SEARCH

SEARCH BY CITATION

Keywords:

  • Fusarium seedling blight;
  • internal transcribed spacer;
  • Microdochium spp.;
  • quantitative PCR;
  • seed-borne disease

Abstract

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

Aims:  To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group.

Methods and Results:  Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed.

Conclusions:  DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment.

Significance and Impact of the Study:  Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.


Introduction

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

Fusarium seedling blight can result in the death of seedlings before or just after seedling emergence (Wiese 1987). Those plants that survive infections at the seedling stage are likely to exhibit symptoms of Fusarium foot rot later in the growing season, the severity of which is affected by environmental conditions (Colhoun 1970; Cook 1980). Although many species of Fusarium have been isolated from wheat, the major pathogens of this disease complex are considered to be Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum and Fusarium poae (Parry et al. 1995; Doohan et al. 2003). Studies have shown that F. culmorum and F. graminearum are the most pathogenic species, whereas F. poae and F. avenaceum are weakly pathogenic (Colhoun et al. 1968; Browne and Cooke 2005; Fernandez and Chen 2005). Fusarium seedling blight can also be caused by the Microdochium spp. pathogens Microdochium nivale (Fr.) Samuels and I. C. Hallett and Microdochium majus (Wollenw.) Glynn and S.G. Edwards, comb. nov. In the cooler regions of northern Europe, M. nivale and M. majus are often the predominant pathogens (Daamen et al. 1991) whereas in warmer regions, F. culmorum and F. graminearum are usually the dominant species (Parry et al. 1995). Seedling blight infections generally arise from seed-borne inoculum (Colhoun 1970). Soil-borne inoculum rarely causes seedling blight although soil-borne Fusarium can damage seedlings in warm, arid soils; M. nivale and M. majus which are less mobile in soil favour cool, dry soils (Millar and Colhoun 1969). Little data exist on the frequency of Fusarium spp. present in UK wheat seed, however, F. culmorum and F. graminearum represented 28% and 69% of 299 Fusarium isolates examined in Dutch wheat seed in 2000 and 2001, F. poae and F. avenaceum made up the remaining 3% of isolates (Waalwijk et al. 2003).

Fungicide seed treatment is the primary control measure for eradicating the seed-borne inoculum leading to Fusarium seedling blight. The performance of fungicides towards pathogens in disease complexes such as Fusarium seedling blight, foot rot and head blight are often difficult to measure. The interpretation of visual disease assessments in field trial experiments is complicated by the significant presence of endogenous disease causing fungi. Visual symptoms on emerged seedlings cannot be used to assess the performance of seed treatments towards individual species unless known species are present alone; this is difficult to achieve under field experiment conditions. Further, the early stages of infection by Fusarium disease-causing pathogens are often symptomless (Parry et al. 1994). Isolation of Fusarium disease-causing pathogens in axenic culture can provide an indication of the incidence of the fungal species involved. However, such isolations cannot quantify the degree of infection or the potential of the infective agent(s) to cause foot rot later in the season (Hare 1997), isolation techniques also favour those species which grow quickest under the culture conditions employed.

PCR can be used to identify pathogens at any taxonomic level. It is useful to detect or quantify the principal fungal taxa or group responsible for disease (Simpson et al. 2001) rather than the individual species which could be present. Competitive PCR allows the determination of the amount of pathogen DNA in infected samples. Relative fungal DNA concentration in plant material has been shown to be a good indicator of future seedling blight disease development (Glynn 2002). In addition, a positive correlation was found between the amount of Microdochium spp. DNA and the number of infected seeds (Cockerell et al. 2004). Competitive PCR has previously been used to study the epidemiology and control of several Fusarium disease complex pathogens of wheat (Doohan et al. 1999; Edwards et al. 2001; Nicholson et al. 2002).

