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Research Article
New Fusarium species from the Kruger National Park, South Africa
expand article infoMarcelo Sandoval-Denis‡§, Wijnand J. Swart§, Pedro W. Crous‡
‡ Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
§ University of the Free State, Bloemfontein, South Africa

Corresponding author: Marcelo Sandoval-Denis ( m.sandoval@westerdijkinstitute.nl )

Academic editor: George Mugambi
© 2018 Marcelo Sandoval-Denis, Wijnand J. Swart, Pedro W. Crous.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Sandoval-Denis M, Swart WJ, Crous PW (2018) New Fusarium species from the Kruger National Park, South Africa. MycoKeys 34: 63-92. https://doi.org/10.3897/mycokeys.34.25974
Open Access

Abstract

Three new Fusarium species, F. convolutans, F. fredkrugeri, and F. transvaalense (Ascomycota, Hypocreales, Nectriaceae) are described from soils collected in a catena landscape on a research supersite in the Kruger National Park, South Africa. The new taxa, isolated from the rhizosphere of three African herbaceous plants, Kyphocarpa angustifolia, Melhania acuminata, and Sida cordifolia, are described and illustrated by means of morphological and multilocus molecular analyses based on sequences from five DNA loci (CAL, EF-1 α, RPB1, RPB2 and TUB). According to phylogenetic inference based on Maximum-likelihood and Bayesian approaches, the newly discovered species are distributed in the Fusarium buharicum, F. fujikuroi, and F. sambucinum species complexes.

Keywords

Natural parks, phylogeny, fungi, multigene, morphology, diversity

Introduction

Fungi are common colonisers of the plant rhizobiome and endosphere, where they play a key role in modulating the interactions between plant roots and soil (Zachow et al. 2009; Visioli et al. 2014). The direct and indirect interaction between fungal growth in the rhizosphere and its effect on plant growth and health is well documented (Havlicek and Mitchell 2014; Hargreaves et al. 2015; Lareen et al. 2016). Such effects include either a positive feedback by producing plant growth promoting factors, solubilising and stimulating nutrient uptake by plant roots or by inhibiting the growth of concomitant pathogenic organisms (Schippers et al. 1987; Mommer et al. 2016). Conversely, deleterious effects have also been observed, either related to the presence of pathogenic fungal species or caused by fungal-induced modifications of plant root functions, impeding root growth or negatively altering nutrient availability (Schippers et al. 1987; Mommer et al. 2016). Likewise, plants can select and harbour a particular fungal community on its roots via root exudates (Lareen et al. 2016; Sasse et al. 2018), while abiotic influences including water availability, climate and season, soil type, grazers and other animals, orchestrate the development of a unique fungal diversity (Philippot et al. 2013; Havlicek and Mitchell 2014; Hargreaves et al. 2015; Lareen et al. 2016).

The genus Fusarium Link (Hypocreales, Nectriaceae) includes a vast number of species, commonly recovered from a variety of substrates including soil, air, water and decaying plant materials; being also able to colonise living tissues of plants and animals, including humans; acting as endophytes, secondary invaders or becoming devastating plant pathogens (Nelson et al. 1994). In addition to their ability to colonise a multiplicity of habitats, Fusarium is a cosmopolitan genus, present in almost any ecosystem in the world, including human-made settings such as air and dust in the indoor environment or even in hospitals (Perlroth et al. 2007; Aydogdu and Asan 2008; Pinheiro et al. 2011).

Being common inhabitants of plant root ecosystems, fusaria and, particularly Fusarium graminearum Schwabe, F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg, F. verticillioides (Sacc.) Nirenberg (Syn. F. moniliforme J. Sheld.), F. oxysporum Schltdl., as well as species recently segregated from Fusarium, including Neocosmospora phaseoli (Burkh.) L. Lombard & Crous (Syn. Fusarium phaseoli Burkh.) and N. virguliforme (O’Donnell & T. Aoki) L. Lombard & Crous (Syn. F. virguliforme O’Donnell & T. Aoki), have been regularly studied for their interactions with the rhizobiome, motivated mainly by the importance of these organisms as soil-borne plant pathogens and the need to develop effective control mechanisms (Larkin et al. 1993; Hassan Dar et al. 1997; Pal et al. 2001; Fravel et al. 2003; Idris et al. 2006; Díaz Arias et al. 2013). Similarly, abundant data is available regarding the ecology and distribution of plant-associated fusaria, particularly related to pathogenic species or commonly isolated endophytes (Leslie and Summerell 2006). Little attention has however been given to the occurrence of non-pathogenic fungal species, including Fusarium spp. in root microbial communities (Zakaria and Ning 2013; Jumpponen et al. 2017; LeBlanc et al. 2017), while comprehensive DNA sequence-based surveys have been directed mostly to the study of highly relevant and abundant rhizosphere fungal genera such as Trichoderma Pers., Verticillium Nees or mycorrhizal fungi (Zachow et al. 2009; Bent et al. 2011; Ruano-Rosa et al. 2016; Saravanakumar et al. 2016).

The Kruger National Park (KNP) in South Africa is one of the largest natural reserves in Africa, encompassing a number of non-manipulated landscapes, with almost no human alteration (Carruthers 2017). Recently, four research “supersites” have been identified and established in KNP, each of these supersites representing unique geological, ecological and climatic features of the park (Smit et al. 2013). A multidisciplinary study was conducted in KNP aimed to determine functioning and interaction between abiotic and biotic components, as well as soil properties, hydrology and other processes that determine the structure, biodiversity and heterogeneity of a catena or hill slope ecosystem on one of these “supersites”, located deep inside the KNP (data not published). In order to assess the microbial soil population and community dynamics, mainly focused on bacteria, several rhizosphere samples were obtained from diverse African plants on one of these exceptional protected savannah landscapes. From these collections, interesting fusaria were isolated from the root ecosystem of three native African herbaceous plants i.e. Kyphocarpa angustifolia (Moq.) Lopr. (Amaranthaceae), Melhania acuminata Mast. (Malvaceae) and Sida cordifolia Linn. (Malvaceae). According to their unique morphological traits and clear phylogenetic delimitations, these isolates are described here as three new Fusarium species.

Methods

Study site and sampling

During March 2015, rhizosphere soil from three herbaceous plants was collected in the Southern Granites “supersite” catena (Stevenson-Hamilton supersite) in the KNP, between 25°06'28.6S, 31°34'41.9E and 25°06'25.7S, 31°34'33.7E (Fig. 1). A catena consists of different soil types observed from a crest to a valley bottom with a wetland or drainage exhibiting different water retention capabilities due to the slope or aspect (topography) and the depth of underlying geological rocks (Brown et al. 2004, Van Zijl and Le Roux 2014). The main characteristics of the Stevenson-Hamilton supersite are described in detail by Smit et al. (2013). Briefly, in this site, a single catena landscape covers approximately 1 km from top to bottom and consists of a hill slope, a sodic site (or grazing lawn), a riparian and floodplain area and a dry drainage line. Three species of plants were selected for sampling occurring at the two extremes of the catena. Two of these species (Kyphocarpa angustifolia and Sida cordifolia) occurred at both top and bottom sites while Melhania acuminata only occurred at the top site. The soil (100 mm depth) at the top of the slope is Clovelly with a high percentage of sand (90%) and a low cation exchange capacity (CEC) (mean sodium concentration of 1062 mg/kg) and pH (mean 5.85). The soil at the bottom of the slope is of the Sterkspruit type, with higher clay content thus higher CEC (mean sodium concentration of 3802 mg/kg) and higher pH (mean 6.4). Rhizosphere soil of 10 plants of the same species occurring at each top or bottom site was sampled using a core soil sampler. A total of 50 samples consisting of ca. 200 g of soil from the roots of each plant were taken, deposited in zip-lock plastic bags and kept on ice in a cool bag at approximately 5 °C until analysed in the laboratory.

