Fusarium species isolated from Pennisetum clandestinum collected during outbreaks of kikuyu poisoning in cattle in South Africa

Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. Kikuyu poisoning occurs sporadically in South Africa. It is of major economic importance, as valuable dairy cows are often poisoned by it, and once affected, the mortality rate is high. Pennisetum clandestinum samples were collected during eight outbreaks of kikuyu poisoning in cattle in the Eastern Cape Province of South Africa from 2008 to 2010. The kikuyu grass samples were submitted specifically for the isolation and molecular identification of Fusarium species, as it was recently suggested that mycotoxins synthesised by Fusarium torulosum could be the cause of this intoxication. Ninety-four Fusarium isolates were retrieved from the grass samples, of which 72 were members of the Fusarium incarnatum/Fusarium equiseti species complex based on morphology and phylogenetic analyses of the translation elongation factor 1α sequence data. The South African isolates from kikuyu identified as members of the F. incarnatum/F. equiseti species complex grouped together in six separate clades. The other isolates were Fusarium culmorum (n = 3), Fusarium redolens (n = 4) and Fusarium oxysporum (n = 15). Although F. torulosum could not be isolated from P. clandestinum collected during kikuyu poisoning outbreaks in South Africa, the mycotoxicosis theory is still highly plausible.


Introduction
Kikuyu poisoning, an intoxication of ruminants, is described as a ruminitis syndrome resulting in, among other manifestations, forestomach necrosis and dehydration (Bourke 2007;Kellerman et al. 2005).Although kikuyu grass (Pennisetum clandestinum Hochst.ex Chiov.) is usually grazed without deleterious effects, it sporadically becomes toxic (Bourke 2007;Kellerman et al. 2005).This usually happens when the kikuyu grass pasture has been subjected to severe stress in some form (like drought or invasion by army worm caterpillars) followed by a growth spurt during warm weather (Bryson 1982;Kellerman et al. 2005;Newsholme et al. 1983).Poisoning has been reported from New Zealand, Australia and eastern and southern Africa (Bourke 2007;Bryson 1982;Martinovich, Mortimer & Di Menna 1972).
Although kikuyu poisoning occurs periodically in South Africa, it is of major economic importance, as valuable dairy cows are often poisoned and, once affected, the mortality rate is high (Bryson 1982).After moving ruminants, especially cattle, to kikuyu pastures there is usually a latent period of 24 h or longer before clinical signs, mainly gastrointestinal and neuromuscular, are observed.Gastrointestinal signs are usually present; these include ruminal atony, distension and colic (kicking at the abdomen and grunting).Ruminal irritation and malabsorption result in the accumulation of excessive fluid in the rumen, leading to sloppy ruminal contents, which may even gush from the mouth and nose at death.Severe dehydration with sunken eyes and an unpliable skin is observed.The neuromuscular signs are distinguished by bulbar paralysis, resulting in an inability to swallow that is characterised by 'sham-drinking' and salivation.Tremors and incoordination have also been described (Bryson 1982;Kellerman et al. 2005;Newsholme et al. 1983).At necropsy, excessive and sloppy, watery ruminal contents (often bright green) are frequently observed.Hyperaemia of the submucosa and necrosis and ulceration of the forestomach mucosa may be seen macroscopically.Severe dehydration is also noticed.On microscopic examination, superficial epithelial necrosis of the rumen, reticulum and omasum are present.Characteristically there is detachment or absence of the stratum corneum, the necrosis only involves the stratum spinosum and stratum granulosum, but with preservation of the stratum basale (Newsholme et al. 1983).
The possibility that kikuyu poisoning is a mycotoxicosis has been mooted in the scientific literature since the first reported outbreaks (Martinovich et al.1972).Although various fungi that are potentially toxic to animals (such as Myrothecium spp.) have been cultured from kikuyu grass

