarged apex, ranging 6.8 × 2.9 μm in average, whereas those of *R. crossotarsa* are globose to ovoid, ranging 6 × 4.9 μm in average.

DISCUSSION {#s4}
==========

A new species of *Raffaelea,* described here as*R. borbonica*, was isolated from dying *L. leucocephala* trees in Réunion. Isolation of this fungus from discoloured wood tissue was associated with ambrosia beetle damage. The known association between *Raffaelea* species and these beetles implies that *R. borbonica* was probably transmitted to the trees by the insects ([@R3], [@R14], [@R35]). However, the absence of the insects in the collected samples precludes the opportunity to link the beetle to *R. borbonica*. To do this, it will be necessary to recollect and determine the species of the beetle responsible for the damage.

*Raffaelea borbonica* bears close morphological resemblance to other species of *Raffaelea s. str*. ([@R3]). It can, however, be distinguished from previously described species based on its oblong to sub-globose conidia. Conidia of *Raffaelea* spp. are more typically ovoid to globose and occasionally T- or Y-shaped ([@R23]). *Raffaelea* now includes 30 described species ([@R3], [@R23], [@R33]) together with many taxa treated only as "*Raffaelea* sp." and yet to be described ([@R33], [@R19]).

In inoculation tests, *R. borbonica* was shown to have a low level of aggressiveness when compared to similar studies done with other *Raffaelea* species. For example, trials with *R. lauricola* were conducted in a similar manner as in this study and resulted in mortality within one month ([@R13], [@R20]). The disparate responses in some but not all of the inoculated saplings in the present study were most likely due to the fact that the saplings were grown from seeds and were thus genetically distinct from each other. The fact that the *L. leucocephala* trees from which *R. borbonica* was isolated in Réunion were wilting could have been due to the physical damage from the beetle infestation, or a combination of this damage and the low aggressiveness of the fungus. Variable levels of susceptibility of individual trees as seen in the pathogenicity tests could also have been a factor. There were no other obvious factors that might have contributed to the death of trees.

A confounding factor in the inoculation trials was that four control saplings developed lesions. *Raffaelea borbonica* was not isolated from three of these plants and we assume that the lesions were due to a host-response to the physical damage of wounding. Re-isolation of the fungus from one of the control plants most likely arose from a contamination event. In this regard, Ophiostomatoid fungi, including *Raffaelea* species, are well-known to be transmitted by arthropods such as mites ([@R28]) that could have moved *R. borbonica* to one of the control plants.

*Leucaena leucocephala* is an aggressive invasive weed in Réunion. A disease of these plants is of interest given that it might provide a means to reduce the invasiveness of the plant. However, results of this study provided no evidence that *R. borbonica* poses a health threat to *L. leucocephala* or that it might offer any opportunity for biological control.

We thank Mr. Seamus Morgan for assistance in maintaining greenhouse plants, Dr Tuan Duong and Prof. Wilhelm de Beer for suggestions regarding the phylogenetic analyses, and Dr Donghyeon Lee and Dr Hiroyuki Suzuki for assistance with the statistical analyses. Dr Dominik Strassberg provided the opportunity to conduct a survey of fungi in Réunion and Prof. David Richardson assisted in providing logistics for this visit. We appreciate the financial support of the University of Pretoria and the DST/NRF Centre of Excellence in Plant Health Biotechnology (CPHB), South Africa.

![**A.** A dead *Leucaena leucocephala* tree observed on Réunion. **B.** Staining of wood associated with insect tunnelling. **C.** Sap stain in the outer growth rings.](fuse-2020-6-16-g001){#F1}

![Three distinct colony morphologies in *Raffaelea borbonica* isolates. Cultures were grown at 25 °C for 12 d in the dark. **A.** CMW 51553. **B.** CMW 51724. **C.** CMW 51555. Scale bars: A--C = 1 cm.](fuse-2020-6-16-g002){#F2}

![Phylogenetic trees resulting from maximum likelihood analyses of the *TUB*, LSU and ITS region for species of *Raffaelea.* Bootstrap support values above 70 are indicated on the nodes. Bayesian inference posterior probability values above 0.9 are indicated by bold lines. Sequences generated for the novel species in this study are indicated in **bold**. Asterisks (\*) indicate where genome data was used to obtain the sequence used in the analysis. T indicates ex-holotype, P indicates ex-paratype. Species of *Ophiostoma* were used to root the tree.](fuse-2020-6-16-g003){#F3}

