Species concepts used in the demarcation of Fusarium species

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CHAPTER 3 A new Fusarium species from Poaceae in South Africa

ABSTRACT

During routine disease surveys, seven isolates representing Fusarium subglutinans sensu lato were obtained from diseased Pinus patula seedlings, as well as the grasses Panicum maximum and Phragmites mauritianus, growing in association with the pine seedlings in South Africa. The presence of curved sterile hyphae, produced on synthetic nutrient agar, and vinaceous gray colonies in young cultures, distinguished them from known species in this complex. The aim of this study was to characterise these isolates using phylogenetic analyses of partial sequences obtained for the ß-tubulin (BT) and translation elongation factor-1a (TEF-1a) genes. Phylogenetic analyses showed that the isolates from Pa. maximum and Ph. mauritianus group in a distinct clade from F. begoniae and F. circinatum. Morphologically, the latter two species are distinguished from each other based on the absence or presence of sterile coils, macroconidial morphology and the nature of the aerial mycelium. The unknown isolates were phylogenetically distinct from other species and mating tests showed that they were sexually compatible with the mating testers strains for F. subglutinans and F. circinatum. They were also not pathogenic to P. patula seedlings in contrast to F. circinatum that caused disease on these plants. The Fusarium isolates from the Poaceae are thus described as new and provided with the name Fusarium ophiodes sp. nov.
Keywords: Fungi, Fusarium, Phylogenetic species, Mating populations, Hybridization

Introduction

Species of fungi in the Gibberella fujikuroi (Sawada) Wollenw. complex include many important pathogens of agricultural and forestry crops. They include eleven biological species or mating populations (Leslie and Summerell, 2006). Three of these mating populations (B, E and H) form part of the so called Fusarium subglutinans morpho-species complex and are pathogens of Saccharum spp., Zea mays and Pinus spp., respectively (Leslie et al., 1992; 2005; Britz et al., 1999) Species accommodated in F. subglutinans sensu lato are morphologically similar. Some of the morphological characters used by Nirenberg and O’Donnell (1998) in their evaluation of these species include the arrangement of conidiophores on the aerial mycelium, the number of conidiogenous openings on the polyphialides, the presence or absence of sterile coils, formation of false chains and macroconidial morphology. The phylogenetic relationships between these species has been determined based on sequence comparisons for the protein coding genes translation elongation factor-1 , b-tubulin, the mitochondrial small subunit, as well as the ITS2 region of the rDNA gene region (O’Donnell et al., 1998a; Aoki et al., 2001).
The pine pitch canker pathogen, F. circinatum, resides in the F. subglutinans sensu lato complex. This fungus has a cosmopolitan distribution and has been reported from the USA, Chile, Japan, Mexico, Spain, Italy and South Africa (Wingfield et al., 2008). In South Africa, the fungus was originally found only in pine nurseries (Wingfield et al., 1998) but pitch canker, as it is known on mature trees, has recently been reported on mature pine stands in the country (Coutinho et al., 2007).
After the first outbreak of F. circinatum in a major pine seedling producing nursery in South Africa (Viljoen et al., 1994), surveys were undertaken and plants growing in the area of the nursery were analysed for infection. These included the native grass, Panicum maximum, and the well-established, non-native reed, Phragmites mauritianus. Isolations from these grasses resulted in a suite of Fusarium isolates of unknown identity. The aim of this study was to identify the unknown Fusarium isolates.

Materials and methods

Isolations and isolates

Isolations were made from Pa. maximum (grass isolates) and Ph. (reed isolates) growing at a forestry nursery near Nelspruit, Mpumulanga, South Africa. Isolations were made by placing small pieces (3 of root tissue onto Fusarium selective medium (20 g Agar, 15 g Peptone, 1 g KH₂PO₄, 0.5 g MgSO₄.7H₂0, 1 g PCNB, 20 mL Streptomycin sulphate in 1 L water) in Petri dishes (Nelson et al., 1983). Petri dishes were incubated at 25 °C under cool-white fluorescent light. The plates were checked regularly and all the colonies with typical Fusarium morphology were transferred to half-strength potato dextrose agar (PDA) (Merck, Germany). Single conidial cultures were made and stored at -70 °C in 15 % glycerol.
All isolates used in this study are maintained in the Fusarium culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa, and in the culture collection of the Medical Research Council (MRC), Tygerberg, Cape Town, South Africa. A representative collection of isolates has also been deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands.

DNA extractions and PCR

Isolates were grown on complete medium (CM) (Correll et al., 1987) at 25 ëC for 7 d and mycelium was harvested by scraping it from the agar surface with a sterile blade. DNA was isolated using the technique described by Jacobs et al. (2007) where ca. 10 g of sterile, chemically treated sand and 500 L extraction buffer [DEB: 200 mM Tris-HCl pH 8, 150 mM NaCl, 25 mM EDTA, 0.59 % SDS] was added to a microcentrifuge tube half filled with fungal mycelium to break open the cell walls. An additional 500 L of phenol and 300 L chloroform was added, mixed and centrifuged for 30 min at 10 000 rpm. The phenol/chloroform step was repeated until the interface was clean. The supernatant was transferred to a new tube and double the volume of 100 % ethanol was added and mixed. The DNA was allowed to precipitate at 4 °C overnight and then pelleted by centrifugation for 30 min at 11 000 rpm. Pellets were washed with 300 L 70 % ethanol, dried and resuspended in 50 L sterile, deionised water and 3 L of RNAse (2.5 M).
Extracted DNA was used as template in PCR reactions to amplify the b-tubulin (BT) and translation elongation factor -1a (TEF-1 ) genes. The TEF-1a gene region was amplified using the primer set EF1 (5′-CGAATCTTTGAACGCACATTG-3′) and EF2 (5′-CCGTGTTTCAAGACGGG-3′) (O’Donnell et al., 1998b). The b-tubulin gene region was amplified using the primer set T1 (5′-TTCCCCCGTCTCCACTTCTTCATG-3′) and T222 (5′-GACCG-GGGAAACGGAGACAGG-3′) (O’Donnell and Cigelik, 1997).
The PCR reactions consisted of 1x Roche Taq Reaction buffer with MgCl₂, dNTPs (200 µM each), primers (0.2 µM each), template DNA (25 ng) and Roche Taq polymerase (0.5 U) (Roche Pharmaceuticals, Germany). The PCR reaction conditions included an initial denaturation at 94 ëC for 2 min. This was followed by 30 cycles of denaturation at 94 ëC for 1 min, annealing at 54 ëC for 1 min and elongation at 72 ëC for 1 min, with a final elongation step at 72 ëC for 5 min. The resulting PCR amplicons were purified using a QIAquick PCR Purification kit (QIAGEN, Germany).