Figure 1. 

Map of the Kruger National Park (KNP) in South Africa. The arrows indicate the location of the four research “supersites” (adapted from Smit et al. 2013). Sampling site is indicated with a black star. The inset shows the location of the KNP within South Africa, indicated by a grey box.

Isolation of Fusarium strains

Soil samples were mixed thoroughly and sieved to remove large elements. Fine soil particles were uniformly spread and distributed over the surface of pentachloronitrobenzene agar (PCNB; also known as the Nash-Snyder medium, recipe in Leslie and Summerell 2006) supplemented with streptomycin (0.3 g/l) and neomycin sulphate (0.12 g/l) and malt-extract agar (MEA; recipes on Crous et al. 2009) on 9 mm Petri dishes and incubated at 24 °C for 10 d under a natural day/night photoperiod. Each soil sample was processed in duplicate. Fungal growth was evaluated daily and growing colonies were transferred to fresh Potato Dextrose Agar (PDA; recipe in Crous et al. 2009). Colonies were evaluated for their macro- and microscopic characteristics and a total of 19 fungal cultures showing features typical of Fusarium were subjected to single spore isolation as described previously (Sandoval-Denis et al. 2018). Single spore isolates were finally transferred and maintained in Oatmeal Agar plates and slants (OA; recipe in Crous et al. 2009). Fungal strains isolated in this study were deposited in the collection of the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, the Netherlands), the working collection of Pedro W. Crous (CPC), held at CBS (Table 1); and voucher specimens were deposited in The South African National Collection of Fungi (NCF) (Mycology Unit, Biosystematics Division, Plant Protection Institute, Agricultural Research Council, Pretoria, South Africa).

Table 1.
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Origin, strain and GenBank/ENA accession number of strains and DNA sequences included in this study.