The method traditionally employed to determine the severity to which individual seed batches or seedlings are infected is to determine the percentage of seeds or stems from which pathogen colonies emanate following surface sterilization and incubation on appropriate media. Although these methods can detect symptomless infection and identify the species present they are unable to determine the amount of pathogen. In the case of seedlings, visual symptoms can also be used to determine the severity of infection, however visual symptoms are unable to differentiate between pathogens.

In this study, we considered the pathogens responsible for Fusarium seedling blight of wheat as being from two groups, namely; Fusarium group (F. culmorum and F. graminearum) and Microdochium group (M. nivale and M. majus) as each group favours separate environmental conditions for disease and have been shown to have differing responses to fungicides (Pettitt et al. 1993; Simpson et al. 2001). Quantification of DNA from the two groups, allows the performance of seed treatments to be determined more efficiently. It is desirable to detect these pathogens in material containing a low amount of inoculum such as in infected seed and in the early stages of seedling infection. Beck (1998) designed a range of genus- and species-specific PCR primer pairs for various plant pathogenic genera and species based on sequences within the internal transcribed spacer (ITS) regions of ribosomal DNA. The numerous identical copies of ribosomal DNA genes increases the sensitivity of the PCR assay over single copy genes. These primers allowed the development of highly sensitive PCR assays specific to a number of important plant pathogenic species and genera which included primers for Fusarium (specifically F. culmorum and F. graminearum) and Microdochium (specifically M. nivale and M. majus) (Beck 1998). Quantitative PCR has the advantage over diagnostic PCR of allowing the amount of a particular pathogen present in infected plant material to be determined.

Aim

(i) To develop a sensitive, quantitative PCR assay to determine the amount of fungal DNA in plant material for the two groups of pathogens responsible for Fusarium seedling blight in wheat: (a) F. culmorum and F. graminearum and (b) M. nivale and M. majus; (ii) to assess the performance of fungicide seed treatments against each group using PCR assays.

Materials and methods

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

Origin of fungal isolates

Fungal strains used in this study and their sources are listed in Table 1.

Table 1.   Source of fungal isolates and reaction obtained using JBF and JBM primers
IsolateSpeciesOriginPCR product*
JBF primersJBM primers
  1. Isolates in bold were used in the quantitative PCR validation.

  2. *+, a positive PCR product using PCR primer combination JBF or JBM; −, no PCR product was present.

  3. †Isolates were from the National Institute of Agrobiological Resources (Kannondai, Japan) and others were from the Harper Adams University College (Shropshire, UK).

95wFusarium culmorumEngland+
017F. culmorumEngland+
302F. culmorumEngland+
421/5F. culmorumFrance+
421/14F. culmorumFrance+
405/11F. culmorumGermany+
NFTPFusarium graminearumEngland+
113F. graminearumEngland+
145F. graminearumEngland+
86F. graminearumFrance+
507F. graminearumFrance+
405/1F. graminearumGermany+
74/1/NMicrodochium nivaleEngland+
2aM. nivaleUK+
SWG052/1/NM. nivaleGermany+
NZ013/5/NM. nivaleNew Zealand+
JP101046dM. nivaleJapan+
NL005/1/NM. nivalethe Netherlands+
24/3/MMicrodochium majusEngland+
NL007/1/MM. majusthe Netherlands+
F060/1/MM. majusFrance+
NZ25/GVR/MM. majusNew Zealand+
JP236880†M. majusJapan+
47bM. majusHolland+

Source of seeds and seedlings

Standard crop husbandry practices were used to maintain 12 plots (10 × 2 m) of wheat cv. Hussar (here on referred to as experiment 1) and cv. Equinox (here on referred to as experiment 2). Four plots were inoculated at mid-anthesis (growth stage 65) (GS 65) (Zadoks et al. 1974) with a conidial suspension from five isolates (2 × 105 spores per ml) of a seedling blight pathogen at a rate of 33 ml m−2 using a knapsack sprayer. In experiment 1, plots were inoculated with F. culmorum, F. graminearum or M. majus; in experiment 2, plots were inoculated with F. culmorum, M. majus or M. nivale. Plots were mist irrigated for 21 days as previously described Hilton et al. 1999) to aid infection. Four uninoculated, non-misted plots were used as guards between each set of inoculated misted plots. Grain was harvested at GS 92 using a Seedmaster Plot combine (Wintersteiger, Austria).