DNA sequencing and sequence comparisons

DNA sequences were determined from PCR amplicons using the ABI PRISM^TM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase (Applied Biosystems, UK), using the same primers as those used in the PCR reactions. All of the sequences generated in this study were deposited in GenBank (Table 1).
DNA sequences were aligned in BioEdit. Gaps were treated as missing data and thus discarded from the subsequent analysis. Phylogenetic analysis was based on parsimony using PAUP* version 4b10 (Phylogenetic Analysis Using Parsimony* and Other Methods version 4, Swofford, 2002). Heuristic searches were done with random addition of sequences (100 replicates), tree bisection-reconnection (TBR) branch swapping, and MULPAR effective and MaxTrees set to auto-increase. Phylogenetic signal in the data sets (g1) was assessed by evaluating tree length distributions over 100 randomly generated trees (Hillis and Huelsenbeck, 1992). The consistency (CI) and retention indices (RI) were determined for all data sets. Phylogenetic trees were rooted to F. oxysporum as monophyletic sister outgroup to the rest of the taxa. Bootstrap analyses were performed to determine the confidence intervals (1 000 replicates) for the branching points for the most parsimonious trees generated from all the data sets. The combinability of the TEF-1 and BT datasets was tested using the partition homogeneity test in PAUP* version 4b10 (Farris et al., 1994).
Bayesian analyses utilised the Metropolis-coupled Markov Chain Monte Carlo search algorithm as implemented in the program MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001). All Bayesian analyses consisted of 1 000 000 generations running one cold and three hot chains, with Bayesian inference posterior probabilities (biPP) calculated after burnin had been determined. BInt analyses utilised the GTR+I+G substitution model with separate parameters for each gene (partition) and an eight-category gamma model.

Morphological comparisons

Fungal strains and culture conditions

Isolates from grass and reed were grown on synthetic low nutrient agar (SNA) (Nirenberg and O’Donnell, 1998) and carnation leaf agar (CLA) (Nelson et al., 1983) for 7 d at 25 ëC, under near ultraviolet light. Fungal structures produced on these media were mounted on microscope slides and used to describe the morphology of the grass and reed isolates. Colony colour was assigned using the colour charts of Rayner (1970). Growth in culture was assessed by placing a single macroconidium (Nelson et al., 1983) at the centre of five PDA plates and incubating these in temperature-controlled incubators at temperatures ranging from 5 to 30 ëC at five degree intervals. Colony diameters were measured after 5 days by means of an electronic ruler and the averages of all measurements for each temperature computed. The standard error was calculated for each isolate at every temperature considered.

Sexual compatibility

To determine the mating types of the seven isolates from grass and reed, the MAT-1 and MAT-2 loci were amplified using PCR, as described by Steenkamp et al. (2000). The MAT idiomorphs were amplified with the primer sets GFmat1a (5’-GTTCATCAAAGGGCAAGCG-3’), GFmat1b (5’-TAAGCGCCTCTTAACGCCTTC-3’), GFmat2c (5’-AGCGTCATTATTCGATCAAG-3’) and GFmat2d (5’-CTACGTTGAGAGCTGTACAG-3’) (Steenkamp et al., 2000). For the PCR reactions, 1 x Roche Taq Reaction buffer with MgCl₂, 200 µM of each dNTP, 0.1 µM of each primer, 25 ng template DNA and 0.5 U Roche Taq polymerase (Roche Pharmaceuticals, Germany) was used. The PCR reaction conditions were an initial denaturation at 92 °C for 1 min, followed by 35 cycles of denaturation at 92 °C for 30 s, annealing at 63 °C for 30 s and elongation at 72 °C for 30 s. A final elongation step was done at 72 °C for 5 min. The products were resolved on a 1 % agarose gel, containing ethidium bromide (0.2 µg/mL) and visualised under UV light.

Acknowledgements 
Publications and presentations resulting from this study 
Preface 
Chapter 1: Species concepts in Fusarium with specific reference to species in Fusarium subglutinans sensu lato
1. Introduction
2. Species concepts used in the demarcation of Fusarium species
3. Conclusions
Literature cited
Chapter 2: Morphological and phylogenetic species concepts to distinguish between species in Fusarium subglutinans sensu lato
Abstract
Introduction
Materials and Methods
Results
Discussion
Chapter 3: A new Fusarium species from Poaceae in South Africa
Abstract
Introduction
Materials and Methods
Results
Discussion
Chapter 4: Characterisation of the pitch canker fungus, Fusarium circinatum, from Chile
Abstract
Introduction
Materials and Methods
Results
Discussion
Chapter 5: Fusarium ananatum sp. nov. in the Gibberella fujikuroi species complex from pineapples with fruit rot in South Africa
Abstract
Introduction
Materials and Methods
Results
Discussion
Summary
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