Species name Strain†‡ Country Host Sequence accession number§
CAL EF-1α RPB1 RPB2 TUB
Fusarium agapanthi NRRL 54463T Australia Agapanthus sp. KU900611 KU900630 KU900620 KU900625 KU900635
Fusarium ananatum CBS 118516T South Africa Ananas comosus fruit LT996175 LT996091 LT996188 LT996137 LT996112
Fusarium andiyazi CBS 119857T = NRRL 31727 South Africa Sorghum bicolor soil debris LT996176 LT996092 LT996189 LT996138 LT996113
Fusarium anthophilum CBS 737.97 = NRRL 13602 Germany Hippeastrum sp. LT996177 LT996093 LT996190 LT996139 LT996114
Fusarium armeniacum NRRL 6227 USA Fescue hay JX171446 JX171560
Fusarium asiaticum CBS 110257 = NRRL 13818 Japan Barley JX171459 JX171573
Fusarium bactridioides NRRL 20476 USA Cronartium conigenum AF158343 AF160290 Not public Not public U34434
Fusarium begoniae CBS 403.97T = NRRL 25300 Germany Begonia elatior hybrid AF158346 AF160293 LT996191 LT996140 U61543
Fusarium buharicum CBS 178.35 = NRRL 25488 USSR Gossypium rotting stem base KX302912 KX302920 KX302928
CBS 796.70 = NRRL 13371 Iran Hibiscus cannabinus stalk JX171449 JX171563
Fusarium bulbicola CBS 220.76T = NRRL 13618 Germany Nerine bowdenii KF466327 KF466415 KF466394 KF466404 KF466437
Fusarium brachygibbosum NRRL 13829 Japan River sediments JX171460 JX171574
Fusarium circinatum CBS 405.97T = NRRL 25331 USA Pinus radiata KM231393 KM231943 JX171510 HM068354 KM232080
Fusarium coicis NRRL 66233T Australia Coix gasteenii LT996178 KP083251 KP083269 KP083274 LT996115
Fusarium concentricum CBS 450.97T = NRRL 25181 Costa Rica Musa sapientum fruit AF158335 AF160282 LT996192 JF741086 U61548
Fusarium continuum F201128 China Zanthoxylum bungeanum stem KM236720 KM520389 KM236780
Fusarium convolutans CBS 144207T = CPC 33733 South Africa Kyphocarpa angustifolia rhizophere LT996094 LT996193 LT996141
CBS 144208 = CPC 33732 South Africa Kyphocarpa angustifolia rhizophere LT996095 LT996194 LT996142
Fusarium culmorum CBS 417.86 = NRRL 25475 Denmark Moldy barley kernel JX171515 JX171628
Fusarium denticulatum CBS 735.97 = NRRL 25302 USA Ipomoea batatas AF158322 AF160269 LT996195 LT996143 U61550
Fusarium dlaminii CBS 119860T = NRRL 13164 South Africa Soil debris in cornfield AF158330 AF160277 KU171681681 KU171701 U34430
Fusarium fracticaudum CBS 137234PT Colombia Pinus maximonoii stem LT996179 KJ541059 LT996196 LT996144 KJ541051
Fusarium fractiflexum NRRL 28852T Japan Cymbidium sp. AF158341 AF160288 Not public LT575064 AF160315
Fusarium fredkrugeri NRRL 26152 Niger Unknown AF160306 AF160321
CBS 144209T = CPC 33747 South Africa Melhania acuminata rhizophere LT996181 LT996097 LT996199 LT996147 LT996117
CBS 144210 = NRRL 26061 Madagascar Striga hermonthica AF158356 AF160303 LT996197 LT996145 AF160319
CBS 144495 = CPC 33746 South Africa Melhania acuminata rhizophere LT996180 LT996096 LT996198 LT996146 LT996116
Fusarium fujikuroi NRRL 13566 China Oryza sativa AF158332 AF160279 JX171456 JX171570 U34415
Fusarium globosum CBS 428.97T = NRRL 26131 South Africa Zea mays KF466329 KF466417 KF466396 KF466406 KF466439
Fusarium goolgardi NRRL 66250T = RBG 5411 Australia Xanthorrhoea glauca KP083270 KP083280
Fusarium graminearum CBS 123657 = NRRL 31084 USA Corn JX171531 JX171644
Fusarium konzum CBS 119849T USA Sorghastrum nuttans LT996182 LT996098 LT996200 LT996148 LT996118
Fusarium kyushuense NRRL 25349 Japan Triticum aestivum GQ915492
Fusarium lactis CBS 411.97NT = NRRL 25200 USA Ficus carica AF158325 AF160272 LT996201 LT996149 U61551
Fusarium langsethiae NRRL 54940 Norway Oats JX171550 JX171662
Fusarium lateritium NRRL 13622 USA Ulmus sp. AY707173 JX171457 JX171571
Fusarium longipes NRRL 13368 Australia Soil JX171448 JX171562
Fusarium mangiferae NRRL 25226 Israel Mangifera indica AF158334 AF160281 JX171509 HM068353 U61561
Fusarium mexicanum NRRL 47473 Mexico Mangifera indica inflorescence GU737389 GU737416 Not public Not public GU737308
Fusarium napiforme CBS 748.97T = NRRL 13604 Namibia Pennisetum typhoides AF158319 AF160266 HM347136 EF470117 U34428
Fusarium nygamai CBS 749.97T = NRRL 13448 Australia Sorghum bicolor necrotic root AF158326 AF160273 LT996202 EF470114 U34426
Fusarium oxysporum CBS 716.74 = NRRL 20433 Germany Vicia faba vascular bundle AF158366 AF008479 JX171469 JX171583 U34435
CBS 744.97 = NRRL 22902 USA Pseudotsuga menziesii AF158365 AF160312 LT996203 LT575065 U34424
Fusarium palustre NRRL 54056T USA Spartina alterniflora KT597718 KT597731
Fusarium parvisorum CBS 137236T Colombia Pinus patula roots LT996183 KJ541060 LT996150 KJ541055
Fusarium phyllophilum CBS 216.76T = NRRL 13617 Italy Dracaena deremensis leaf KF466333 KF466421 KF466399 KF466410 KF466443
Fusarium poae NRRL 13714 Unknown Unknown JX171458 JX171572
Fusarium proliferatum CBS 217.76 = NRRL 22944 Germany Cattleya pseudobulb, hybrid AF158333 AF160280 JX171504 HM068352 U34416
Fusarium pseudocircinatum CBS 449.97T = NRRL 22946 Ghana Solanum sp. AF158324 AF160271 LT996204 LT996151 U34427
Fusarium pseudograminearum CBS 109956T = NRRL 28062 Australia Hordeum vulgare crowns JX171524 JX171637
Fusarium pseudonygamai CBS 417.97T = NRRL 13592 Nigeria Pennisetum typhoides AF158316 AF160263 LT996205 LT996152 U34421
Fusarium ramigenum CBS 418.98T = NRRL 25208 USA Ficus carica KF466335 KF466423 KF466401 KF466412 KF466445
Fusarium sacchari CBS 223.76 = NRRL 13999 India Saccharum officinarum AF158331 AF160278 JX171466 JX171580 U34414
Fusarium sambucinum NRRL 22187 = NRRL 20727 England Solanum sp. JX171493 JX171606
Fusarium sarcochroum CBS 745.79 = NRRL 20472 Switzerland Viscum album JX171472 JX171586
Fusarium sibiricum NRRL 53430T Russia Avena sativa HQ154472
Fusarium sororula CBS 137242T Colombia Pinus patula stems LT996184 KJ541067 LT996206 LT996153 KJ541057
Fusarium sp. NRRL 66179 USA Hibiscus moscheutos KX302913 KX302921 KX302929
NRRL 66180 USA Hibiscus moscheutos KX302914 KX302922 KX302930
NRRL 66181 USA Hibiscus moscheutos KX302915 KX302923 KX302931
NRRL 66182 USA Hibiscus moscheutos KX302916 KX302924 KX302932
NRRL 66183 USA Hibiscus moscheutos KX302917 KX302925 KX302933
NRRL 66184 USA Hibiscus moscheutos KX302918 KX302926 KX302934
CBS 201.63 = NRRL 36351 Portugal Arachis hypogaea stored nut GQ915484
Fusarium sporotrichioides NRRL 3299 USA Corn JX171444 HQ154454
Fusarium sterilihyphosum NRRL 25623 South Africa Mango AF158353 AF160300 Not public Not public AF160316
Fusarium stilboides NRRL 20429 Nyasaland Coffee bark JX171468 JX171582
Fusarium subglutinans CBS 747.97 = NRRL 22016 USA Corn AF158342 AF160289 JX171486 JX171599 U34417
Fusarium sublunatum CBS 190.34 = NRRL 20897 Unknown Unknown KX302919 KX302927 KX302935
CBS 189.34T = NRRL 13384 Costa Rica Soil of banana plantation JX171451 JX171565
Fusarium succisae CBS 219.76 = NRRL 13613 Germany Succisa pratensis flower AF158344 AF160291 LT996207 LT996154 U34419
Fusarium sudanense CBS 454.97T = NRRL 25451 Sudan Striga hermonthica LT996185 KU711697 LT996208 LT996155 KU603909
Fusarium temperatum NRRL 25622 = NRRL 26616 South Africa Zea mays AF158354 AF160301 Not public Not public AF160317
Fusarium terricola CBS 483.94T Australia Soil KU603951 KU711698 LT996209 LT996156 KU603908
Fusarium thapsinum CBS 733.97 = NRRL 22045 South Africa Sorghum bicolor LT996186 AF160270 JX171487 JX171600 U34418
Fusarium tjaetaba NRRL 66243T Australia Sorghum interjectum LT996187 KP083263 KP083267 KP083275 LT996119
Fusarium torreyae NRRL 54149 USA Torreya sp. HM068337 JX171548 HM068359
Fusarium transvaalense CBS 144211T = CPC 30923 South Africa Sida cordifolia rhizosphere LT996099 LT996210 LT996157 LT996120
CBS 144212 = CPC 30929 South Africa Melhania acuminata rhizophere LT996100 LT996211 LT996158 LT996121
CBS 144213 = CPC 33751 South Africa Melhania acuminata rhizophere LT996159 LT996122
CBS 144214 = CPC 30946 South Africa Sida cordifolia rhizosphere LT996101 LT996212 LT996160 LT996123
CBS 144215 = CPC 33723 South Africa Sida cordifolia rhizosphere LT996102 LT996161 LT996124
CBS 144216 = CPC 30918 South Africa Sida cordifolia rhizosphere LT996103 LT996213 LT996162 LT996125
CBS 144217 = CPC 30919 South Africa Sida cordifolia rhizosphere LT996104 LT996214 LT996163 LT996126
CBS 144218 = CPC 30922 South Africa Sida cordifolia rhizosphere LT996105 LT996215 LT996164 LT996127
CBS 144219 = CPC 30926 South Africa Sida cordifolia rhizosphere LT996106 LT996216 LT996165 LT996128
CBS 144220 = CPC 30927 South Africa Sida cordifolia rhizosphere LT996107 LT996217 LT996166 LT996129
CBS 144221 = CPC 33740 South Africa Kyphocarpa angustifolia rhizophere LT996167 LT996130
CBS 144222 = CPC 30939 South Africa Kyphocarpa angustifolia rhizophere LT996108 LT996218 LT996168 LT996131
CBS 144223 = CPC 30941 South Africa Kyphocarpa angustifolia rhizophere LT996109 LT996169 LT996132
CBS 144224 = CPC 30928 South Africa Melhania acuminata rhizophere LT996110 LT996219 LT996170 LT996133
CBS 144496 = CPC 33750 South Africa Melhania acuminata rhizophere LT996171 LT996134
NRRL 31008 Australia Soil JX171529 JX171642
Fusarium tupiense NRRL 53984 Brazil Mangifera indica GU737377 GU737404 Not public Not public GU737296
Fusarium udum CBS 178.32 = NRRL 22949 Germany Lactarius pubescens AF158328 AF160275 LT996220 LT996172 U34433
Fusarium venenatum CBS 458.93T Austria Winter wheat halm base KM232382
Fusarium verticillioides CBS 734.97 = NRRL 22172 Germany Zea mays AF158315 AF160262 LT996221 EF470122 U34413
Fusarium xanthoxyli F201114 China Zanthoxylum bungeanum KM236706 KM520380 KM236766
Fusarium xylarioides CBS 258.52 = NRRL 25486 Ivory Coast Coffea sp. trunk AY707136 JX171517 HM068355 AY707118

†CBS: Westerdijk Fungal Biodiversity Institute. CPC: Collection of Pedro W. Crous, held at CBS. F: College of Forestry, Northwest A&F University, Taicheng Road, Yangling, Shaanxi China. NRRL: Agricultural Research Service, Peoria, IL, USA.

‡ IT: ex-isotype culture. PT: ex-paratype culture. T: ex-type culture. NT: ex-neotyype culture.

§CAL: Calmodulin. EF-1α: Translation elongation factor 1-alpha. RPB1: RNA polymerase largest subunit. RPB2: RNA polymerase second largest subunit. TUB: Tubulin. New sequences are shown in bold. Sequences marked as “Not public” were obtained from Kerry O’Donnell’s alignment datasets.