Four seed lots in each experiment 1–4 (experiment 1), 5–8 (experiment 2) were used in fungicide seed treatment efficacy experiments. Seed lots 1 and 5 were commercial seed, seed lots 2 and 6 were from field plots inoculated with F. culmorum, seed lot 3 F. graminearum, seed lots 4 and 7 M. majus and seed lot 8 M. nivale. Seed was treated with either Beret Gold (Syngenta) (a.i. fludioxonil: 24·3 g l−1) or Sibutol (Bayer) (a.i. bitertanol + fuberidazole: 375 + 23 g l−1) at the label recommended rate, untreated seed was used as a control. In both experiments 1 and 2, seed was drilled according to a randomized block design with four replicates at Edgmond (Shropshire, UK). At GS 13, 30 seedlings were removed from each plot, the roots, remaining seed coat and any soil debris were removed and the seedlings were cut to 4 cm in length from the base of the stem, washed, placed in plastic kartell tubes (Fisher, Loughborough, UK) and freeze dried prior to DNA extraction.

DNA extraction

The fungi were grown in 150 ml potato dextrose broth inoculated with mycelial fragments from potato dextrose agar cultures. Cultures were incubated in an orbital incubator at 28°C, 100 rev min−1 for 7–11 days. Fungal mycelia were pelleted by centrifugation and ground in liquid nitrogen. Total genomic DNA was extracted from the ground mycelia using the protocol of Lee and Taylor (1990). DNA for fungal standards used in quantitative PCR reactions was extracted from 7-day-old cultures and diluted to 1 ng μl−1 in Tris–EDTA (TE) buffer according to the methods described previously (Glynn et al. 2005).

DNA extracted from seed was performed as described in Edwards et al. (2001). Extraction of DNA from seedlings was performed using a modified method of Edwards et al. (2001) described for seed material. Seedlings were freeze dried and then milled to a fine powder by adding three sterile 8-mm diameter steel ball bearings and shaken for 1 h using a soil mill (Griffin, London, UK). Plant material was transferred to a sterile 50-ml centrifuge tube and 10 ml of cetyl trimethyl ammonium bromide extraction buffer was added. Tubes were incubated at 65°C for 1 h, cooled to 20°C and 3·3 ml of potassium acetate (5 mol l−1) was added. Total DNA was purified, quantified and diluted to 40 ng μl−1.

Development of quantitative PCR assays for Fusarium and Microdochium seedling blight pathogen DNA

Oligonucleotide primers designed on regions of rDNA for Fusarium seedling blight pathogens; F. culmorum and F. graminearum (JB566, 5′-GTTTTTAGTGGAACTTCTGAGT-3′ and JB572, 5′-AAGTTGGGGTTTAACGGC-3′ here on referred to as JBF) and Microdochium seedling blight pathogens; M. nivale and M. majus (JB612, 5′-GGTGCTGTCTCTCGGGAC-3′ and ITS4, 5′-TCCTCCGCTTATTGATATGC-3′ here on referred to as JBM) as described previously (Beck 1998) were used. DNA from all isolates detailed in Table 1 was amplified with the JBM and JBF assays. Diagnostic PCR amplifications were carried out using JBM or JBF primers in total reaction volumes of 25 μl containing 100 μmol l−1 of each nucleotide, 100 nmol l−1 of each forward and reverse primer, 20 units ml−1 of Super Taq polymerase (Kramel Biotech, Cramlington, UK), 10 mmol l−1 Tris–HCl, 1·5 mmol l−1 MgCl2, 50 mmol l−1 KCl, 0·1 mg ml−1 of gelatin (Sigma), 0·5 mg ml−1 of Tween 20 (Sigma) and 0·5 mg ml−1 of Nonidet P-40 (Sigma) and 5 μl of sample DNA. Positive controls contained either 5 μl of M. nivale or F. culmorum DNA (1 ng μl−1) instead of sample DNA and negative controls contained 5 μl of water. Thermocycling consisted of an initial denaturation step of 95°C for 75 s followed by 35 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 45 s and a final extension step of 72°C for 4 min and 15 s. Electrophoresis was performed using 2% agarose gels containing 0·5 μg of ethidium bromide per ml in TAE buffer (40 mmol l−1 Tris-acetate, 1 mmol l−1 EDTA, pH 8·0), amplified products were observed under UV light.

