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Keywords:

  • deoxynivalenol;
  • Fusarium graminearum;
  • nivalenol;
  • Tri3;
  • Tri7;
  • Tri13

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fusarium graminearum, one of the causal agents of fusarium head blight (FHB) of wheat in the UK, can be broadly divided into two chemotypes based on the production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). DON-producing isolates can be further distinguished on the basis of the predominant acetyl DON derivative that they produce; 3-acetyl DON (3-AcDON) or 15-acetyl DON (15-AcDON). Functional Tri13 and Tri7 are required for the production of NIV and 4-acetyl NIV, respectively, whereas, in isolates that produce only DON and its acetylated derivatives, these genes are nonfunctional or absent. Infections caused by F. graminearum are becoming more frequent in the UK; however, it is unknown whether these represent more than one chemotype. In this study, polymerasae chain reaction (PCR) assays specific for functional and nonfunctional/deleted versions of Tri7 and Tri13 were used to characterize 101 single-spore isolates of Fusarium graminearum as DON or NIV chemotypes. Primer sets developed to Tri3 were used to classify DON chemotypes further by the acetyl derivative produced (3-AcDON or 15-AcDON). Isolates were collected from 65 fields located around England and Wales between 1997 and 2002. All three chemotypes were identified from the F. graminearum population of England and Wales, with 15-AcDON chemotypes predominating overall. All isolates characterized as 3- or 15-AcDON chemotypes had nonfunctional versions of both genes. Where multiple isolates were collected from a field, mixed-chemotype populations were identified. Variation in the number of 11-bp repeats in Tri7 was detected among 3- and 15-AcDON chemotypes. Seventy-two of the 76 DON chemotypes (95%) were classified as 15-AcDON producers and the remaining four isolates (5%) as 3-AcDON producers. In all four isolates with a 3-AcDON chemotype, Tri7 was deleted from the trichothecene gene cluster. There was no evidence of regional variation between 3-AcDON, 15-AcDON or NIV chemotypes within the F. graminearum population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Traditionally, there have been four pathogens commonly associated with fusarium head blight (FHB) of wheat crops in England and Wales: Fusarium culmorum, F. poae, F. avenaceum and Microdochium nivale. Of these, F. culmorum has been regarded as potentially the most serious both in terms of yield loss and mycotoxin production (Parry et al., 1995; Jennings & Turner, 1996; Turner & Jennings, 1997). The occurrence of mycotoxins in grain is of particular importance due to their potentially harmful effects on humans and livestock. Isolation of Fusarium species from infected wheat ears, carried out between 1998 and 2003, has indicated that the proportion of F. graminearum in the Fusarium population has increased year on year (Turner et al., unpublished data). The proportion of F. graminearum was greater than that of F. culmorum for the first time in 2002 and continued to be higher in 2003. As F. graminearum is generally regarded to be a more damaging pathogen than F. culmorum, in terms of both yield loss and mycotoxin production, this may have significant implications for the future. Similar changes in the Fusarium population have also been reported in parts of Germany (Obst et al., 1997) and the Netherlands (Waalwijk et al., 2003). In both instances the switch in predominance was associated with changes in farming practices, in particular increased maize cultivation.

The principal toxins produced by F. graminearum are the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV), with NIV being reported as more toxic than DON (Ryu et al., 1988). Based on the production of different trichothecenes, Ichinoe et al. (1983) split Japanese isolates of F. graminearum into two chemotypes. The DON type produced DON and 3-acetyldeoxynivalenol (3-AcDON) while the NIV type produced NIV and 4-acetylnivalenol. Miller et al. (1991) further divided the DON chemotype depending on where it was acetylated, with 3-AcDON producers termed IA and 15-acetyldeoxynivalenol (15-AcDON) producers IB. Their work indicated that 15-AcDON producers were prevalent in the USA and 3-AcDON producers in Asia. This is probably a reflection of the lineage of the F. graminearum isolates as described by O'Donnell et al. (2000), where isolates from the USA were lineage 7 and those from Asia lineage 6. Several surveys of cereals grown in the UK have shown that both DON and NIV were present in harvested grain (Tanaka et al., 1986; Polley et al., 1991; Turner et al., 1999; Prickett et al., 2000). A recent report has shown that DON and NIV chemotypes are present in the UK population of F. culmorum (Jennings et al., 2004). Over recent years F. graminearum has become the predominant FHB pathogen isolated from the south-west of England and south Wales (Turner et al., unpublished data). These areas are where a high proportion of the F. culmorum NIV chemotype has been reported (Jennings et al., 2004). To date, only a very limited number of F. graminearum isolates from the UK have been chemotyped and all these proved to be of the DON chemotype (Carter et al., 2002). If only DON chemotypes of F. graminearum exist in the UK, as is the case in the USA (Mirocha et al., 1994; Abramson et al., 2001), and F. graminearum eventually replaces F. culmorum, as in Germany, it is possible that the levels of NIV currently found in UK wheat may reduce.