Morphological characterisation

Fusarium isolates were characterised morphologically according to procedures described elsewhere (Aoki et al. 2013; Leslie and Summerell 2006, Sandoval-Denis et al. 2018). Colonial growth rates and production of diffusible pigments were evaluated on PDA, colony features were also recorded on corn-meal agar (CMA; recipe in Crous et al. 2009) and OA. Colour notations followed those of Rayner (1970). For the study of micro-morphological features, cultures were grown for 7–10 d at 24 °C, using a 12 h light/dark cycle with near UV and white fluorescent light. Aerial and sporodochial conidiophores and conidia and formation of chlamydospores were evaluated on Synthetic Nutrient-poor Agar (SNA; Nirenberg 1976) and on Carnation Leaf Agar (CLA; Fisher et al. 1982). Measurements and photomicrographs were recorded from a minimum of 30 elements for each structure, using sterile water as mounting medium and a Nikon Eclipse 80i microscope with Differential Interference Contrast (DIC) optics and a Nikon AZ100 dissecting microscope, both equipped with a Nikon DS-Ri2 high definition colour digital camera and the Nikon software NIS-elements D software v. 4.30.

DNA isolation, amplification and sequencing

Isolates were grown for 7 d on MEA at 24 °C using the photoperiod described above. Fresh mycelium was scraped from the colony surface and subjected to total DNA extraction using the Wizard® Genomic DNA purification Kit (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Fragments of five DNA loci were amplified using primers and PCR conditions described by O’Donnell et al. (2009) for calmodulin (CAL), O’Donnell et al. (2010) for the RNA polymerase largest subunit (RPB1) and second largest subunit (RPB2), O’Donnell et al. (1998) for the translation elongation factor 1-alpha (EF-1α) and Woudenberg et al. (2009) for beta-tubulin (TUB). Sequencing was made in both strand directions using the same primer pairs as for PCR amplification on an Applied Biosystems, Hitachi 3730xl DNA analyser (Applied Biosystems Inc., Foster City, California, USA). Consensus sequences were assembled using Seqman Pro v. 10.0.1 (DNASTAR, Madison, WI, USA). All DNA sequences generated in this study were lodged in GenBank and the European Nucleotide Archive (ENA) (Table 1).

Molecular identification and phylogenetic analyses

A first analysis was based on pairwise alignments and blastn searches on the Fusarium MLST (http://www.westerdijkinstitute.nl/fusarium/) and NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) databases, respectively, using EF-1α and RPB2 sequences in order to resolve the position of the KNP isolates amongst the different species complexes recognised in Fusarium (O’Donnell et al. 2013). Sequences from individual loci were aligned using MAFFT (Katoh and Standley 2013), on the web server of the European Bioinformatics Institute (EMBL–EBI; http://www.ebi.ac.uk/Tools/msa/mafft/) (Li et al. 2015).

Phylogenetic analyses were based on Maximum-likelihood (ML) and Bayesian (B) analyses, both algorithms run on the CIPRES Science Gateway portal (Miller et al. 2012). Evolutionary models were calculated using MrModelTest v. 2.3 using the Akaike information criterion (Nylander 2004; Posada and Crandall 1998). For ML, RAxML-HPC2 v. 8.2.10 on XSEDE was used (Stamatakis 2014), clade stability was tested with a bootstrap analysis (BS) using the rapid bootstrapping algorithm with default parameters. The B analyses were run using MrBayes v. 3.2.6 on XSEDE (Ronquist and Huelsenbeck 2003) using four incrementally heated MCMC chains for 5M generations, with the stop-rule option on and sampling every 1000 trees. After convergence of the runs (average standard deviation of split frequencies below 0.01) the first 25% of samples were discarded as the burn-in fraction and 50% consensus trees and posterior probabilities (PP) were calculated from the remaining trees.

Phylogenies were first made individually for each locus dataset and visually compared for topological incongruence amongst statistically supported nodes (ML-BS ≥ 70% and B-PP ≥ 0.95) (Mason-Gamer and Kellogg 1996, Wiens 1998), before being concatenated for multi-locus analyses using different locus combinations according to strains and DNA sequences currently available in public databases, in addition to previously published phylogenies (O’Donnell et al. 2000, 2013; Herron et al. 2015; Lupien et al. 2017; Moussa et al. 2017, Sandoval-Denis et al. 2018). A further 232 sequences representing 72 taxa were retrieved from GenBank and included in the phylogenetic analyses, while an additional 58 DNA sequences were obtained from 24 fungal strains requested from the CBS and NRRL (Agricultural Research Service, Peoria, IL, USA) culture collections (Table 1). All alignments and trees generated in this study were uploaded to TreeBASE (https://treebase.org).

Results

Phylogenetic analyses

Pairwise DNA alignments and BLAST searches using EF-1α and RPB2 sequences showed that the 19 isolates from KNP belonged to three different species complexes of the genus Fusarium i.e. the F. buharicum Jacz. ex Babajan & Teterevn.-Babajan species complex (FBSC; two isolates), the F. fujikuroi Nirenberg species complex (FFSC; two isolates) and the F. sambucinum Fuckel species complex (FSAMSC; 15 isolates). According to these results, sequences of related taxa and lineages were retrieved from GenBank and incorporated into individual phylogenetic analyses for each species complex.

Multi-locus analyses were carried out in order to further delimit the KNPFusarium isolates amongst the known diversity in their respective species complexes. With the exception of the FFSC, the topologies observed from ML and B analyses of single and multi-locus datasets were highly congruent, with only minor differences affecting unsupported nodes on the trees (all trees available in TreeBASE). The characteristics of the different alignments and tree statistics for all the species complexes are shown in Table 2.

Table 2.
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Characteristics of the different datasets and statistics of phylogenetic analyses used in this study.

Analysis† Locus‡ Number of Sites§ Evolutionary model| Number of trees sampled in B Maximum-likelihood statistics
Total Conserved Phylogenetically informative B unique patterns Best tree optimised likelihood Tree length
Fusarium buharicum SC EF-1α 495 300 119 198 GTR+G 414 -11313.23702 0.598675
RPB1 930 682 203 211 SYM+G
RPB2 1663 1251 330 310 GTR+I+G
Fusarium fujikuroi SC CAL 545 423 67 167 SYM+G 282 -20603.30043 0.567054
EF-1α 677 428 127 295 GTR+I+G
RPB1 1534 1219 185 137 SYM+I+G
RPB2 1551 1211 227 315 GTR+I+G
TUB 488 351 66 336 SYM+G
Fusarium sambucinum SC RPB1 854 594 201 213 SYM+I+G 241 -9871.793718 0.740271
RPB2 1580 1128 346 396 GTR+G

† SC: Species complex.

‡CAL: Calmodulin. EF-1α: Translation elongation factor 1-alpha. RPB1: RNA polymerase largest subunit. RPB2: RNA polymerase second largest subunit. TUB: Tubulin.

§ B: Bayesian inference.

| G: Gamma distributed rate variation among sites. GTR: Generalised time-reversible. I: Proportion of invariable sites. SYM: Symmetrical model.

The analysis of the FBSC included sequences of EF-1α, RPB1 and RPB2 loci from 18 isolates representing 10 taxa, including members of the Fusarium torreyae T. Aoki, J.A. Sm., L.L. Mount, Geiser & O’Donnell species complex (FTYSC) and Fusarium lateritium Nees species complex (FLSC) as outgroup (Fig. 2). The four ingroup taxa resolved with high statistical support. Two KNP isolates from K. angustifolia obtained from the bottom site of the catena (CBS 144207 and 144208) clustered in a sister relationship with the clade representing Fusarium sublunatum Reinking, but were genetically clearly delimited.