Internal standards (JBMIS and JBFIS) were constructed for competitive PCR assays based on the method of Edwards et al. (2001) from a 1·2-kb fragment of the onion (Allium cepa) gene alliinase (EMBL accession code L48614). The fragment was used as a template and amplified using primers ONI/F (5′-TGCTCTGCTGATGTTGCCAG-3′) and ONI/R (5′-TACATGGGGATGGAGGTCTC-3′). Reaction conditions, thermocycling and electrophoresis were as described above with the exception that an annealing temperature of 58°C was used. The amplicon was excised from the gel following electrophoresis, placed in 1 ml of TE buffer and incubated at 4°C for 16 h. A 5-μl aliquot served as template DNA in a PCR reaction with the linker primers (Microdochium: MNIV/FL (5′-CTCTCGGGACGTTGCTCATGCCCC-3′), NUC4/RL (5′-ATTGATATGCTCTCGGGAAGTGCC-3′) or Fusarium: JBF3/FL (5′-ACTTCTGAGTAGGAAATGCAGCGG-3′), JBF4/RL (5′-GTTTAACGGCTGAGGTCGCGCATG-3′). The concentrations of reaction ingredients were as described above, the thermocycling programme used consisted of 10 cycles with an annealing temperature of 38°C followed by 20 cycles with an annealing temperature of 55°C. Amplified fragments (628 bp Microdochium and 534 bp Fusarium) here on referred to as JBM and JBF internal standard DNA (JBMIS and JBFIS) were cloned using the pGEM®-T vector system (Promega, Southampton, UK) according to the standard protocol suggested by the manufacturer.

Quantitative PCR

Internal standard DNA (JBMIS and JBFIS) and DNA from M. nivale and F. culmorum were diluted and amplified together with JBM or JBF primers to determine the concentration giving the greatest sensitivity and dynamic range. Stock internal standard DNA (2·9 fg μl−1 JBMIS; 38·3 fg μl−1 JBFIS) was prepared in TE buffer in the presence of 10 ng μl−1 carrier (herring sperm) DNA to improve stability during storage at −20°C. A standard curve was generated for each primer pair, M. nivale DNA was diluted two-fold over the range 0·003–1·6 pg μl−1 for use with JBM primers. Fusarium culmorum DNA was diluted twofold over the range of 0·0008–6·4 pg μl−1 for use with JBF primers. Ten microlitres of each dilution was amplified in the presence of 10 μl of the respective internal standard in a final volume of 50 μl. PCR reaction conditions were as described previously for the diagnostic PCR protocol. To quantify the amount of Microdochium or Fusarium DNA in total DNA extracted from seed and seedlings, 50 μl of competitive PCR reactions were setup each containing 10 μl of sample DNA and 10 μl of JBMIS or JBMIS respectively. The concentrations of reaction ingredients and thermocycling conditions were as described above.

Gels were viewed under UV light on a Gel Doc 1000 fluorescent gel documentation system (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) following electrophoresis. Unsaturated images were analysed using Molecular Analyst software (Bio-Rad). PCR product ratios were determined for each standard and sample by dividing the band intensity of the JBM or JBF product (472 or 346 bp) by that of the JBMIS or JBFIS product (628 or 534 bp). Quantification of amplified fungal DNA was determined by comparison of sample ratio to the respective standard curve and was expressed as concentration of fungal DNA as a proportion of total DNA.