The usual method used for chemotyping Fusarium isolates is by HPLC or GC/MS analysis of extracts from cultures inoculated onto substrates such as wheat, maize or rice (Sugiura et al., 1990; Miller et al., 1991; Muthomi et al., 2000). Chemotyping by these methods can be difficult and time-consuming. Two genes, Tri7 and Tri13, have been found to be nonfunctional in all DON-producing isolates of F. graminearum studied to date (Brown et al., 2002; Lee et al., 2002; Chandler et al., 2003). Furthermore, the Tri7 gene and flanking sequence is deleted in 3-AcDON isolates (Chandler et al., 2003; Kimura et al., 2003). Lee et al. (2001) developed a method allowing F. graminearum isolates to be chemotyped using polymerase chain reaction (PCR) analysis based on size polymorphisms between functional and nonfunctional Tri7 genes in DON and NIV chemotypes of F. graminearum. Recently, other PCR assays have been developed to both the Tri7 and Tri13 genes to enable chemotype determination of F. graminearum, F. culmorum and F. cerealis (Chandler et al., 2003). Primers developed to the Tri3 gene (Jennings et al., 2004) have enabled the acetyl derivative of DON (3- or 15-AcDON) to be determined.

This study aimed to characterize UK isolates of F. graminearum, principally through PCR analysis of the Tri7 and Tri13 regions in order to determine the chemotypes present and establish whether any differences existed in chemotype distribution. The study also sought to establish whether, as is observed in the USA, only chemotype IB (15-AcDON) isolates were found among the DON producers.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation and identification

Wheat ears showing classic symptoms of fusarium head blight were collected from England and Wales between 1997 and 2002. In 1997, samples were taken from the random samples that arrived at the laboratory; however, from 1998 onwards, infected ears were collected from the annual survey of winter wheat diseases funded by Defra (Department for Environment Food and Rural Affairs) as described by Jennings et al. (2004). All Fusarium isolates were purified by single-spore isolation and identified to species level. A total of 101 isolates were identified as F. graminearum from 65 different fields around England and Wales. These isolates were used throughout this study.

DNA extraction

Isolates of F. graminearum were grown on potato dextrose agar (PDA) plates for 5–6 days. DNA was extracted using a method adapted from Chang et al. (1993).

F. graminearum species-specific PCR

Polymerase chain reaction analysis specific to F. graminearum was performed using primer pair Fg16F/R which produces polymorphic products with DNA from F. graminearum lineages, but no products with DNA from any other fungal species (Nicholson et al., 1998). The sequence of the Fg16F/R product is diagnostic of lineage and may be used simultaneously to detect F. graminearum and determine lineage (Carter et al., 2002; Nicholson et al., 2004). Amplification and PCR reactions were as described by Jennings et al. (2004). PCR products were separated by electrophoresis in 2% agarose gels and DNA visualized with 1 µg mL−1 ethidium bromide solution.

Determination of F. graminearum chemotype

PCR analysis

The chemotype of individual F. graminearum isolates was determined using the primer set GzTri7/f1 and GzTri7/r1 described by Lee et al. (2001). This primer set produced a PCR product ranging in size from 173 to 327 bp for a DON chemotype or a product 161 bp long for a NIV chemotype. The amplification reaction used was described by Jennings et al. (2004). The cycling protocol used was described by Lee et al. (2001) and consisted of denaturation at 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. PCR products were analysed as described above.

Where no PCR product was detected using the GzTri7/f1 and GzTri7/r1 primer set, and the isolate had been confirmed as F. graminearum, the primer set Minus Tri7F and Minus Tri7R was used (Chandler et al., 2003). This produced a PCR product 483 bp long and indicated a DON chemotype in which the entire Tri7 gene and flanking regions were deleted (Chandler et al., 2003). The amplification reaction used was as described by Jennings et al. (2004). The cycling protocol was 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and a final extension step of 72 °C for 10 min.