Figure 2. 

Maximum-likelihood (ML) phylogram obtained from combined EF-1α, RPB1 and RPB2 sequences of 18 strains belonging to the Fusarium buharicum (FBSC), Fusarium tricinctum (FTSC) and Fusarium lateritium (FLSC) species complexes. Numbers on the nodes are ML bootstrap values above 70% and Bayesian posterior probability values above 0.95. Branch lengths are proportional to distance. Ex-type strains are indicated with T. Strains corresponding to new species described here are shown in bold.

The phylogeny of the FFSC included sequences of CAL, EF-1α, RPB1, RPB2 and TUB loci from 48 strains and 44 taxa, including two outgroups (F. oxysporumCBS 716.74 and 744.97) (Fig. 3). The phylogeny showed a clear delimitation between the biogeographic clades recognised in this species complex (African, American and Asian clades sensuO’Donnell et al. 1998). Both American and Asian clades where shown as monophyletic with high ML-BS and B-PP support; in contrast, the African clade was resolved as polyphyletic, comprising two distinct and highly supported lineages. A terminal, speciose clade (African A) encompassing 17 taxa and a basal clade (African B), close to the American clade which included the ex-type of Fusarium dlaminii Marasas, P.E. Nelson & Toussoun (CBS 119860) and a sister terminal clade (ML-BS=100, B-PP=1) comprising two KNP isolates from M. acuminata (CBS 144209 and 144495) and two unidentified African Fusarium isolates (CBS 144210 and NRRL 26152). From the loci used here, only TUB resolved both African clades as sister groups; however, its monophyly was not supported by clade stability measurements (data not shown). Conversely, individual CAL, EF-1α and RPB2 phylogenies resolved African B as basal to the ingroup, while RPB1 allocated this clade as basal to the American clade. Nonetheless, all the individual phylogenies, in addition to the combined dataset, clearly demonstrated genealogical uniqueness of the terminal clade encompassing KNP isolates.

Figure 3. 

Maximum-likelihood (ML) phylogram obtained from combined CAL, EF-1α, RPB1, RPB2 and TUB sequences of 48 strains belonging to the Fusarium fujikuroi (FFSC) and Fusarium oxysporum (FOSC) species complexes. Numbers on the nodes are ML bootstrap values above 70% and Bayesian posterior probability values above 0.95. Branch lengths are proportional to distance. Ex-type, ex-neotype and ex-paratype strains are indicated with T, NT and PT, respectively. Strains corresponding to new species described here are shown in bold.

The FSAMSC was studied using combined RPB1 and RPB2 sequences. The phylogeny included 35 isolates from 20 taxa, including the two outgroups Fusarium circinatum Nirenberg & O’Donnell (CBS 405.97) and Fusarium fujikuroi Nirenberg (NRRL 13566) (Fig. 4). Fifteen KPN Fusarium isolates from the three sampled plant species (three isolates from K. angustifolia, four isolates from M. acuminata and eight isolates from S. cordifolia), all obtained from the top site of the catena, clustered with an unidentified Fusarium isolate (NRRL 31008) in a distinct clade (ML-BS=100, B-PP=1), close to Fusarium brachygibbosum Padwick (strain NRRL 13829).

Figure 4. 

Maximum-likelihood (ML) phylogram obtained from combined RPB1 and RPB2 sequences of 35 strains belonging to the Fusarium sambucinum (FSAMSC) and Fusarium fujikuroi (FFSC) species complexes. Numbers on the nodes are ML bootstrap values above 70% and Bayesian posterior probability values above 0.95. Branch lengths are proportional to distance. Ex-type strains are indicated with T. Strains corresponding to new species described here are shown in bold.

The clades including KNP isolates and corresponding to previously undisclosed lineages of Fusarium are described in the taxonomy section as the three novel species, F. convolutans, F. fredkrugeri and F. transvaalense.

Taxonomy

Fusarium convolutans Sandoval-Denis, Crous & W.J. Swart, sp. nov.

MycoBank No: MB825102
Fig. 5

Diagnosis

Different from F. circinatum, F. pseudocircinatum O’Donnell & Nirenberg and F. sterilihyphosum Britz, Marasas & M.J. Wingf. by the absence of aerial conidia (microconidia) and the presence of chlamydospores. Different from F. buharicum Jacz. ex Babajan & Teterevn.-Babajan and F. sublunatum by its shorter, less septate and less curved conidia and by the presence of sterile hyphal coils.

Type

South Africa, Kruger National Park, Skukuza, Granite Supersite, 25°06'33.9"S, 31°34'40.9E, from rhizosphere soil of Kyphocarpa angustifolia, 23 Mar 2015, W.J. Swart, holotype CBS H-23495, dried culture on OA, ex-holotype strain CBS 144207 = CPC 33733.

Description

Colonies on PDA growing in the dark with an average radial growth rate of 2.1–4.8 mm/d, 4.4–5.8 mm/d and 4.6–6.3 mm/d at 24, 27 and 30 °C, respectively; reaching 11–28 mm diam. in 7 d at 24 °C and a maximum of 23–37 mm diam. in 7 d at 30 °C. Minimum temperature for growth 12 °C, maximum 36 °C, optimal 27–33 °C. Colony surface white to cream coloured, flat and highly irregular in shape, velvety to felty, with scant and short aerial mycelium; colony margins highly irregular to rhizoid, with abundant white to grey submerged mycelium. Reverse white, straw to yellow diffusible pigment produced between 21–33 °C, scarcely produced and turning luteous to orange at 36 °C. Colonies on CMA and OA incubated in the dark reaching 40–48 mm diam. in 7 d at 24 °C. Colony surface white to cream coloured, flat or slightly elevated at the centre, velvety to dusty; aerial mycelium abundant, short and dense, concentrated on the colony centre; margins membranous and regular, buff to honey coloured, without aerial mycelium. Reverse ochreous without diffusible pigments. Sporulation scant from conidiophores formed on the aerial mycelium, sporodochia not formed. Conidiophores on the aerial mycelium straight or flexuous, smooth- and thin-walled, simple, mostly reduced to conidiogenous cells borne laterally on hyphae or up to 50 μm tall, bearing terminal single or paired monophialides; phialides subulate to subcylindrical, smooth- and thin-walled, 15.5–22 μm long, (3.5–)4–5 μm at the widest point, with inconspicuous periclinal thickening and a short- flared collarette; conidia clustering in discrete false heads at the tip of monophialides, lunate to falcate, curved or somewhat straight, tapering gently toward the basal part, robust; apical cell often equal in length or slightly shorter than the adjacent cell, blunt to conical; basal cell papillate to distinctly notched, (1–2–)3-septate, hyaline, thin- and smooth-walled. One-septate conidia: 24 × 4.5 μm; two-septate conidia: 24.5 × 6 μm; three-septate conidia: (25.5–)29–36.5(–38.5) × (4–)5–6.5(–7.5) μm. Chlamydospores abundantly formed, globose to subglobose, smooth- and thick-walled, (9.5–)11–13.5(–14) μm diam.; terminal or intercalary in the hyphae or conidia, often borne laterally at the tip of elongated, cylindrical, stalk-like projections, solitary or in small clusters. Sterile, coiled, sometimes branched hyphal projections abundantly formed laterally from the substrate and aerial mycelium.

Figure 5. 

Fusarium convolutans sp. nov. A–D Colonies on PDA, SNA, OA and CMA, respectively, after 7 d at 24 °C in the dark E–IConidiophores, phialides and conidia J–MChlamydosporesN–P Sterile hyphal projections Q Conidia. Scale bars: 20 μm (E, F); 5 μm (G–I); 10 μm (J–Q).