Quantitative PCR validation

To validate that each species was amplified with equal efficiency within the quantitative PCR, DNA from three isolates of F. culmorum, F. graminearum, M. nivale and M. majus (detailed in Table 1) was extracted and diluted to 1 pg μl−1 and quantified using the corresponding assays detailed above. Isolates were amplified in quadruplicate to provide adequate degrees of freedom for analysis.

Statistical analysis

Results were analysed by anova using Genstat 5·4.1 (Lawes Agricultural Trust, Harpenden, UK). Where necessary, data were transformed to obtain normal distributions. Individual treatments were compared using the least significant difference at the 5% significance level. All statistical significance quoted is at the 5% level unless stated otherwise.

Results

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

Diagnostic and quantitative PCR validation

All isolates tested amplified with the correct primer pair and no misamplification was detected (Table 1). anova of the DNA concentration of the three isolates of M. nivale and M. majus indicated that there was no difference in the amplification of the two species using the JBM assay (P = 0·80). The average concentration of the 1 pg μl−1 stocks as determined by the JBM PCR assay was 1·26 pg μl−1. anova of the DNA concentration of the three isolates of F. culmorum and F. graminearum indicated that there was no difference in the amplification of the two species using the JBF assay (P = 0·86). The average concentration of the 1 pg μl−1 stocks as determined by the JBF PCR assay was 1·06 pg μl−1.

Quantification of pathogens in seed

Pathogen DNA from each Fusarium seedling blight group was detected and quantified in all eight seed lots. The commercial seed lots in both experiments (lots 1 and 5) had the lowest amount of Microdochium group DNA (Fig. 1). In experiment 1, the commercial seed lot (lot 1) had the lowest amount of Fusarium group DNA and in experiment 2, it contained the second lowest amount of Fusarium group pathogen (lot 5). Seed produced from field plots inoculated with either a Microdochium or a Fusarium species contained high amounts of Microdochium or Fusarium group DNA, respectively, with the exception of the F. graminearum inoculated plots. The amount of Microdochium DNA was significantly different between all experiment 1 seed lots, ranging from 0·19 pg ng−1 total DNA in seed lot 1 to 6·1 pg ng−1 total DNA in seed lot 4 (Fig. 1a). With the exception of seed lots 7 (5·9 pg ng−1 total DNA) and 8 (6·8 pg ng−1 total DNA) which contained the most, the amount of Microdochium DNA in each experiment 2 seed lot was significantly different (Fig. 1b). Quantification of Fusarium DNA showed significant differences between all seed lots in experiment 1, seed lot 2 contained the most (17 pg ng−1 total DNA) followed by seed lots 3, 4 and 1 (Fig. 1c). Seed lot 6 contained the most Fusarium DNA in experiment 2, significantly more than seed lot 8. No significant difference was observed in the amount of Fusarium group DNA in seed lots 5 and 7 although they did contain significantly less Fusarium DNA than seed lot 8 (Fig. 1d).

image

Figure 1.  Quantification of Microdochium seedling blight pathogen DNA in seed lots for: (a) experiment 1 (cv. Hussar) seed and (b) experiment 2 (cv. Equinox) seed; Fusarium seedling blight pathogen DNA in seed lots for: (c) experiment 1 seed and (d) experiment 2 seed. Key to seed lots: 1 and 5, commercial seed; 2 and 6, from field plots inoculated with Fusarium culmorum; lot 3, Fusarium graminearum; lots 4 and 7, Microdochium majus; lot 8, Microdochium nivale. Bars indicate standard errors of the mean values.