Two primer sets (Tri13F/Tri13DONR and Tri13NIVF/Tri13R) (Chandler et al., 2003) were used to characterize the Tri13 gene. Isolates produced a PCR product from either the primer set Tri13F/Tri13DONR (282 bp) or Tri13NIVF/Tri13R (312 bp) but not from both. The cycling protocol for all primer sets consisted of denaturation at 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. PCR products were analysed as described above.

In summary, isolates that produced a PCR product of 161 bp with GzTri7f1/r1 and a product with Tri13NIVF/Tri13R indicated a NIV producer. Isolates that produced a product of greater than 173 bp with GzTri7f1/r1 or with Minus Tri7F/Minus Tri7R, and a product with Tri13F/Tri13DONR indicated a DON producer.

Two primer sets [Tri303F/Tri303R (586 bp) and Tri315F/Tri315R (864 bp)] developed to the Tri3 gene (Jennings et al., 2004) were used to further characterize the DON chemotype of F. graminearum into either 3- or 15-AcDON. The amplification reaction and cycling protocol used were as described by Jennings et al. (2004). PCR products were analysed as described above.

Cloning and sequencing

To confirm the size of the PCR products using the primer set GzTri7/f1 and GzTri7/r1 PCR products of two F. graminearum strains, one DON (FC168) and one NIV (FC306) chemotype, were cloned in pGEM-T (Promega, Southampton, UK). Sequencing of the PCR products was carried out on plasmid minipreps (Wizard preps, Promega) using an ABI automated sequencer (Sequiserve, Vaterstetten, Germany) and the sequencing primers SP6 and T7. These strains were used as the DON/NIV controls for all analyses using GzTri7 f1/r1.

Toxin analysis

Five F. graminearum isolates (FC133, 168, 233, 253 and 306) were grown in rice culture (Evans et al., 2000) and mycotoxin extracted using a method adapted from that of Cooney et al. (2001). Rice culture (5 g) from each sample was placed in 40 mL of acetonitrile/methanol (14:1) for 12 h; 2 mL was taken for DON and NIV analysis and passed through a clean-up cartridge consisting of a 2 mL syringe (Fisher Ltd, Nepean, Ontario, Canada) packed with a filter-paper disc (No.1 Whatman International Ltd, Maidstone, UK), a 5 mL lug of glass wool and 300 mg of alumina/activated carbon (20:1). The sample was allowed to seep by gravity feed through the cartridge and residues in the cartridge were washed out with acetonitrile/methanol/water (80:5:15; 500 µL). The combined eluate was evaporated (compressed air, 50 °C) and then resuspended in methanol/water (5:95; 500 µL). Quantification of DON and NIV was by HPLC, using a luna C18 reverse phase column (100 mm × 4·6 mm i.d.) (Phenomenex, Macclesfield, UK). Separation was achieved using an isocratic mobile phase of methanol/water (12:88) at 1·5 mL min−1. Eluates were detected using an ultraviolet detector set at 220 nm with an attenuation of 0·01 absorption units full scale (AUFS). The retention times for NIV and DON were 3·4 and 7·5 min, respectively. External standards were used to quantify DON and NIV. The minimum level of quantification was 5 ng g−1 for DON and 2·5 ng g−1 for NIV.

Statistical analysis of chemotype distribution

The distribution of DON and NIV chemotypes was analysed by splitting England and Wales into regions of approximately equal area roughly based on county boundaries. Chi-squared analysis was then carried out to test the proportion of DON and NIV chemotypes in each area.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Species identification

A total of 101 isolates from 65 different field locations were identified as F. graminearum using traditional identification techniques. All were confirmed as F. graminearum by species-specific PCR analysis (Table 1). All F. graminearum isolates produced a PCR product 420 bp long, characteristic of lineage 7/RAPD group C isolates, as shown in Figs 1(a) and 4(a).

Table 1.  PCR products from Fusarium graminearum identification and chemotyping
Culture numberOther ref.aF. graminearum- specific PCRbF. graminearum chemotype
GzTri7 f1/r1cMinus Tri7F/RdTri13 primersTri3 primers
DONeNIVf3-AcDONg15-AcDONhNIVi
  • a

    The first two digits indicate the year of isolation. Cultures with the same code were isolated from the same field.

