Characterisation and pathogenicity of Fusarium taxa isolated from ragwort (Jacobaea vulgaris) roots

Fungal Ecology 20 (2016) 186e192

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Characterisation and pathogenicity of Fusarium taxa isolated from ragwort (Jacobaea vulgaris) roots K.A. Pearson a, 1, A.F.S. Taylor a, b, R.M.E. Fuchs c, S. Woodward a, * a University of Aberdeen, Institute of Biological and Environmental Sciences, Department of Plant and Soil Science, Cruickshank Building, St. Machar Drive, Aberdeen, AB24 3UU, Scotland, UK b James Hutton Institute, Craigiebuckler, Aberdeen, AB15 8QH, Scotland, UK c SRUC, Craibstone Estate, Aberdeen, AB21 9YA, Scotland, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2013 Received in revised form 23 November 2015 Accepted 31 December 2015 Available online xxx

We characterised the fungi isolated from necrotic lesions observed on roots of the noxious weed Jacobaea vulgaris and assessed their host range. ITSerDNA sequence data identified twenty-one isolates of Fusarium and TEF-1a sequencing separated these isolates into four clades namely Fusarium avenaceum/ Fusarium acuminatum, Fusarium redolens, Fusarium culmorum/Fusarium cerealis and Fusarium solani. Representatives of each clade were tested for virulence against ragwort seedlings. All isolates in the F. avenaceum/F. acuminatum clade caused visible disease symptoms. Host range of three isolates was tested using six pasture grass species along with Trifolium pratense and Trifolium repens. No disease symptoms were detected on the grasses. All three isolates, however, caused disease symptoms on T. repens, and two isolates also attacked T. pratense. The results demonstrate that several species of Fusarium can colonise ragwort roots but only isolates from one clade cause significant disease symptoms on ragwort seedlings. © 2016 Elsevier Ltd and The British Mycological Society. All rights reserved.

Corresponding Editor: Gareth W. Griffith Keywords: Jacobaea vulgaris Senecio jacobaea Root pathogens Fusarium Ragwort

1. Introduction Ragwort (Jacobaea vulgaris syn. Senecio jacobaea; common ragwort; Asteraceae; Senecioneae) is a common weed of temperate areas worldwide that can cause serious liver damage on ingestion by susceptible animals. A native of Europe and East Asia, it spread to the New World in the mid to late 19th Century (Harper, 1958). Ragwort is usually a biennial weed, producing a flat rosette of leaves in the first year of growth and generally flowering in the second year; however, if a plant is cut, the lifecycle can be prolonged by several years (Defra, 2004). It is an effective coloniser of bare ground and is particularly associated with poorly managed pasture, roads and railways where disturbed ground is available for invasion (Harper, 1958). One of the reasons that ragwort is such a successful coloniser is the high number of seeds produced, with one plant capable of producing several thousand seeds (Cameron, 1935; Tutin et al., 1976; Wardle, 1987). The seeds also have

* Corresponding author. E-mail address: [email protected] (S. Woodward). 1 Present address: Science and Advice for Scottish Agriculture, Roddinglaw Road, Edinburgh, EH12 9FJ, Scotland, UK. http://dx.doi.org/10.1016/j.funeco.2015.12.011 1754-5048/© 2016 Elsevier Ltd and The British Mycological Society. All rights reserved.

extended persistence in the seed bank, remaining viable for up to 10 yr (Harper, 1958). Both fresh and dried ragwort plants may contain between 10 and 12 pyrrolizidine alkaloids (PAs), which are toxic to grazing livestock, particularly horses (Witte et al., 1992; Prakash et al., 1999). Consumption of PAs results in severe liver damage in susceptible animals and may lead to death (Mattocks, 1986). Consequently, legislation to encourage the control of ragwort on land used for grazing is in place both within the native range of ragwort (e.g. Anon, 1937; Defra, 2004; Leiss, 2011) and in its introduced range, e.g. Australia and New Zealand (Wardle, 1987; Roberts and Pullin, 2007). Despite the recognised animal health problems associated with ragwort, there are currently no entirely satisfactory control methods for this plant. Hand pulling is regularly used to manage the weed in small areas but this is too labour intensive to be used on a large scale. Control by the application of either general or broadleaf specific herbicides is prohibitively expensive due to the extensive nature of the weed; moreover, widespread chemical application to pasture areas may be ecologically undesirable. An environmentally benign and time effective alternative control method would, therefore, be preferable to wide-scale chemical