Distribution

South Africa.

Etymology

From Latin, “convolutans”, participle of convolutare, coiling, in reference to the abundant sterile, coiled lateral hyphal projections.

Additional isolate examined

South Africa, Kruger National Park, Skukuza, Granite Supersite, 25°06'33.9"S, 31°34'40.9E, from rhizosphere soil of Kyphocarpa angustifolia, 23 Mar 2015, W.J. Swart, CBS 144208 = CPC 33732.

Notes

The main morphological feature of F. convolutans, namely the production of sterile, coiled hyphal projections, grossly resembles other Fusarium species producing similar structures i.e. F. circinatum, F. pseudocircinatum and F. sterilihyphosum. The three latter species, however, are genetically unrelated to F. convolutans, being allocated in the FFSC; and are also easily differentiable by the characteristics of the aerial conidia (typical Fusarium microconidia are absent in the new species) and the lack of chlamydospores (present in the new species) (Leslie and Summerell 2006). Fusarium convolutans can be easily differentiated morphologically from their phylogenetically closely related species, F. buharicum and F. sublunatum. It has relative simple conidiophores and shorter, less septate and markedly less curved conidia (up to 38.5 μm long and 1–3-septate vs. up to 87 and 81 μm long, 0–8-septate in F. buharicum and F. sublunatum, respectively) (Gerlach and Nirenberg 1982). Fusarium buharicum and F. sublunatum also lack sterile hyphal coils.

Fusarium fredkrugeri Sandoval-Denis, Crous & W.J. Swart, sp. nov.

MycoBank No: MB825103
Fig. 6

Diagnosis

Differs from Fusarium dlaminii Marasas, P.E. Nelson & Toussoun by producing only one type of aerial conidia, shorter sporodochial conidia and the absence of chlamydospores.

Type

South Africa, Kruger National Park, Skukuza, Granite Supersite, 25°06'48.6"S, 31°34'36.5"E, from rhizosphere soil of Melhania acuminata, 23 Mar 2015, W.J. Swart, holotype CBS H-23496, dried culture on OA, culture ex-holotype CBS 144209 = CPC 33747.

Description

Colonies on PDA growing in the dark with an average radial growth rate of 4.7–5.8 mm/d and reaching 22–35 mm diam. in 7 d at 24 °C, filling an entire 9 cm Petri dish in 7 d at 27 and 30 °C. Minimum temperature for growth 12 °C, maximum 36 °C, optimal 27–30 °C. Colony surface at first white to cream coloured, later turning bay to chestnut with pale luteous to luteous periphery; flat, felty to cottony with abundant erect- aerial mycelium forming white patches; colony margins regular and filiform with abundant submerged mycelium. Reverse pale luteous, a blood sepia to chestnut coloured diffusible pigment is scarcely produced at 24 °C, pigment production is markedly enhanced at 27–30 °C, becoming greyish-sepia at 33 °C. Colonies on CMA and OA incubated at 24 °C in the dark reaching 65–67 mm diam. or occupying an entire 9 cm Petri dish in 7 d, respectively. Colony surface pale bay coloured, flat, felty to velvety, aerial mycelium scant, forming white to cream patches; margins regular. Reverse pale bay to pale vinaceous. Sporulation abundant from conidiophores formed on the substrate and aerial mycelium and from sporodochia. Conidiophores on the aerial mycelium straight or flexuous, erect or prostrate, septate, smooth- and thin-walled, often appearing rough by accumulation of extracellular material, commonly simple or reduced to conidiogenous cells borne laterally on hyphae or up to 200 μm tall and irregularly branched at various levels, branches bearing lateral and terminal monophialides borne mostly single or in pairs; phialides subulate, ampulliform, lageniform to subcylindrical, smooth- and thin-walled, (8.5–)9.5–17.5(–24.5) μm long, 2–3(–3.5) μm at the widest point, without periclinal thickening, collarets inconspicuous; conidia formed on aerial conidiophores, hyaline, obovoid, ellipsoidal to slightly reniform or allantoid, smooth- and thin-walled, 0-septate, (4.5–)5–8.5(–12.5) × (1.5–)2–3.5(–6) μm, clustering in discrete false heads at the tip of monophialides. Sporodochia pale orange to pink coloured, often somewhat translucent, formed abundantly on the surface of carnation leaves and on the agar surface. Conidiophores in sporodochia 26–46 μm tall, densely aggregated, irregularly and verticillately branched up to three times, with terminal branches bearing 2–3 monophialides; sporodochial phialides doliiform to subcylindrical, (9–)11.5–15.5(–18.5) × (2.5–)3–4(–4.5) μm, smooth- and thin-walled, with periclinal thickening and an inconspicuous apical collarette. Sporodochial conidia falcate, tapering toward the basal part, robust, moderately curved and slender; apical cell more or less equally sized than the adjacent cell, blunt to slightly papillate; basal cell papillate to distinctly notched, (1–)3–4-septate, hyaline, thin- and smooth-walled. One-septate conidia: 13–17(–18) × (2.5–)3–4 μm; two-septate conidia: 15 × 4.5 μm; three-septate conidia: (16–)28.5–39(–45) × (3–)4–5(–5.5) μm; four-septate conidia: 39.5–40(–41) × 4.5–5 μm; overall (13–)27.5–39.5(–45) × (3–)3.5–5.5 μm. Chlamydospores absent.

Figure 6. 

Fusarium fredkrugeri sp. nov. A–D Colonies on PDA, SNA, OA and CMA, respectively, after 7 d at 24 °C in the dark E–GSporodochia formed on the surface of carnation leaves H–N Aerial conidiophores, phialides and conidia O, P Aerial conidia Q Sporodochial conidiophores and phialides R Sporodochial conidia. Scale bars: 100 μm (E–G); 10 μm (H–R).

Distribution

Madagascar, Niger and South Africa.

Etymology

In honour and memory of Dr. Frederick J. Kruger, pioneer of forest hydrology, fynbos ecology and invasive species and fundamental for the collections included in this study.

Additional isolates examined

Madagascar, from Striga hermonthica, unknown date, A.A. Abbasher, CBS 144210 = NRRL 26061 = BBA 70127. South Africa, Kruger National Park, Skukuza, Granite Supersite,25°06'48.6"S, 31°34'36.5"E, from rhizosphere soil of Melhania acuminata, 23 Mar 2015, W.J. Swart, CBS 144495 = CPC 33746.

Notes

This species is genetically closely related to F. dlaminii, both species having similar colonial morphology, optimal growth conditions and biogeography. Moreover, both species exhibit relatively short aerial phialides producing conidia in heads, somewhat resembling those produced by F. oxysporum rather than most members of the FFSC (Leslie and Summerell 2006; Marasas et al. 1985). However, besides exhibiting much faster growth rates, F. fredkrugeri presents clearly distinctive morphological features such as the production of only one type of aerial conidia (vs. two types in F. dlaminii: allantoid to fusiform and 0-septate; and napiform 0–1-septate); orange to pink sporodochia, produced on carnation leaves but also abundantly on the agar surface (vs. orange sporodochia, produced only on the surface of carnation leaves in F. dlaminii) (Leslie and Summerell 2006). Additionally, F. fredkrugeri produces shorter and less septate sporodochial conidia ((1–)3–4-septate and up to 45 μm long in the latter species vs. mostly 5-septate and up to 54 μm long in F. dlaminii) while chlamydospores are not produced. The latter feature, coupled with the somewhat more complex conidiophores also clearly differentiates F. fredkrugeri from F. oxysporum.