Download figure to PowerPoint

Quantification of Microdochium seedling blight pathogen DNA in seedlings

Microdochium seedling blight pathogen DNA was detected in all seedling samples in both experiments (Fig. 2a,b). Seedlings produced from commercial seed (lots 1 and 5) contained significantly less fungal DNA than seedlings from untreated seed from the three infected seed lots in each experiment. In experiment 1, no significant difference was observed between Microdochium DNA in seedlings produced from untreated seed from seed lots 3 and 4 although this was significantly more than from seed lot 2. In experiment 2, no significant difference was observed in the amount of Microdochium DNA detected in seedlings from untreated seed from lots 6 and 7 and, this was less than that from untreated seed lot 8. In experiment 1, the quantity of Microdochium DNA was reduced significantly using bitertanol + fuberidazole for seedlings from seed lots 2, 3 and 4 (98%, 70% and 92% reduction respectively) although not for seed lot 1 which had the lowest amount of Microdochium infection (19% reduction). Treatment with fludioxonil resulted in significant reductions in Microdochium DNA in seedlings from seed lots 2, 3 and 4 (99·5%, 87% and 93% reduction respectively) although not for seed lot 1 (48% reduction). In experiment 2, the quantity of Microdochium DNA was reduced significantly in seedlings from all seed lots treated with either bitertanol + fuberidazole (seed lot 5: 99·6% reduction, seed lot 6: 84·5% reduction, seed lot 7: 69·7% reduction and seed lot 8: 93% reduction) or fludioxonil (seed lot 5: 95% reduction, seed lot 6: 99·9% reduction, seed lot 7: 99·5% reduction and seed lot 8: 99·9% reduction). Fludioxonil showed a significant reduction in Microdochium DNA compared with bitertanol + fuberidazole in seedlings produced from seed lot 7.

image

Figure 2.  Quantification of Microdochium seedling blight pathogen DNA in experiment 1 (cv. Hussar) (a) and experiment 2 (cv. Equinox) (b), and Fusarium seedling blight pathogen DNA in experiment 2 (cv. Equinox) (c) in seedlings from four seed lots treated with (bsl00001) fludioxonil (48·6 g ai/100 kg seed), (bsl00004) bitertanol + fuberidazole (375 g ai + 23 g ai/100 kg seed) and (□) untreated control. Key to seed lots: 1 and 5, commercial seed; 2 and 6, from field plots inoculated with Fusarium culmorum; lot 3, Fusarium graminearum; lots 4 and 7, Microdochium majus; lot 8, Microdochium nivale. Bars indicate standard errors of the mean values.

Download figure to PowerPoint

Quantification of Fusarium seedling blight pathogen DNA in seedlings

In experiment 1, Fusarium seedling blight pathogen DNA was only detected in seedlings produced from untreated seed lot 2 (34·0 pg ng−1). In experiment 2, Fusarium DNA was detected in all samples (Fig. 2c). No significant difference was observed in the amount of Fusarium DNA detected in untreated seedlings from seed lots 6 and 8 and these samples contained significantly more Fusarium DNA than seedlings from untreated seed lots 5 and 7. Fusarium DNA was reduced significantly for all four seed lots using bitertanol + fuberidazole (seed lot 5: 94% reduction, seed lot 6: 99·7% reduction, seed lot 7: 64% reduction and seed lot 8: 89% reduction) or fludioxonil (seed lot 5: 85% reduction, seed lot 6: 99·9% reduction, seed lot 7: 91% reduction and seed lot 8: 94% reduction). No significant difference was observed between seed treatments.