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application or labour intensive hand pulling. Classical biological control tends to deal only with an introduced pest species, whereas mycoherbicides, or biological control based on inundative application of fungal spores, may be highly effective within the native range of a weed (Weaver et al. 2007). Effectiveness is dependent, however, on the virulence and specificity of the fungal species selected. There are few records of fungi infecting ragwort. Ellis and Ellis (1985) described six fungal pathogens on ragwort foliage: the rusts Coleosporium tussilaginis and Puccinia dioicae var. schoeleriana, the leaf spots Alternaria dennisii, Ramularia pruinosa and Septoria senecionis-silvatici, and the powdery mildew Podosphaera xanthii. In addition, a phytoplasma has been reported causing stunting and leaf chlorosis in ragwort (Reeder and Arocha, 2008). Alber et al. (1986) reported infection of ragwort by the rust Puccinia expansa, and demonstrated a 60% reduction in dry weight of ragwort plants in a glasshouse trial of pathogenicity. Field experiments were suggested for the further testing of P. expansa as a biological control agent of ragwort but this work was not carried out. Root pathogens may be far more damaging to their host plants than foliar pathogens through disruption of vascular system (Boyette et al., 1993; Thomas et al., 1999; Alabouvette et al., 2009). However, little work has so far been done on using root pathogens as biocontrol agents of weeds. The aim of the present work was, therefore, to investigate the occurrence, identity and pathogenicity of fungi isolated from lesions on roots of ragwort plants growing in the UK. In addition, the virulence of fungal isolates against common pasture grass and clover species was tested to determine the host range.

2. Materials and methods 2.1. Isolation of root fungi from ragwort Two hundred and fifty Pony Clubs distributed throughout the UK were contacted by post and asked to collect the root systems of five separate ragwort plants in their local area, rinse off adhering soil under running tap water and post the roots back to the laboratory in Aberdeen as soon as possible using an enclosed pre-paid first class postage envelope. On arrival at the laboratory, the root systems were washed thoroughly under running tap water and examined under a dissecting microscope for lesions, which were recognised by the presence of well-established necrotic host tissues around an infection point. The necrotic areas were excised, surface sterilized in 10% sodium hypochlorite for 5 min and rinsed in three changes of sterile distilled water before placing onto potato dextrose antibiotic agar (PDA, Oxoid Ltd., Basingstoke, Hampshire, England) containing (per litre) 100,000 units penicillin and 100 mg streptomycin (SigmaeAldrich, Gillingham, Dorset, England), Corn Meal antibiotic agar (CMA, Oxoid Ltd.) containing the same antibiotics, or PARPH V8 agar containing (per litre) 20 g agar no. 1 (Oxoid, address as above), 200 ml filtered V8 juice (Campbell's Soup Company UK, Cambourne, Cambridgeshire, England), 50 mg hymexazol (SigmaeAldrich), 5 mg pimaricin (SigmaeAldrich), 10 mg rifampicin (SigmaeAldrich), 250 mg ampicillin (SigmaeAldrich) and 125 mg pentachloronitrobenzene (PCNB, SigmaeAldrich). PDA and CMA are routinely used to isolate both Fusarium spp. and Rhizoctonia spp. and PARPH V8 is a selective medium developed to isolate Phytophthora (Ferguson and Jeffers, 1999). Cultures were incubated at 20
C in the dark and inspected daily for emergence of hyphae. Any fungal hyphae growing from the root tissues were carefully excised and sub-cultured onto fresh media without antibiotics.