Fusarium transvaalense Sandoval-Denis, Crous & W.J. Swart, sp. nov.

MycoBank No: MB825104
Fig. 7

Diagnosis

Different from most species in FSAMSC by its slender sporodochial conidia with tapered and somewhat rounded apex; its smooth- to tuberculate, often pigmented chlamydospores and the formation of large mycelial tufts on OA.

Type

South Africa, Kruger National Park, Skukuza, Granite Supersite, 25°06'45.5"S, 31°34'35.0"E, from rhizosphere soil of Sida cordifolia, 23 Mar 2015, W.J. Swart, holotype CBS H-23497, dried culture on SNA, culture ex-holotype CBS 144211 = CPC 30923.

Description

Colonies on PDA growing in the dark with an average radial growth rate of 8.5–9.3 mm/d, reaching 34–37 mm diam. in 7 d at 24 °C, filling an entire 9 cm Petri dish in 7 d at 27–33 °C. Minimum temperature for growth 12 °C, maximum 36 °C, optimal 27–30 °C. Colony surface at first white, turning coral to dark vinaceous with white periphery and abundant yellow hyphae at the centre; flat, velvety to woolly, with abundant aerial mycelium and erect hyphal strings reaching several mm tall; colony margins regular and filiform. Reverse with yellow, coral or dark vinaceous patches, coral diffusible pigments strongly produced between 15–30 °C, turning scarlet to orange at 33–36 °C. Colonies on CMA and OA incubated at 24 °C in the dark occupying an entire 9 cm Petri dish in 7 d. Colony surface coral, rust to chestnut coloured in irregular patches, flat, felty to woolly, aerial mycelium scarce on CMA, mostly as radially dispersed white patches, on OA aerial mycelium abundant, especially on the periphery of the colony, forming dense, pustule-like, white mycelial tufts, formed by abundant intermingled hyphae and chlamydospores, 1–1.5 cm tall, with flesh to coral coloured stipes; margins on CMA and OA regular. Reverse pale luteous with red to coral periphery. Sporulation abundant from conidiophores formed on the aerial mycelium, at the agar level and from sporodochia. Conidiophores on the aerial mycelium straight or flexuous, septate, smooth- and thin-walled, up to 150 μm tall, sometimes emerging from irregular, swollen, pigmented and rough-walled cells on the hyphae; simple or sparingly and irregularly branched, branches bearing terminal, rarely lateral monophialides or reduced to conidiogenous cells borne laterally on hyphae; phialides on the aerial conidiophores short ampulliform, subulate to subcylindrical, smooth- and thin-walled, (7–)9–14(–15) μm long, (3–)4–5 μm at the widest point, without periclinal thickening and with a minute, inconspicuous collarette; conidia formed on aerial conidiophores of two types: a) hyaline, obovoid, ellipsoidal to clavate, smooth- and thin-walled, 0–1-septate, 2–14 × 2–4 μm; b) lunate to short falcate with a pointed apex and a somewhat flattened base, smooth- and thin-walled, 3–5-septate. Three-septate conidia: (16–)18–27(–29) × 5–6 μm; four-septate conidia: 21–24(–25) × 5–6 μm; five-septate conidia: (25–)27–33 × 5–6 μm. Sporodochia cream to orange coloured, formed abundantly on the surface of carnation leaves and rarely on the agar surface, at first very small and sparse later becoming aggregated. Conidiophores in sporodochia 22–31 μm tall, irregularly branched, bearing clusters of 3–6 monophialides; sporodochial phialides doliiform to ampulliform, (5–)9–14(–18) × (3–)4–5 μm, smooth- and thin-walled, with periclinal thickening and a short apical collarette. Sporodochial conidia falcate, wedge-shaped, tapering towards both ends, markedly curved and robust; apical cell longer than the adjacent cell, pointed; basal cell distinctly notched, sometimes somewhat extended (1–)3–5(–6)-septate, hyaline, smooth- and thick-walled. One-septate conidia: 19 × 4 μm; three-septate conidia: 20–27(–28) × 5–7 μm; four-septate conidia: (29–)30–32 × 5–7 μm; five-septate conidia: (26–)29–41(–53) × 4–5(–6) μm; six-septate conidia: 36 × 7 μm; overall (19–)25.9–40(–53) × (3.5–)4–6(–7) μm. Chlamydospores abundant, hyaline or pigmented, smooth- to rough-walled or tuberculate, 7–8 μm diam., terminal or intercalary, solitary, in chains or in clusters.

Figure 7. 

Fusarium transvaalense sp. nov. A–D Colonies on PDA, SNA, OA and CMA, respectively, after 7 d at 24 °C in the dark E Pustule-like growth on OA F, GSporodochia formed on the surface of carnation leaves H–L Aerial conidiophores phialides and conidia M Aerial conidia N, OChlamydosporesP Sporodochial conidiophores and phialides Q Sporodochial conidia. Scale bars: 2 mm (E); 20 μm (F–J); 5 μm (K); 10 μm (L–Q).

Distribution

Australia and South Africa

Etymology

After Transvaal, the name of a former colony and Republic located between the Limpopo and Vaal rivers, currently a province of South Africa and where this species was found. From Latin trans meaning “on the other side of” and Vaal a South African river.

Additional isolates examined

South Africa, Kruger National Park, Skukuza, Granite Supersite, 25°06'48.6"S, 31°34'36.5"E, from rhizosphere soil of Melhania acuminata, 23 Mar 2015, W.J. Swart, CBS 144224 = CPC 30928, CBS 144212 = CPC 30929); 25°06'45.6"S, 31°34'37.7"E, CBS 144496 = CPC 33750, CBS 144213 = CPC 33751; 25°06'48.8"S, 031°34'36.6"E, from rhizosphere soil of Sida cordifolia, 23 Mar 2015, W.J. Swart, CBS 144214 = CPC 30946; 25°06'45.7"S, 31°34'35.1"E, CBS 144215 = CPC 33723; 25°06'45.5"S, 31°34'35.0"E, CBS 144216 = CPC 30918, CBS 144217 = CPC 30919, CBS 144218 = CPC 30922, , CBS 144219 = CPC 30926, CBS 144220 = CPC 30927); 25°06'51.4"S, 31°34'37.5"E, from rhizosphere soil of Kyphocarpa angustifolia, 23 Mar 2015, W.J. Swart, CBS 144221 = CPC 33740; 25°06'51.8"S, 31°34'38.1"E, CBS 144222 = CPC 30939, CBS 144223 = CPC 30941.

Notes

Fusarium transvaalense exhibits a sporodochial conidial morphology typical of members of FSAMSC with marked dorsiventral curvature and tapered ends. Several species in FSAMSC form comparable conidia in culture i.e. F. crookwellense L.W. Burgess, P.E. Nelson & Toussoun, F. sambucinum, F. sporotrichioides Sherb., F. venenatum Nirenberg and F. culmorum (Wm.G. Sm.) Sacc. However, with the exception of F. sporotrichioides, the conidia of most species above-mentioned, differ by being more robust and often more pointed apically. Fusarium transvaalense differs from F. sporotrichioides by the absence of pyriform aerial conidia.