Fusarium species isolated from Pennisetum clandestinum collected during outbreaks of kikuyu poisoning in cattle in South Africa
collected during outbreaks, they were not consistently present and there was little evidence of heavy fungal infestation of the pastures (Bourke 2007;Kellerman et al. 2005;Newsholme et al. 1983).Thus, the mycotoxicosis theory was placed on hold.However, more recently, Australian researchers reported that an endophytic fungus, Fusarium torulosum, was consistently isolated from kikuyu grass collected during an outbreak (Bourke 2007;Ryley et al. 2007).
During the summers and late summers of 2008-2010 various reports were received of large numbers of cattle dying as a result of kikuyu poisoning in the Eastern Cape Province of South Africa (F.van Niekerk, Humansdorp, personal comm., 2009).Sods of kikuyu grass (roots and base of plant) were collected during eight outbreaks; in five outbreaks, the clinical diagnosis was confirmed by the characteristic histopathology of lesions in the forestomach.The kikuyu grass was submitted particularly for the isolation and molecular identification of Fusarium species.The specific objective was to verify if F. torulosum was also consistently present in kikuyu grass during outbreaks of intoxication in South Africa.If this endophytic fungus is always present in poisonous kikuyu grass on two different continents, it could direct future research to identify an aetiological agent.

Isolations
From 2008 to 2010, sods of kikuyu grass were collected from various localities in the Eastern Cape Province during eight outbreaks of kikuyu poisoning in cattle.Isolations were made from leaf and stem material.Small pieces (4 mm) of plant tissue were surface disinfected with 1% sodium hypochlorite, rinsed twice with sterile water, blotted dry and plated onto Fusarium selective medium (20.0 g agar, 15.0 g peptone, 1.0 g KH 2 PO 4 , 0.5 g MgSO 4 .7H 2 O, 1 g PCNB, 20 mL streptomycin sulphate in 1 L water) in Petri dishes (Nelson, Tousson & Marasas 1983).Petri dishes were incubated for 7-10 days at 25 °C under coolwhite fluorescent light.The plates were checked regularly and all colonies with typical Fusarium morphology were transferred to half-strength potato dextrose agar (PDA) (Merck, Germany  1976).Colony colour and morphology were compared with that stipulated by Leslie and Summerell (2006).Morphological characters were described from structures produced on SNA, except the macroconidia morphology.
Ten measurements per isolate of the macroconidia and microconidia were also taken and the averages computed.

DNA extraction and amplification
Isolates were grown on half-strength PDA at 25 °C for 7 days.DNA was isolated using the DNeasy plant mini extraction kit (Qiagen, Valencia, CA) and following the manufacturer's protocol after the mycelium was placed in Eppendorf tubes and ground with approximately 10 µg sterile, chemically treated sand.
Extracted DNA was used as the template in polymerase chain reactions (PCR).Part of the translation elongation factor (TEF) 1-α gene was amplified using the primer set EF1 (5'-CGAATCTTTGAACGCACATTG-3') and EF2 (5 Nirenberg 1998).The PCR consisted of 1 x SuperTherm Taq reaction buffer with MgCl 2 , dNTPs (250 µM each), primers (0.2 µM each), template DNA (25 ng) and SuperTherm Taq polymerase (0.5 U) (Southern Cross, South Africa).The PCR conditions for the TEF gene region were amplified by initial denaturation at 94 °C for 2 min.This was followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min and elongation at 72 °C for 1 min, with a final elongation step at 72 °C for 5 min (Jacobs et al. 2010).The resulting PCR amplicons were visualised on a 1% agarose gel under ultraviolet (UV) light and purified using a QIAquick PCR Purification kit (QIAGEN, Hilden, 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, Warrington, United Kingdom) using the primers EF1 and EF2.Sequences generated in the present study have been deposited in GenBank (Table 1).
The partial sequence data for TEF were compared against both the Fusarium multilocus sequence typing (MLST) database and the Fusarium database (Geiser et al. 2004).Complete dataset and alignments for previously published phylogenetic relationships within the Fusarium incarnatum/Fusarium equiseti species complex (FIESC) were used (O'Donnell et al. 2009).Gaps were treated as missing data in the subsequent analysis.Phylogenetic analysis was based on parsimony using PAUP 4.0* (Swofford 2002).Heuristic searches were done with random addition of sequences (100 replicates), tree bisectionreconnection (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 & Huelsenbeck 1992).The consistency (CI) and retention (RI) indices were determined for the TEF data set.Phylogenetic trees were rooted with Fusarium concolor as the monophyletic sister outgroup to the rest of the taxa.Bootstrap analyses were performed to determine branching point confidence intervals (1000 replicates) for the most parsimonious trees generated for the TEF data set (also see Jacobs & Van Heerden 2012).
The F. equiseti isolates were characterised by the absence of microconidia and macroconidia with elongated apical cells.Chlamydospores were present in all isolates.The  colony colour ranged from white to brownish with age.
Isolates identified as F. incarnatum were characterised by morphology typical of Fusarium semitectum.These included macroconidia with a curved apical cell and pyriform to obovate microcondia borne on monophialides and polyphialides.No chlamydospores were observed in these cultures.The F. oxysporum isolates were characterised by purple colonies, with chlamydospores forming after 4 weeks.The macroconidia were straight to slightly curved, whilst the microconidia were oval to elliptical, borne on short monophialides.Fusarium redolens isolates formed oval to cylindrical microconidia on monophialides, whilst the macroconidia were thick-walled with hooked apical cells.Isolates of F. culmorum were characterised by the absence of microconidia and robust, short, thick-walled macroconidia.