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2.2. Morphological identification of isolates Initial identification of isolates involved examination of hyphae under a compound microscope at magnifications from  40 to  1000, with hyphae mounted in water or stained with lactophenol-cotton blue. 2.3. Molecular identification The single-hyphal tip method (Nelson et al., 1983) was used to ensure that all fungal isolates were single cultures prior to species identification using molecular means. Pure isolates were subcultured into 100 ml potato dextrose broth (Oxoid Ltd.) in 250 ml conical flasks and incubated on a rotary shaker at 150 r.p.m, at room temperature for 7 d. DNA was extracted using a Qiagen DNeasy Plant Minikit (Qiagen UK, Crawley), following the manufacturer's instructions. For identification to the level of genus, primers ITS1 and ITS4 were used (White et al., 1990). A 50 ml reaction was run in a PCR machine (PTC200 Peltier Thermal Cycler, MJ Research, Waltham, Massachusetts, USA or PTC-220 Peltier Thermal Cycler, MJ Research) with the following programme: Initial denaturation at 95
C for 5 min; thirty cycles of 30 s at 95
C (denaturation); 30 s at 52
C (annealing) and 1 min at 72
C (extension) followed by 10 min final extension at 72
C. The ITS region is recognised to be unreliable for distinguishing taxa at the species level in Fusarium (Geiser et al., 2004). Therefore, specific identification was carried out using the translation elongation factor 1a (TEF-1a), a conserved DNA region regularly used for sequencing for identification and phylogenetic analysis of the genus Fusarium (Seifert, 2009). Around 700 base pairs of TEF-1a were amplified with primers EF1 and EF2 (O'Donnell et al., 1998). A 50 ml reaction was carried out using the conditions described above for ITS amplification with an amended annealing temperature of 53
C (Geiser et al., 2004). PCR products from both primer sets were purified with a QIAquick PCR purification kit (Qiagen UK) and visualised on 1% agarose gels. Sequencing was set up in 20 ml reaction volume with the DNA adjusted to 100 ng ml1, 1 ml primer and 4 ml big dye (ABI Prism BigDye Terminator v3.1 Cycle Seq Kit, Applied Biosystems, Foster City, CA, USA) per reaction. The sequencing program SEQ1 consisted of 25 cycles of 96
C for 15 s, 55
C for 15 s and 60
C for 4 min. This was followed by purification by ethanol precipitation and sequencing (ABI 3130 XL Genetic Analyzer, Applied Biosystem, Foster City, CA, USA). Sequences were compared to Genbank and Fusarium-ID (TEF-1a only; http://isolate.fusariumdb.org/; Geiser et al., 2004) for isolate identification. A Neighbour-Joining dendrogram with the Maximum Composite Likelihood method and complete deletion with 1000 replicates for bootstrap analysis was prepared to compare the similarity in DNA sequences between the isolates (Mega 4.1, Tamura et al. 2007). 2.4. Plant materials Ragwort seed (“S. jacobaea”, catalogue number 31735, Herbiseed, Twyford, England) was surface sterilized in 10% sodium hypochlorite solution for 5 min and rinsed in 3 changes of sterile distilled water. Seeds were plated onto 1% distilled water agar (DWA) in 9 cm diam. Petri dishes and incubated at 21
C under a light intensity of 88.4 mmol m2 s1 with a 16 h photoperiod. After 7 d, ungerminated seeds were discarded and germinated seedlings allowed to develop for a further 7 d under the same conditions. The 14 d old seedlings were transferred to clear round pots (diameter 7.25 cm, depth 6 cm; TQPL, Brookside, Southern Lane, New Milton, Hampshire, UK) containing mixed sterilized horticultural grade