![Lesions associated with inoculation on *Leucaena leucocephala* and discoloration of the vascular plant tissue. **A.** Control using clean agar. **B.** Isolate CMW 51553. **C.** Isolate CMW 51724 (arrows in A, B and C indicate limits of lesions originating from the agar plug). **D, F.** Inoculated with isolate CMW 51724. **E, G.** A control inoculation.](fuse-2020-6-16-g004){#F4}

![Bar graph showing mean lesion lengths on inoculated *Leucaena leucocephala* trees. Based on the results of the ANOVA, both inoculated treatments (CMW 51553 and CMW 51724) were significantly different from the control as denoted by **a** and **b**. Inoculated treatments were not significantly different from one another as denoted by **a**. Protruding lines indicate standard deviation.](fuse-2020-6-16-g005){#F5}

![Morphological characteristics of *Raffaelea borbonica* (ex-type, CMW 51553 = PPRI 27953). **A.** Culture grown at 25 °C in the dark for 12 d and 120 d. **B.** Mycelial cluster formed in older culture. **C--E.** Conidiophores (arrows). **F.** Conidiogenous cells showing percurrent growth (arrows). **G.** Conidia. **H, I.** Early stage of budding conidium. **J, K.** Later stage of budding conidium. Scale bars: B = 100 μm, C, G = 10 μm, D, E = 5 μm, F, H--K = 2.5 μm.](fuse-2020-6-16-g006){#F6}

###### 

Isolates used in this study, including multiple isolates of *Raffaelea borbonica* *sp. nov*.