  • b

    +/− indicates the presence/absence of F. graminearum DNA using primer pair Fg16F/R.

  • c

    Primer pair GzTri7 f1/r1 yield PCR products of differing size dependent on the isolate chemotype: nivalenol (NIV) chemotypes with a 161-bp product were scored 1, deoxynivalenol (DON) chemotypes with three 11-bp repeats scored 3, four repeats scored 4 and five repeats scored 5. Where no PCR product was produced isolates were scored as −.

  • d

    Primer pair Minus Tri7F/R yield product for DON production where the Tri7 gene is deleted from the trichothecene gene cluster (scored as +).

  • e

    +/− indicates the presence/absence of PCR product from the primer pair Tri13F/Tri13DONR. The presence of a PCR product indicates an active Tri13 gene in a DON producer.

  • f

    +/− indicates the presence/absence of PCR product from the primer pair Tri13NIVF/Tri13R. The presence of a PCR product indicates an active Tri13 gene in a NIV producer.

  • g

    +/− indicates the presence/absence of PCR product from the primer pair Tri303F/Tri303R. The presence of a PCR product indicates a 3-acetyl DON producer.

  • h

    +/− indicates the presence/absence of PCR product from the primer pair Tri315F/Tri315R. The presence of a PCR product indicates a 15-acetyl DON producer.

  • i

    +/− indicates the presence/absence of PCR product from the primer pair Tri3NIVF/Tri3NIVR. The presence of a PCR product indicates a NIV producer.

FC1997rick+3 ++
FC2097harp+3 ++
FC11397corn+4 ++
FC12097420+3 ++
FC14598394+1 ++
FC14898386+3 ++
FC15798394+1 ++
FC15998394+1 ++
FC16898355+3 ++
FC16998355+3 ++
FC17898451+1 ++
FC17998442+1 ++
FC22698268+4 ++
FC23200410+3 ++
FC23300390+1 ++
FC23400167+3 ++
FC23500445+1 ++
FC23600400+3 ++
FC23700438+1 ++
FC23800452+3 ++
FC23900377+3 ++
FC25100232+3 ++
FC25200309+3 ++
FC25300058++++
FC25400144+1 ++
FC25500218+3 ++
FC25600308+3 ++
FC25700453+3 ++
FC26000217+4 ++
FC30401391+4 ++
FC30501433+3 ++
FC30601124+1 ++
FC30701376+3 ++
FC30801433+3 ++
FC31001143+3 ++
FC31101373+5 ++
FC33401084+3 ++
FC33601-2+3 ++
FC33801-5+3 ++
FC33901-rmd+3 ++
FC34001-rmd++++
FC34101-5+3 ++
FC34201-1+3 ++
FC34301-2+4 ++
FC34401-1+3 ++
FC34501-rmd+4 ++
FC34601-5+1 ++
FC34701-5-4-14-C+3 ++
FC34801-5-4-14-B+3 ++
FC34901-1-2-28-B+4 ++
FC35001-2+4 ++
FC39701113+3 ++
FC41502cull+4 ++
FC41602cull+3 ++
FC41902cull+3 ++
FC42002cull+3 ++
FC42302cull+3 ++
FC42602064++++
FC42702064++++
FC44002425+3 ++
FC44102425+3 ++
FC44602366+3 ++
FC44702366+3 ++
FC46002394+3 ++
FC46102407+3 ++
FC46202407+3 ++
FC46302216+1 ++
FC46402216+1 ++
FC46502137+3 ++
FC46602137+3 ++
FC46702414+3 ++
FC46802414+3 ++
FC46902360+3 ++
FC47002360+3 ++
FC47202281+1 ++
FC47502373+3 ++
FC47602285+3 ++
FC47702285+1 ++
FC48002287+1 ++
FC48102287+1 ++
FC48302395+3 ++
FC48402395+3 ++
FC48502327+3 ++
FC48602327+3 ++
FC49602373+3 ++
FC49802268+4 ++
FC49902268+3 ++
FC50202283+1 ++
FC50902258+1 ++
FC51002358+1 ++
FC51102375+1 ++
FC51702344+3 ++
FC51802344+3 ++
FC52302286+1 ++
FC52402286+1 ++
FC52902249+1 ++
FC53402371+3 ++
FC53502371+3 ++
FC54002389+1 ++
FC56202365+3 ++
FC56502987+4 ++
image

Figure 1. PCR products from five Fusarium graminearum isolates from the same field using polymorphic F. graminearum-specific primer set Fg16F/R (a) and primer set GzTri7 f1/r1 (b) to determine deoxynivalenol (DON)/nivalenol (NIV) chemotype.