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sand and perlite (3:1 v/v) to a depth of 1.5 cm, watered with 10 ml Phostrogen™ (500 mg l1 ; N:P:K, 14:10:27) solution. Pots were covered with close-fitting clear plastic lids fitted with a 0.45 mm vent (provided by the same supplier), and placed in a Phytotron (Fison Fi-trotron 600H) at 18
C under a light intensity of 88.4 mmol m2 s1 with a 16 h photoperiod. The seedlings were raised under these conditions for 3, 7 or 11 weeks prior to fungal inoculation when 16, 12 and 12 seedlings were inoculated, respectively. 2.5. Inoculum production and inoculation Fungal isolates were sub-cultured onto PDA and incubated at 20
C in the dark for 7 d. Plant inoculations were carried out by placing a 2 mm diam. disk of fungal mycelium, excised from the edge of an actively growing colony, onto the surface of the sandperlite substratum in contact with the root collar of the plant. In total, between 31 and 40 seedlings were inoculated with 19 fungal isolates. Control plants were inoculated with a 2 mm disc of sterile PDA. Inoculated and control pots were returned to the Phytotron and maintained as described above. 2.6. Disease assessment All ragwort seedlings were scored daily for 14 d after inoculation using the following scale of disease symptoms: 1. No symptoms; 2. Signs of necrosis on cotyledons; 3. Necrosis spreading through the plant vascular system; 4. Advanced necrosis; 5. Plant death. Isolates with a median disease score of 3 or greater after 14 d were considered to be virulent. 2.7. Host range Seed of several pasture species commonly included in agricultural or amenity seed mixtures (personal communication, Russell Thomson, Science and Advice for Scottish Agriculture, Roddinglaw, Edinburgh) were germinated and tested for susceptibility to infection by three of the fungal isolates e two Fusarium avenaceum/ Fusarium acuminatum isolates and one Fusarium culmorum isolate. Pasture species included Cynosurus cristatus (crested dogs tail), Phleum pratense (Timothy), Festuca rubra spp. litoralis (creeping red fescue), Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass), Trifolium pratense (red clover) and Trifolium repens (white clover). Grass and clover seed was surface sterilized in 15% sodium hypochlorite for 5 min and rinsed in three changes of 20 ml sterile distilled water. Sterilized seeds were placed on 1% DWA in 9 cm diam. Petri dishes and incubated at 20
C under the conditions described above. After 7 d, germinated seedlings were transferred into P40 multi-cell trays (LBS Polythene, Lancashire, UK) which had been prepared by placing a double layer of sterilized muslin cloth in the bottom of each cell. Cells were filled to 75% capacity with twice autoclaved (2  1 h at 105 kPa) sand e perlite mix (3:1 v/v) then placed into a basin containing sterile Phostrogen™ solution (500 mg l1) until it had soaked through the hole in the bottom of each cell and saturated the substratum. Ten seedlings of each species were used for inoculation with each fungal isolate and the experiment repeated twice under identical conditions. Seedlings were inoculated in the same way as the ragwort seedlings described above, using a 2 mm diam. core of mycelium and agar. The multi-cell trays were placed in polythene bags supported above the plants with a wire frame and the bag opening sealed around a 90 mm nylon mesh filter. The nylon mesh was sandwiched between two plastic rings cut from a 100 ml plastic beaker. This filter system allowed air flow but prevented transfer of fungal spores into the bags.

Disease symptoms were scored using the scale described above for ragwort seedlings. 2.8. Statistical analyses The statistical analysis of the disease score data required nonparametric techniques to account for the ordinal nature of the score values. The effect of seedling age at inoculation on disease score after 14 d was analysed with Friedman's test with age at inoculation as a blocking factor (Townend, 2002). Having established that there was no age effect, a series of KruskaleWallis tests was used to determine relative isolate virulence. All analyses were carried out in Minitab 16 (Minitab® Statistical Software). 3. Results 3.1. Isolation of fungi from root lesions Fifty-five replies were received from pony clubs across the UK, each of which included 5 ragwort root systems. Depending on the number and severity of lesions present on the root systems, up to 6 lesions per root system were plated onto PDA and CMA media. In total, approximately 1100 lesions were plated. A majority of lesions did not produce fungal growth; 36 separate isolates were obtained from the ragwort root systems. 3.2. Morphological identification of isolates To allow easier comparison of different cultures, any isolates growing on CMA were subcultured onto PDA. Each of the 36 fungal isolates obtained was initially examined in culture for morphological characters characteristic of Pythium, Phytophthora, Rhizoctonia and Fusarium. Examination of micro- and macroscopic culture features allowed the provisional identification of 22 potential Fusarium spp. cultures by characteristics including production of a pink coloured colony on PDA and production of sickle shaped macro-conidia (Booth, 1971). The remaining fourteen isolates were provisionally identified as members of the genera Aureobasidium, Mucor, Penicillium and Phoma. These were discounted from further study. Despite the use of selective media, no potential Pythium, Phytophthora or Rhizoctonia isolates were recovered from the plant tissue collected. 3.3. Molecular identification of isolates Sequencing of the ITS and blasting against GenBank confirmed that 21of the 22 isolates examined were in the genus Fusarium: the remaining isolate was identified as Neonectria and omitted from subsequent phylogenetic study. The TEF-1a sequences of the Fusarium isolates were deposited in GenBank (JF430772eJF430794). Comparative analysis using the TEF-1a sequence data segregated the isolates into four clear groups, each of which aligned with a reference sequence from either Fusarium-ID or Genbank (Fig. 1). Clade 1 contained isolates of F. avenaceum or the closely related F. acuminatum. Clade 2 was identified as the only example of F. redolens found in the study, whereas clade 3 isolates aligned with F. cerealis or the closely related F. culmorum. The single isolate in clade 4 was a representative of F. solani. The identification of F. redolens, F. cerealis, F. culmorum and F. solani based on TEF-1a data in GenBank was supported by TEF-1a sequences contained in the Fusarium-ID database. However, no representatives of F. avenaceum or F. acuminatum were present in Fusarium-ID. No attempt was made to further resolve the withineclade relationships in these isolates of Fusarium.