DNA extraction and amplification
Amplicons of the TEF gene region were 640 bp in size.

DNA sequencing and sequence comparisons
Parsimony analysis of the TEF gene region was performed to determine the phylogenetic placement of the kikuyu isolates within the FIESC.Alignment of the TEF data set by inserting gaps resulted in a total of 587 characters used in the comparison of the different species.All parsimonyuninformative and constant characters were excluded, resulting in 164 parsimony-informative characters.Heuristic searches on the data set generated one most parsimonious tree.
In the TEF data set (Figure 1), the South African isolates from kikuyu identified as members of the FIESC grouped together in six separate clades (A-F), of which four were supported by high bootstrap values (> 85%

Discussion
Contrary to the findings in Australia, F. torulosum was not isolated from the P. clandestinum collected during eight outbreaks of kikuyu poisoning in South Africa.Nonetheless, a number of other Fusarium species were isolated from kikuyu grass collected from toxic pastures during 2008 to 2010.
The genus Fusarium is recognised for the taxonomic difficulties associated with it and, based on the classification system used, the total number of species in this genus has ranged from 9-1000 (Summerell & Leslie 2011).Fusarium isolates from toxic kikuyu grass pastures in South Africa mainly represent species in the FIESC.The DNA sequence comparisons based on the TEF gene formed the most important basis for distinguishing these Fusarium species.
The TEF 1 α gene was also the most informative of the four gene regions used by O'Donnell et al. (2009) to distinguish the 28 phylogenetic species in the FIESC.The grouping of the South African isolates from P. clandestinum with five of these species, based on sequence data for the TEF gene, confirms that the majority of the Fusarium isolates obtained from P. clandestinum associated with kikuyu poisoning in cattle form part of the FIESC.Historically, the genus Fusarium has been known as a plant pathogen, but since the 1960s has also been reported to be associated with secondary metabolites responsible for Fusarium-related mycotoxicoses in humans and animals (Summerell & Leslie 2011).In many of the Fusarium-associated intoxications, the mycotoxin(s) are unknown but the toxins of veterinary importance synthesised by some of the strains include fumonisins, zearalenone and trichothecenes such as diacetoxyscirpenol, deoxynivalenol and T2-toxin (Desjardins 2006;Kellerman et al. 2005;Marasas, Nelson & Tousson 1984).Other lesser-known Fusarium mycotoxins include beauvericin, enniatins and fusarochromanone (Altomare et al. 1995;Bryden et al. 2004;Logrieco et al. 1998).
The mycotoxicosis theory is thus still very plausible and is supported by the periodic occurrence of kikuyu poisoning usually during warm, humid weather when fungal growth in a pasture is likely to be optimal (Martinovich et al.1972).Fusarium torulosum is known to produce mycotoxins such as wortmannin and butenolide (Bourke 2007;Ryley et al. 2007).
Oral administration of wortmannin to rats is toxic at a dose as low as 4 mg/kg, causing gastric and myocardial haemorrhage (Gunther, Abbas & Mirocha 1989).Butenolide is reported to induce acute inflammation in the forestomachs of cattle (Tookey & Grove 1972).In addition, Brazilian researchers described a ruminitis syndrome very similar to kikuyu poisoning (Riet-Correa et al. 2009).Unlike kikuyu poisoning, this intoxication is caused by members of the Asteraceae family, Baccharis coridifolia and Baccharis megapotamica, but the toxins in the plants are a range of trichothecenes synthesised by soil fungi (Myrothecium spp.) and absorbed by the roots of the plant (Riet-Correa et al. 2009).If kikuyu poisoning is a mycotoxicosis, assays could be developed to be able to forecast risk and to warn farmers that intoxication may occur.