Discussion

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

Numerous molecular systematic studies have utilized the coding and non-coding sequence of rDNA to distinguish fungal species. Areas of rDNA that were highly conserved between many species of filamentous fungi have been reported (White et al. 1990) and primers designed in these regions have proved useful for taxonomic studies within Fusarium (Bateman et al. 1996; Waalwijk et al. 1996). In the present study, primers based on rDNA were used to separately detect the two groups of Fusarium disease-causing pathogens in wheat. Ribosomal genes represent good targets for PCR assay development due to their high copy number. Previous quantitative PCR assays for the individual Fusarium disease-causing pathogens were developed using primers and or probes derived from RAPD fragments (Nicholson et al. 1996, 1998; Parry and Nicholson 1996; Schilling et al. 1996; Waalwijk et al. 2004) or low copy number functional genes (Glynn et al. 2005). The quantitative PCR assays described in this study had limits of detection of 0·0008 pg μl−1 (JBF) and 0·003 pg μl−1 (JBM). This represents between a five- and 50-fold greater sensitivity than previous competitive PCR assays for the individual species; (0·02 pg μl−1) F. culmorum and F. graminearum (Nicholson et al. 1998) and (0·02 pg μl−1) M. nivale (Nicholson et al. 1996) and (0·2 pg μl−1) M. majus (Nicholson et al. 1996). Greater sensitivity is advantageous as it allows detection of low amounts of pathogen DNA in planta during initial stages of colonization. The greater sensitivity we observed likely stems from the high copy number of the target rDNA genes in fungal genomes in contrast to the likely low copy number of the RAPD fragments. The validation of the quantitative assays using three isolates of each species showed that there was no difference in the concentration of any species as determined by either assay.

The development and control of seedling blight of spring wheat caused by F. graminearumsensu stricto (syn. F. graminearum lineage 7) has been described previously (Jones 1999). As far as we know, no data are available on the epidemiology of Fusarium seedling blight of wheat caused by other lineages within the F. graminearum clade. Ear infections by separate species have been reported as optimal at 28–29°C for F. graminearum, 26·5°C for F. culmorum and 18°C for Microdochium sp. (Rossi et al. 2001). The mean maximum daily temperature during the mist irrigation process in our experiment was 18·2°C (minimum 13·2°C, maximum 24·4°C), which may explain the limited infection by F. graminearum and the higher amount of Microdochium quantified in seed.

In both experiments, those seed lots that contained the most Microdochium or Fusarium group DNA produced seedlings with the most Microdochium or Fusarium group DNA indicating effective transmission from seed to seedlings. Infection by the Microdochium seedling blight pathogens was more severe in untreated seedlings in experiment 2 than in experiment 1. For Fusarium seedling blight pathogens, however, infection in untreated seedlings was more severe in experiment 1 than experiment 2 despite more seed-borne Fusarium DNA in the experiment 2 seed. This could be a result of separate environmental conditions that favoured infection by each group of pathogens (Millar and Colhoun 1969; Parry et al. 1994). The mean daily soil temperature between drilling and sampling for experiment 1 was 12·2°C (minimum 8·0°C maximum 14·6°C) whereas for experiment 2 the mean soil temperature was 9·5°C (minimum 6·5°C maximum 11·7°C) between drilling and sampling. The lower soil temperature in experiment 2 may account for the higher level of infection by Microdochium compared with Fusarium pathogens. A further explanation may be that when severe disease caused by Microdochium seedling blight pathogens occur, infected seedlings may be more susceptible to infection by seed-borne Fusarium. Millar and Colhoun (1969) stated that when applied to the seed surface, high spore loads of F. culmorum could act as a substitute for unfavourable environmental conditions in determining seedling disease. Under favourable environmental conditions, however, Microdochium seedling blight pathogens can cause severe infection even if the number of spores per seed was extremely low (Colhoun 1970). The amount of Fusarium detected in the eight seed lots was much greater than Microdochium spp., in general, more Microdochium spp. than Fusarium was detected in infected seedlings except when Fusarium was present in the seed at the highest level. The conclusions of the earlier workers, based on the number of spores artificially applied to the surface of wheat seeds, may be corroborated by results from this study where the amount of pathogen DNA contained within a sample of seed has been quantified.