Two strains NRRL 13829 and NRRL 31008, previously identified as F. brachygibbosum Padwick showed different degrees of genetic similitude with the new species. While NRRL 31008 clustered within F. transvaalense, NRRL 13829 formed a clearly delimited sister linage. Morphologically, F. transvaalense exhibits significant differences allowing its separation from F. brachygibbosum. Both species produce sporodochial conidia with similar septation and sizes; however, F. brachygibbosum commonly exhibits a bulge in the middle portion of the conidia (Padwick 1945), a feature not present in F. transvaalense. In addition, the latter species produces comparatively larger sporodochial conidia, when elements with the same degree of septation are compared; its chlamydospores are smaller, smooth-walled to markedly tuberculate and pigmented (7–8 μm vs. 10.7–15.3 μm, smooth-walled and hyaline in F. brachygibbosum) and has a distinctive colonial growth on OA, forming large, pustule-like hyphal tufts, a feature not reported for F. brachygibbosum (Padwick 1945).

Discussion

In this study, three new Fusarium spp. were introduced, isolated from rhizosphere soils of three native African shrubs in a protected savannah ecosystem deep inside the Kruger National Park, South Africa.

Some remarkable differences were noted regarding the distribution of the novel fungal species and their respective hosts on this particular site. For instance, F. transvaalense, which exhibited the greatest relative abundance, was found in high quantities from the rhizospheres of the three hosts sampled, showing a considerable genetic diversity. Interestingly, this species was only on the top of the catena, even when two of its hosts, K. angustifolia and S. cordifolia, were found and sampled either at the top and bottom sites. Similarly, F. fredkrugeri was recovered only from soils under M. acuminata, a host species which occurred only at the top location. In contrast, F. convolutans was found in the rhizosphere of K. angustifolia, occurring only at the bottom of the catena, while none of the three fungal species was found associated with S. cordifolia at the bottom of the site. Nevertheless, not being an objective of this work, it was not possible to categorically assign these new species to specific hosts or locations. Likely, these fungi could be in low abundance and thus not detectable using the current methods. However, plant species composition varies considerably through a catena ecosystem, in relation to the different soil characteristics, pH gradient and water availability, which also greatly influence microbial and animal biodiversity (Lareen et al. 2016; Mohammadi et al. 2017). However, the full patterns of variation between locations on this particular catena still need to be systematically assessed and compared. As evidenced here, certain differences do exist between the soils at the upper and bottom locations of the Stevenson-Hamilton supersite, which might explain the fungal diversity variation observed here. The cation exchange capacity (CEC; capacity of a soil to hold exchangeable cations) varies considerably between sampling sites, basically depending on the proportion of sand versus clay content of each soil type (Ketterings et al. 2007; Van Zijl and Le Roux 2014). It is known that CEC greatly impacts the soil’s ability to retain essential nutrients and prevents soil acidification (Ketterings et al. 2007). Nutrient content also increased from the top to the bottom of the slope which is consistent with the increase in CEC. Nutrient poor soils are also a driver of biological diversity and most likely influenced fungal diversity in these particular locations (Havlicek and Mitchell 2014, Mapelli et al. 2017).

The three Fusarium species, described here, were not associated with any visible symptomatology on their hosts. However, they cannot be ruled out as pathogens since they were not assessed for pathogenicity against the sampled plants nor any other putative host species at the same locations. Likewise, it is unknown if these fungi exert any beneficial or deleterious effect on their ecosystems. These are important unsolved questions that need further evaluation. However, as shown by phylogenetic analyses, each of the three new species was in close genetic proximity with well-known plant pathogenic Fusarium spp. on their respective species complexes, which could suggest a potential pathogenic role. Fusarium convolutans clustered within the FBSC, together with three known plant pathogenic Fusarium spp. i.e. F. buharicum, a pathogen of Hibiscus cannabinus L. and Gossypium L.; F. sublunatum, known to affect banana and Theobroma cacao L. in Central America (Gerlach and Nirenberg 1982, Leslie and Summerell 2006) and a newly discovered although unnamed phylogenetic species causing wilt, crown and root rot of Hibiscus moscheutos L. (Lupien et al. 2017). Fusarium transvaalense belonged to the FSAMSC, a genetically diverse group common in temperate and subtropical zones (Leslie and Summerell 2006). Fusarium sambucinum, the conserved type species of the genus (Gams et al. 1997) being an aggressive plant pathogen and one of the most important agents of potato dry rot (Peters et al. 2008); while the latter species and several others in the complex have been reported causing disease on diverse crops, including many cereals and fruits (Leslie and Summerell 2006).

Fusarium fredkrugeri is here recognised and formally proposed as a new species. Although the clade representing this taxon had already been identified as a distinct unnamed phylogenetic species by O’Donnell et al. (2000), it had not been given a formal description pending the collection of additional isolates. Two other African isolates previously determined to belong to this clade i.e. CBS 144210 from Striga hermonthica (Del.) Benth. in Madagascar and NRRL 26152 from an unknown substrate in Niger, were incorporated into the analyses, although the latter strain is not viable anymore (NRRL, pers. comm.), thus not available for morphological assessment. Strain CBS 144210, however, is known as a pathogen of the ‘purple witchweed’, a parasite plant common to sub-Saharan Africa and known to devastate Sorghum bicolor (L.) Moench and Oryza sativa L. plantations (O’Donnell et al. 2000; Yoshida et al. 2010). As previously demonstrated by O’Donnell et al. (2000), our phylogenetic results showed that the clade comprising F. fredkrugeri and its sister species F. dlaminii does not cluster within the main African core of species in the FFSC. Thus, despite the African origin of our isolates, the predicted biogeographic patterns did not match the observed phylogeny. It has been hypothesised that this should not be the result of genetic markers tracing different phylogenies, but the consequence of losing the phylogenetic signal due to saturated sites and introns (O’Donnell et al. 2000). However, the inclusion in our analysis of additional, highly informative and slowly evolving loci such as RPB1 and RPB2 yielded similar results, which points out the need to re-evaluate the phylogeographic arrangement of this important species complex including the vast new data generated during the last 20 years that challenges the established assumptions (Kvas et al. 2009; Walsh et al. 2010; O’Donnell et al. 2013; Laurence et al. 2015). Nevertheless, although rather unlikely, alternative factors such as anthropogenic dispersion of F. fredkrugeri, its host or additional invasive alternative hosts, cannot be rejected as an explanation for the discordance between biogeography and phylogenetic results. However, these scenarios are difficult to imagine given the characteristics of the sampled site, not being an agroecosystem but a protected, isolated zone, with minimal human intervention (Smit et al. 2013).

This study is a new example of how easily new Fusarium spp. can be found when mycological studies are directed to neglected natural ecosystems of minimal anthropogenic disturbance (Phan et al. 2004; Leslie and Summerell 2011; Summerell et al. 2011; Burgess 2014, Laurence et al. 2015). Although irrelevant for some researchers, finding and properly describing new species, regardless of whether they have little or no pathogenic or mycotoxigenic potential, is of utmost importance to improve our understanding on the diversity, biogeographic and phylogeographic patterns of such a complex and heterogeneous genus as Fusarium. In addition, this study remarks on the significance and need to further stimulate the exploration of conserved, non-manipulated natural environments (supersites) and their potential impact on biodiversity research on the fungal kingdom.

Aknowledgments

Todd J. Ward and James Swezey (Agricultural Research Service, Peoria, IL, USA) are thanked for providing strains. We kindly thank Kerry O’Donnell (Mycotoxin Prevention and Applied Microbiology Research Unit, Agricultural Research Service, US Department of Agriculture, Peoria, IL, USA) for providing DNA sequence datasets. Mercia Coetzee (Central University of Technology, Bloemfontein, South Africa) is thanked for her technical support in the field. Alejandra Giraldo (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) is thanked for her assistance with fungal isolation. Eddie Riddell and Navashni Govender (SANParks) are acknowledged for their research support in the Kruger National Park. We also thank Konstanze Bensch (Mycobank curator) and Uwe Braun (Geobotanik und Botanischer Garten, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany) for their help regarding Latin names.

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