Conclusion
Although F. torulosum could not be isolated from P. clandestinum when collected during eight outbreaks of kikuyu poisoning in South Africa, various other Fusarium isolates (n = 94) were retrieved from the toxic kikuyu grass.These isolates were predominantly members of the FIESC; however, the possibility that kikuyu poisoning is a mycotoxicosis should be further investigated and toxic grass samples could be subjected to analytical screening for fungal metabolites.

TABLE 1 :
Fusarium isolates obtained from Pennisetum clandestinum collected during kikuyu poisoning outbreaks in the Eastern Cape Province of South Africa.
FIESC, Fusarium incarnatum/Fusarium equiseti species complex; PPRI, Plant Protection Research Institute; N/I, not included in phylogenetic analysis.a, PPRI culture collection of the National Collections of Fungi, Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council, Pretoria, South Africa.

TABLE 1 (
Fusarium isolates obtained from Pennisetum clandestinum collected during kikuyu poisoning outbreaks in the Eastern Cape Province of South Africa.Fusarium incarnatum/Fusarium equiseti species complex; PPRI, Plant Protection Research Institute; N/I, not included in phylogenetic analysis.a, PPRI culture collection of the National Collections of Fungi, Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council, Pretoria, South Africa. continues...): FIGURE 1: Phylogenetic tree produced using parsimony of the translation elongation factor gene with Fusarium concolor as outgroup.

Sequence Type associated with the particular isolate Fusarium ID database BLAST result
Plant Protection Research Institute; NRRL, Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America; MLST, multi loci sequence type; CBS, culture collection of Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht, The Netherlands; FIESC, Fusarium incarnatum/Fusarium equiseti species complex; BLAST, Basic Local Alignment Search Tool.
†, PPRI refers to the PPRI culture collection of the National Collections of Fungi, Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council (ARC-PPRI), Pretoria, South Africa.

TABLE 2 :
Molecular identification of Fusarium isolates based on translation elongation factor sequence data.

Table 2
continues on next page → Culture

number Multi Loci Sequence Type associated with the particular isolate Fusarium ID database BLAST result
Plant Protection Research Institute; NRRL, Agricultural Research Service culture collection, United States Department of Agriculture, Illinois, United States of America; MLST, multi loci sequence type; CBS, culture collection of Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht, The Netherlands; FIESC, Fusarium incarnatum/Fusarium equiseti species complex; BLAST, Basic Local Alignment Search Tool.†, PPRI refers to the PPRI culture collection of the National Collections of Fungi, Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council (ARC-PPRI), Pretoria, South Africa.

TABLE 2 (
continues...): Molecular identification of Fusarium isolates based on translation elongation factor sequence data.