  **Species**                                               **CMW**[^1^](#tfn1){ref-type="table-fn"}   **CBS or others**   **GenBank Accession Numbers**[^2^](#tfn2){ref-type="table-fn"}                  
  --------------------------------------------------------- ------------------------------------------ ------------------- ---------------------------------------------------------------- -------------- --------------
  *R. aguacate*                                                                                        Raff. sp. 272 ^G^   **MT633065**                                                     **MT629748**   --
                                                            38067^T^                                   141672              --                                                               --             KJ909297
  *R. albimanens*                                           25532^T^                                   271.70 ^G^          **MT633066**                                                     **MT629749**   **MT644111**
  *R. amasae*                                               25542^T^                                   116694              --                                                               **MT629750**   EU977470
  *R. ambrosiae*                                            25533^T^                                   185.64 ^G^          **MT633067**                                                     **MT629751**   **MT644094**
                                                            25533^T^                                   185.64 ^G^          **MT633068**                                                     **MT629752**   **MT644095**
  *R. arxii*                                                25534                                      273.70              --                                                               **MT629754**   --
                                                            25534                                      273.70 ^G^          MH859604                                                         **MT629753**   --
  ***R. borbonicasp. nov.***                                51548                                      IV4a M2             **MT633054**                                                     **MT629736**   **MT644100**
                                                            51549                                      IV4a M3             **MT633055**                                                     **MT629737**   **MT644109**
                                                            51719                                      IV4a M4             **MT633056**                                                     **MT629738**   **MT644108**
                                                            51550                                      IV4a M5             **MT633057**                                                     **MT629739**   **MT644107**
                                                            51720                                      V1b M7              **MT633080**                                                     **MT629770**   **MT644098**
                                                            51551                                      V1b M8              **MT633081**                                                     **MT629771**   **MT644097**
                                                            51722                                      V1b M9              **MT633082**                                                     **MT629772**   **MT644103**
                                                            51552                                      V1b M10             **MT633079**                                                     --             --
                                                                                                       IV4b M11            --                                                               **MT629740**   **MT644110**
                                                                                                       IV4b M12            **MT633058**                                                     **MT629741**   **MT644104**
                                                            51553^P^                                   IV4b M13            **MT633059**                                                     **MT629742**   **MT644106**
                                                            51723                                      IV4b M14            **MT633060**                                                     **MT629743**   **MT644105**
                                                            51724^T^                                   IV4b M15            **MT633061**                                                     **MT629744**   **MT644099**
                                                            51554                                      IV2c M22            **MT633052**                                                     **MT629734**   **MT644102**
                                                            51555                                      IV2c M23            **MT633053**                                                     **MT629735**   **MT644101**
                                                            51556                                      V3a M28             **MT633083**                                                     **MT629773**   **MT644096**
  *R. brunnea*                                                                                         378.68              --                                                               EU177457       --
  *R. campbellii*                                           44800^T^                                   139943              --                                                               KR018414       KR018444
  *R. cf. campbellii*                                                                                  Hulcr7355           --                                                               KX267101       KX267112
  *R. canadensis*                                           25536^T^                                   168.66              GQ225699                                                         **MT629755**   EU977473
  *R. crossotarsa*                                          44793^T^                                   141675              KX267138                                                         **MT629756**   KX267135
  *R. cyclorhipidia*                                        44790^T^                                   141676              **MT633069**                                                     **MT629757**   --
  *R. ellipticospora*                                       38056^T^                                   121569              **MT633070**                                                     **MT629758**   --
                                                                                                       C2345               --                                                               --             KJ909298
  *R. fusca*                                                38798^T^                                   121570              --                                                               EU177449       KJ909301
  *R. lauricola*                                            36261                                      PL159               --                                                               **MT629760**   --
                                                                                                       Raff. sp. 570 ^G^   **MT633071**                                                     **MT629759**   **MT644093**
  *R. montetyi*                                             25537^T^                                   463.94              --                                                               **MT629761**   --
                                                                                                       451.94              --                                                               --             EU977475
  *R. quercivora*                                           36263                                      122982 ^G^          **MT633072**                                                     **MT629762**   **MT644090**
                                                                                                       122982              --                                                               --             GQ225691
                                                                                                       Hulcr7167           --                                                               --             KX267119
                                                                                                       Hulcr7176           --                                                               --             KX267120
  *R. quercus-mongolicae*                                   37749^P^                                   KACC44403           **MT633073**                                                     **MT629764**   --
                                                            37751                                      KACC44405 ^G^       **MT633074**                                                     **MT629763**   **MT644091**
  *R. rapaneae*                                             40357^T^                                   140084              KT192596                                                         KT182930       --
  *R. santoroi*                                             25539^T^                                   399.67              **MT633075**                                                     **MT629765**   EU977476
  *R. seticollis*                                           1031^T^                                    634.66              **MT633076**                                                     **MT629766**   --
  *R. subalba*                                              38797^T^                                   121568              --                                                               **MT629767**   --
                                                                                                       C2401               --                                                               --             KJ909305
  *R. cf. subalba*                                                                                     Hulcr7375           --                                                               KX267102       KX267113
  *R. subfusca*                                                                                        Hulcr4717           KX267138                                                         --             KX267122
  *R. sulcati*                                              25540^T^                                   806.70              MH859951                                                         EU177462       EU977477
  *R. sulphurea*[^1^](#tfn1){ref-type="table-fn"}           25529                                      380.68 ^G^          **MT633077**                                                     **MT629768**   **MT644092**
  *R. xyleborina*                                           45859^T^                                   Hulcr6099           **MT633078**                                                     **MT629769**   KX267124
  *Raffaelea* sp. A (PL1001)                                38062                                                          --                                                               KJ909293       KJ909295
  *Raffaelea* sp*.*                                                                                    Hulcr7507           KX267141                                                         --             KX267128
  *Ophiostoma piliferum*[^3^](#tfn3){ref-type="table-fn"}   JGI                                                            **MT633063**                                                     **MT629746**   **MT644089**
  *O. quercus*                                              2465                                       117912              **MT633064**                                                     **MT629747**   **MT644088**
  *O. piceae*                                                                                          UAMH11346           **MT633062**                                                     **MT629745**   **MT644087**

^1^ T = ex-holotype, P = ex-paratype, G = DNA data extracted from whole genomes.

^2^ Sequences generated in this study are in bold.

^3^ The genome sequence for *Ophiostoma piliferum* is not yet published, but has been made available on <https://mycocosm.jgi.doe.gov/Ophpi1/Ophpi1.home.html>.

[^1]: **Citation:** Procter M, Nel WJ, Marincowitz S, Crous PW, Wingfield MJ (2020). A new species of *Raffaelea* from beetle-infested *Leucaena leucocephala*. *Fungal Systematics and Evolution* **6**: 305--314. doi: 10.3114/fuse.2020.06.16

[^2]: **Corresponding editor:** A.J.L. Phillips

[^3]: **Conflict of interest:** The authors declare that there is no conflict of interest.