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Fig. 1. Dendrogram based on TEF-1a sequences derived from Fusarium strains isolated from root lesions of Jacobaea vulgaris and assessed for their virulence towards ragwort seedlings. Reference isolates taken from Genbank to allow clade identification. Bootstrap value from 1000 reps.

3.4. Disease assessment Comparison of median disease scores 14 d after inoculation of plants at 5, 9 or 13 weeks old, using a Friedman's test, showed no significant effect of plant age at inoculation on susceptibility to disease (P ¼ 0.798, data not shown). Subsequently, results for inoculation with each isolate were combined for further analyses (Table 1). All of the eleven isolates in clade 1 of the dendrogram, the F. avenaceum/F. acuminatum group, were virulent on ragwort when virulence was defined as a median disease score of 3 or greater after 14 d. In addition, one isolate from clade 3 caused a median disease score of 2 after 14 d. Due to time constraints, no attempts could be made to satisfy Koch's fourth postulate, re-isolation of the causal agent. 3.5. Host range The three Fusarium isolates tested showed no virulence to any of the six grass species when compared to control plants. The white

clover plants, however, developed disease symptoms with both F. avenaceum (isolates JF430774 and JF430776) giving median disease scores of 3.5 and 4 respectively 14 d after inoculation and with F. culmorum (isolate JF430789) giving a median disease score of 3.5, whereas red clover was only affected by the two F. avenaceum isolates (median disease scores of 4 and 3 respectively) and not by F. culmorum. 4. Discussion The work reported here is the first to characterise fungal pathogens from roots of the toxic weed species, common ragwort (J. vulgaris), although previous work has isolated fungal pathogens from ragwort roots to use in work on the inhibitory effects of pyrrolizidine alkaloids on plant pathogens (Hol and van Veen, 2002; Bezemer et al., 2013). The work focused on isolating species of Pythium, Phytophthora, Rhizoctonia and Fusarium as many of the major soil borne diseases of plants worldwide are placed in these genera (Sneh et al., 1994; Booth, 1971). Isolates of Aureobasidium, Mucor, Penicillium and Phoma were also recovered from lesions but

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Table 1 Median and interquartile ranges of disease scores on ragwort following inoculation with one of 18 Fusarium isolates. Isolate

Clade

n

Q1

Median

Q3

JF430783 JF430786 JF430784 JF430779 JF430787 JF430780 JF430782 JF430774 JF430776 JF430777 JF430790 JF430775 JF430789 JF430788 JF430794 JF430785 JF430781 JF430792 Neonectria sp.

1 1 1 1 1 1 1 1 1 1 1 2 3 3 3 3 3 4

39 34 31 37 33 38 34 33 38 40 40 34 40 39 35 35 40 40 40

2 1 2 3 2 2.25 3 3 3 2 2 1 1 1 1 1 1 1 1

3 3 3 3 3 3 4 3 4 3 4 1 2 1 1 1 1 1 1

4 4 4 4 4 4 4 4 4 4 4 1 2 1 1 1 1 1 1

Q1 ¼ lower quartile score, Q3 ¼ upper quartile score, n ¼ number of replicates. Clade ¼ the clade each isolate is from (see Fig. 1), Clade 1 represents Fusarium avenaceum/F. acuminatum, clade 2 F. redolens, clade 3 F. culmorum, clade 4 F. solani. Score ¼ 1. No effect on plant, 2. Signs of necrosis on lowest leaves, 3. Necrosis spreading through the plant vascular system, 4. Advanced necrosis, 5. Plant death. The Neonectria isolate was included in the virulence test but not placed within the Fusarium dendrogram. The median and interquartile ranges represent the disease symptoms 14 days after inoculation.