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image

Figure 4. In-field variation (01-rmd) between deoxynivalenol (DON) chemotypes of Fusarium graminearum. (a) Polymorphic F. graminearum-specific primer set Fg16F/R; (b) primer set GzTri7 f1/r1; and (c) primer set Minus Tri7F/R. Lanes 1 and 7, 100-bp DNA ladder; lane 2, DON chemotype control (FC168); lane 3, nivalenol (NIV) chemotype control (FC306); lane 4, isolate FC339 containing three 11-bp repeats; lane 5, isolate FC340 in which Tri7 is deleted; lane 6, isolate FC345 containing four 11-bp repeats.

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Mapping the location of fields from which F. graminearum had been isolated showed that all but three of them were located south of a line from the Wash in the east to the north Pembrokeshire coast in the west (Fig. 2).

image

Figure 2. Regional distribution of Fusarium graminearum chemotype between 1997 and 2002. Fields containing deoxynivalenol (DON) chemotypes (inline image), nivalenol (NIV) chemotypes (○), mixed population of DON and NIV chemotypes (•), DON chemotype (Tri7 minus) (▴) and mixed population of DON chemotype (including Tri7 minus) (▵).

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F. graminearum chemotype

Rice cultures of five F. graminearum strains were analysed by HPLC to determine the type B trichothecenes produced. Three of the strains analysed were identified as DON chemotypes and two as NIV chemotypes (Table 2). Direct sequencing of the PCR products from two isolates, one DON chemotype and one NIV chemotype (determined using the primer set GzTri7 f1/r1), indicated a product size of 161 bp for the NIV chemotype and 194 bp for the DON chemotype. This indicated there were three 11-bp repeats in the DON isolate examined.

Table 2.  Chemotype of Fusarium graminearum strains indicated by trichothecene concentrations in rice culture and PCR analysis
F. graminearum isolateTrichothecene (µg g−1)F. graminearum chemotype using primer pair
DeoxynivalenolNivalenolGzTri7 f1/r1Minus Tri7F/R
  1. NP, no product detected; NA, not analysed.

Fc113 16 0DeoxynivalenolNA
Fc168206 0DeoxynivalenolNA
Fc233  0 8NivalenolNA
Fc253 26 0NPDeoxynivalenol
Fc306  012NivalenolNA

Of the 101 F. graminearum isolates analysed, the primer set GzTri7 f1/r1 identified 71% (72/101) as DON chemotypes and 25% (25/101) as NIV chemotypes (Table 1). Four isolates (FC253, FC340, FC426 and FC427) did not produce a PCR product with the GzTri7 f1/r1 primer set; however, they did produce PCR products with the Minus Tri7F/R primer set (Table 1), indicating that these strains were also DON chemotypes. Genetic variation within the F. graminearum DON population was shown through examination of the number of 11-bp repeats, using primer set GzTri7 f1/r1, and the acetyl derivative produced using the primer sets developed to Tri3. The majority of isolates (83%) contained three 11-bp repeats; however, four (15%) and five repeats (2%) were also present (Table 1 and Fig. 3). Analysis of Tri3 indicated that 95% (72/76) of the DON chemotypes produced 15-AcDON (Table 1). The remaining 5% of isolates produced 3-AcDON; these were the same isolates that produced a PCR product with the Minus Tri7F/R primer set. The results of PCR chemotyping using the Tri7 and Tri13 assays were always in agreement (Table 1). Chemotyping by PCR analysis of five F. graminearum strains was confirmed by HPLC analysis (Table 2).

image

Figure 3. Variation in the deoxynivalenol (DON) chemotype population of Fusarium graminearum in the UK. Lanes 1 and 16, 100-bp DNA ladder; lane 2, DON chemotype control (FC168); lane 3, nivalenol (NIV) chemotype control (FC306); lanes 4–9, DON chemotype containing three 11-bp repeats; lanes 10–14, DON chemotype with four repeats; lane 15, DON chemotype with five repeats.

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There were 25 fields from which more than one strain of F. graminearum was isolated. A mixed DON and NIV population (Fig. 1b) was identified in two of these fields (Fig. 2). One field (01-rmd) contained a mixed 3-/15-AcDON population, with two strains (15-AcDON) producing PCR products with GzTri7 f1/r1, indicating three (FC339) and four (FC345) 11-bp inserts, and one strain (FC340) producing a PCR product with Minus Tri7F/R indicative of a 3-AcDON producer (Fig. 4).

Geographical and seasonal variation in F. graminearum chemotype

Mapping field locations of F. graminearum chemotypes (Fig. 2) indicated some clustering of NIV chemotypes in the east and south-west of England, and Wales; however, no overall regional differences in 3-/15-AcDON and NIV chemotype were observed. Fields containing 3-AcDON chemotypes (in which the Tri7 gene is deleted) were found at the northernmost reaches of F. graminearum distribution (Fig. 2).

Little seasonal variation in the frequency of 3-/15-AcDON and NIV chemotypes was observed (Table 3), although a consistent increase in the proportion of NIV to 3-/15-AcDON chemotypes was seen in the east of England, culminating in NIV chemotypes outnumbering 3-/15AcDON by almost 3:1 in 2002. In contrast, while fields containing NIV chemotypes were more frequent than those with 3- or 15-AcDON in Wales in 1998 and 2000, the 15-AcDON chemotype was predominant in 2001 and 2002.

Table 3.  Seasonal variation in isolation of Fusarium graminearum chemotypes from wheat crops in England and Wales
Year of isolationNorthEastSouth-eastSouth-westMidlands and westWales
DONNIVDONNIVDONNIVDONNIVDONNIVDONNIV
  1. −, indicates no crop sampled.

  2. DON, deoxynivalenol; NIV, nivalenol.

19972020
1998001010110002
2000105100311013
2001003131202010
2002103830731030

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Using PCR analysis to chemotype isolates of F. graminearum from 65 separate field locations in England and Wales indicated that both DON and NIV chemotypes were present. Previously it has been reported that NIV chemotypes of F. culmorum exist in the UK (Jennings et al., 2004); however, this is the first report to show that the NIV chemotype of F. graminearum is also present. Overall, the number of fields containing DON chemotypes outnumbered those with the NIV chemotype. This is consistent with previous studies that have shown that in Europe and north and south America, the DON chemotypes of F. graminearum are more frequently isolated than the NIV chemotype (Miedaner et al., 2000; Waalwijk et al., 2003). The proportion of NIV chemotypes found in the F. graminearum (25%) population in the UK was lower than that in the F. culmorum (43%) population; however, levels were still relatively high. Given the high proportion of NIV chemotypes in the F. graminearum population in the UK, it is likely that NIV contamination of grain will occur even if the current trend continues and F. graminearum replaces F. culmorum as the predominant type B trichothecene producer in the UK (Turner et al., unpublished data).

With the exception of 1997, samples were taken from a fully stratified survey based on the acreage of wheat grown in a specified region, and thus should give a good representation of the current F. graminearum distribution in England and Wales. In general, occurrence of F. graminearum was limited to southern and eastern areas of England, and south Wales. No regional differences were seen in the distribution of F. graminearum 15AcDON and NIV chemotypes. This is in contrast to F. culmorum where NIV chemotypes mainly predominated in the south-west and 3-AcDON chemotypes in the east (Jennings et al., 2004).

Carter et al. (2002) characterized isolates of F. graminearum using RAPDs and the primer pair Fg16F/R, which produces a polymorphic PCR product (Nicholson et al., 1998). Isolates that produced a sequence characterized amplified region (SCAR) product 420 bp long were also placed in RAPD group C; isolates placed in this group were from north-west Europe and North America. Subsequent sequence analysis of Tri101 (Nicholson et al., unpublished data) has demonstrated that RAPD group C is congruent with lineage 7, as described by O'Donnell et al. (2000). In the current study, primer pair Fg16F/R was used to confirm the identity of F. graminearum isolates and showed that all 101 F. graminearum isolates tested produced a PCR product 420 bp long. Thus, all F. graminearum isolates used in the current study appear to be from lineage 7/RAPD group C. Fusarium graminearum isolates categorized as RAPD group C by Carter et al. (2002) and lineage 7 by O'Donnell et al. (2000) were predominately 3-/15AcDON chemotypes, but the results presented here indicate that the occurrence of NIV chemotypes within the RAPD C/lineage 7 grouping may be more frequent than the data previously reported suggest.