these were discounted from further study because although representatives of these genera are plant pathogens, they are rarely considered important pathogens of higher plants and far more frequently exhibit saprotrophic or endophytic habits (Ingold, 1978; Pitt, 1979; Frankland, 1998; Macia-Vicente et al., 2008). Of the four pathogen genera targeted in this work, only representatives of the genus Fusarium were identified. Fusarium is arguably the most common genus of soil-borne pathogenic fungi (Booth, 1971), causing diseases such as vascular wilts, damping off, root rot and stem rot on most genera of cultivated plants. Fusarium spp. may be primary plant pathogens, or may be components in disease complexes (Parry et al., 1995). In the present study, all isolates of F. avenaceum/F. acuminatum tested caused disease symptoms in ragwort, suggesting that other Fusarium species isolated from lesions may have been secondary infections, establishing in damage arising from other causes. Insect larvae were observed feeding on ragwort roots on several occasions (data not presented) and it is possible that some of the root lesions observed may have resulted from insect feeding rather than fungal infection per se. This observation could explain the low rate of recovery of pathogens from lesions. The presence of lesions may have allowed endophytes, weak pathogens or primary saprotrophs, which could not penetrate the root cortex independently, to establish in the dead and dying tissues in the lesion (Wang and Jeffers, 2000). It is possible that the three Fusarium species isolated in the present study that caused no disease symptoms in inoculated ragwort, had colonized the original lesions in this manner. An isolate was deemed to be virulent if it caused an infection that had spread into the vascular system and through the plant 14 d after seedling inoculation (indicated by a disease score of 3 or more). Because scores reflecting percentage disease cover are readily converted into a disease severity index suitable for direct statistical comparisons, they are often used in assessments of the virulence of plant pathogens (Dissanayake et al., 2009; Dervis et al.,

2010; Imathiu et al., 2010). In this work, however, the use of a percentage based system was not appropriate as infections spread initially through the vascular system of inoculated plants, before breaking out into the surrounding tissues. For this reason, a scale that tracked the progress of the disease was more appropriate. Although this method limited the subsequent analyses to nonparametric statistical techniques, it gave a clear division between isolates that caused disease symptoms and those that did not, and also allowed some differentiation between the virulence of pathogenic isolates. All eleven representatives of F. avenaceum and F. acuminatum isolated from ragwort roots were virulent on ragwort seedlings. This result contrasts with similar work where some isolates of the same Fusarium species were pathogenic and others non-pathogenic to host plants, e.g. rice (Oryza sativa, Amatulli et al., 2010); red clover (T. pratense, Yli-Mattila et al., 2009); asparagus (Asparagus officinalis, Wong and Jeffries, 2006); field pea (Pisum sativum, Feng et al., 2010); Welsh onion (Allium fistlosum, Dissanayake et al., 2009). It is possible, however, that if more isolates from the F. avenaceum/F. acuminatum complex were obtained from ragwort and tested, a proportion could prove to be non-pathogenic. The study focused on root pathogens of ragwort because, although foliar pathogens such as rusts, mildews and leaf spots are known on various species of ragwort, serious damage by aerial pathogens is rarely reported (Harper, 1958; Ellis and Ellis, 1985; Wardle, 1987). Moreover, root pathogens have marked disruptive effects on nutrient and water transfer through the plant, and therefore may be more effective than foliar pathogens when the objective is control of the whole plant (Boyette et al., 1993; Thomas et al., 1999; Alabouvette et al., 2009). Although the provision of a genetic comparison of species within the genus Fusarium was not the objective of the work reported here, the clade groupings determined in this work for the isolates from ragwort were supported by previously published work on differentiation within the Fusarium complex (Kristensen et al., 2005; Nitschke et al., 2009; Harrow et al., 2010). Identification to the species level in the genus Fusarium using only cultural characteristics is difficult due to the lack of morphological distinction between species (Booth, 1971; Aoki et al., 2003; Geiser et al., 2004; Kristensen et al., 2005). Molecular approaches have enabled a re-analysis of species interrelationships within Fusarium based on the highly informative translation elongation factor 1-a (TEF 1-a; Geiser et al., 2004; Seifert and Levesque, 2004). In the present work, although ITS clearly demonstrated that the isolates obtained from ragwort were Fusarium spp., this marker did not separate isolates at the species level (O'Donnell and Cigelnik, 1997; O'Donnell et al., 1998; O'Donnell, 2000). TEF 1-a, however, distinguished between four clades within the isolates obtained here, enabling the identification of the virulent isolates as F. avenaceum and F. acuminatun. Accurate identifications were essential prior to any further analysis of the virulence of these isolates. Mycoherbicide research began over 40 yr ago when this developing science was believed to be of great promise in weed management (Boyetchko et al., 2007; Charudattan, 2010). However, research in the field has “languished in recent years” (Hallett, 2005). The main problems in mycoherbicide development are the limitations of the active organisms, costs associated with formulating a product and the development of a large enough market to justify production and registration costs (Weaver et al., 2007). With this in mind, ragwort remains an excellent candidate for biological control with the huge global market for an effective, targeted control. Fusarium sp. would be easy to produce in bulk and there would be a significant market both in the UK and overseas for a ragwort control product as long as a strain which was specific could