Furthermore, results here confirm those of Lee et al. (2001), which showed that primer set GzTri7 f1/r1 produced a PCR product 161 bp long with all NIV strains of F. graminearum. However, several isolates tested in this study did not produce a PCR product using this primer set. These isolates were subsequently shown to produce PCR products with the primer set Minus Tri7F/R. This primer set indicates DON chemotypes when the Tri7 gene has been deleted from the trichothecene gene cluster (Chandler et al., 2003). Deletion of Tri7 is the norm for isolates of F. culmorum (Chandler et al., 2003; Jennings et al., 2004); however, it is very rare for the deletion to be detected in lineage 7 isolates of F. graminearum. Deletion of Tri7 has previously been reported in only four European F. graminearum isolates (Chandler et al., 2003). Deletion of Tri7, however, is relatively common among lineage 6/RAPD group A isolates; for example it has been observed in a F. graminearum isolate from Japan (Kimura et al., 2001) and many isolates from China (Chandler et al., 2003; Nicholson, unpublished data). Those isolates in which the Tri7 gene was deleted were producers of 3-AcDON, similar to F. culmorum, whereas all other isolates were producers of 15-AcDON, similar to lineage 7/RAPD group C isolates from the USA. These results are in accordance with the findings of Kimura et al. (2003) who determined that deletion of Tri7 is associated with 3-AcDON chemotypes.

Analysis of individual field populations of F. graminearum indicated that mixed DON and NIV chemotype populations were present in some fields. Similar mixed-chemotype populations have previously been reported in F. graminearum (Miedaner et al., 2000) and F. culmorum (Jennings et al., 2004). Lee et al. (2001) found that using primer pair GzTri7 f1/r1, the size of the PCR product obtained from DON chemotypes of F. graminearum varied between 173 and 327 bp depending on the number of 11-bp inserted repeats in the Tri7 sequence. The isolates examined by Lee et al. (2001) were primarily from Asia and, as such, are probably of lineage 6/RAPD group A. This contrasts with the lineage/grouping determined for the F. graminearum isolates tested in this study, although variations in the number of 11-bp inserts were identified. Isolates mostly contained three 11-bp repeats; however, four and five repeats were also seen, but at much lower levels. Results from the Netherlands have shown that up to 10 11-bp repeats were detected in the F. graminearum population using the GzTri7 primer set (Waalwijk et al., 2003). No pattern in regional distribution for the different number of 11-bp inserts was detected. The variation in DON chemotype was particularly noteworthy in one field (01-rmd) where isolates producing PCR products indicating Tri7 minus, and three and four 11-bp repeats were all present. Such high levels of genetic variability in a single field are consistent with results from Schilling et al. (1997), who reported the genotypic diversity in one field population, measured by PCR-based molecular markers, to be 60% of that found within a worldwide F. graminearum collection.

Considering that F. graminearum, until recently, was only rarely isolated from wheat crops grown in the UK, a surprising degree of variability has been demonstrated within the population. Unless hybridization between F. graminearum and F. culmorum is very high, this suggests that F. graminearum was not introduced from a single source but from several sources containing a high level of diversity.

Historically the predominant type B trichothecene producer in the UK has been F. culmorum; this population comprises a high proportion of NIV chemotypes (Jennings et al., 2004). The recent increase in the prevalence of F. graminearum in the UK could potentially have resulted in reduced levels of NIV contamination in grain depending on the toxin profile of the F. graminearum population. However, the high proportion of the NIV chemotype found within the F. graminearum population, as reported here, combined with the lack of regional chemotype distribution associated with F. culmorum, implies that NIV contamination of grain in the UK may become more widespread in the future.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to thank Moray Taylor, Jackie Stonehouse and Jean Slough (CSL) and Duncan Simpson and Elizabeth Chandler (JIC) for their invaluable input into this study. We would also like to thank Professor Naresh Magan and Dra. Laura Ramirez at Cranfield University for their help with the toxin analysis. PN gratefully acknowledges the support of Defra to the study of facultative pathogens of cereals within the JIC. Chemotype primer development was supported by EU project DETOXFUNGI (project code QLK1-CT-1999-01380).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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