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be identified. The most virulent isolates found in this work caused considerable disease symptoms on ragwort, suggesting that they could have potential as biological control agents. However, in host range tests, the two most virulent F. avenaceum isolates also caused significant damage to both red and white clover (T. pratense and T. repens, respectively) and this result raises the need for further specificity testing. It is vital that safety and specificity of a biological control agent is thoroughly tested and proven before any organism is released (Barton, 2004). Indeed, in many studies assessing the biological control potential of fungi, more resources are spent confirming the host range of the candidate fungal species than assessing the effectiveness of the candidate biological control agent in suppressing the target weed (Morin et al., 2009). 5. Conclusions In the current study, isolates of Fusarium originating from the roots of ragwort could be divided into four species complexes within the Fusarium genus. All isolates within the F. avenaceum and F. acumination clade proved virulent towards ragwort, with those isolates tested also showing some virulence to clover but not to pasture grass species. A range of further work would allow testing the isolates against a wider host range to allow a true assessment of any potential for the development of a biological control agent for ragwort. It may also be possible to sample for further isolates of F. avenaceum and F. acumination from ragwort roots which may have even greater virulence towards ragwort. Acknowledgements This work was supported by a generous grant from the Bransby Home of Rest for Horses, Bransby, Lincolnshire and formed part of the research towards the PhD of KP. Janis Brodie (UOA) and Alison Williams (JHI) are thanked for technical support. We are grateful to Russell Thomson at SASA for provision of grass and clover seed. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2015.12.011. References Alabouvette, C., Olivain, C., Migheli, Q., Steinberg, C., 2009. Microbiological control of soil-borne phytopathogenic fungi with special emphasis on wilt-inducing Fusarium oxysporum. New Phytol. 184, 529e544. Alber, G., Defago, G., Kern, H., Sedlar, L., 1986. Host range of Puccinia expansa a possible fungal biocontrol agent against Senecio weeds. Weed Res. 26, 69e74. Amatulli, M.T., Spadaro, D., Gullino, M.L., Garibaldi, A., 2010. Molecular identification of Fusarium spp. associated with bakanae disease of rice in Italy and assessment of their pathogenicity. Plant Pathol. 59, 839e844. Anon, 1937. Noxious Weeds (Thistle, Ragwort, and Dock) Order, 1937. Statutory Instrument No. 103/1937. Irish Office of the Attorney General, Dublin. Aoki, T., O'Donnell, K., Homma, Y., Lattanzi, A.R., 2003. Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex e F. virguliforme in North America and F. tucumaniae in South America. Mycologia 95, 660e684. Barton, J., 2004. How good are we at predicting the field host range of fungal pathogens used for classical biological control of weeds? Biol. Control 31, 99e122. Bezemer, T.M., van der Putten, W.H., Martens, H., van de Voorde, T.F.J., Mulder, P.P.J., Kostenko, O., 2013. Above and below ground herbivory effects on below-ground plant-fungus interactions and plant-soil feedback responses. J. Ecol. 101, 325e333. Booth, C., 1971. The Genus Fusarium. CAB International, Wallingford, UK. Boyetchko, S.M., Bailey, K.L., Hynes, R.K., Peng, G., 2007. Development of the mycoherbicide BioMal. In: Vincent, C., Goettel, M.S., Lazarovits, G. (Eds.), Biological Control: A Global Perspective. CAB International, Wallingford, UK. Boyette, C.D., Abbas, H.K., Connick, W.J., 1993. Evaluation of Fusarium oxysporum as a

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