Beret Gold (a.i. fludioxonil) and Sibutol (a.i. bitertanol + fuberidazole) were chosen in this investigation as in vitro, they are the most active fungicide seed treatments against isolates of M. nivale and M. majus (Glynn 2002). We found that fludioxonil alone showed better performance than bitertanol + fuberidazole towards Microdochium. This was significantly better when the pressure from infection was particularly high in experiment 2. The level of control for the F. culmorum-infected seed was high (>90%) for both fungicides. No significant benefit was apparent in our experiments for the commercial seed with low infection. This is not unexpected as working with treated and untreated seeds with low Microdochium infection, Paveley and Davies (1994) found no benefit from seed treatments when seed was sown at a number of field sites in the UK.

There are conflicting reports on the severity of infection of seedlings surviving pre- or post-emergence seedling blight death. Humphreys et al. (1995) reported that in field studies and in trays of compost, seedlings that survived pre-emergence mortality caused by Microdochium or Fusarium spp. were largely free from infection following establishment. Hare (1997) however, reported that all seedlings surviving pre-emergence death caused by seed-borne Microdochium spp. in experiments performed at 6°C exhibited stem-base disease symptoms. Severe symptoms were also reported on seedlings that survived pre- and post-emergence death caused by F. graminearum (Kane and Smiley 1987). In our experiments, treatments with the lowest emergence scores (data not shown) had higher pathogen DNA contents and are consistent with the findings of Hare (1997) and Kane and Smiley (1987). The contradiction to the findings of Humphreys et al. (1995) may be a result of the detection of both symptom-causing and symptom-less pathogen infections in our study. The seed treatments used in our experiments were effective in reducing pathogen infection, as measured by reduction in fungal DNA, during the early stages of plant development. Further experiments that relate the amount of pathogen DNA at the seedling stage to the amount present later in crop development and at harvest are warranted in order to determine the overall effectiveness of the treatments used.

The competitive PCR assays for seedling blight pathogens in the genera Microdochium and Fusarium described here present significant advantages over traditional methods as they are able to detect symptomless pathogen infection and assess the quantity of fungi present. The assays we describe are also more sensitive than previously described PCR-based methods of quantification and will prove useful for future studies on the epidemiology and control of Fusarium complex diseases of small grain cereals.

Acknowledgements

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

NCG and SGE acknowledge Syngenta Crop Protection for funding and Syngenta Biotechnology, Inc. for technical support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Bateman, G.L., Kwasna, H. and Ward, E. (1996) Relationships among Fusarium spp estimated by comparing restriction fragment length polymorphisms in polymerase chain reaction-amplified nuclear rDNA. Can J Microbiol 42, 12321240.
  • Beck, J.J. (1998) Detection of Fungal Pathogens Using the Polymerase Chain Reaction. US Patent 5 814 453.
  • Browne, R.A. and Cooke, B.M. (2005) Resistance of wheat to Fusarium spp. in an in vitro seed germination assay and preliminary investigations into the relationship to Fusarium head blight resistance. Euphytica 141, 2332.
  • Cockerell, V., Kenyon, D.M., Mulholland, V., Bates, J.A., McNeil, M., Law, J.R., Handy, C.L., Roberts, A.M.I. et al. (2004) Cereal Seed Health and Seed Treatment Strategies: Exploiting New Seed Testing Technology to Optimise Seed Health Decisions for Wheat. Home Grown Cereals Authority Project Report No. 340.
  • Colhoun, J. (1970) Epidemiology of seed borne Fusarium diseases of cereals. Ann Acad Sci Fenn Ser 168, 3136.
  • Colhoun, J., Taylor, G.S. and Tomlinson, R. (1968) Fusarium diseases of cereals II. Infection of seedlings by Fusarium culmorum and Fusarium avenaceum in relation to environmental factors. Trans Br Mycol Soc 51, 397404.
  • Cook, R.J. (1980) Fusarium foot rot of wheat and its control in the Pacific Northwest. Plant Dis 64, 10611066.
  • Daamen, R.A., Langerak, C.J. and Stol, W. (1991) Surveys of cereal diseases and pests in the Netherlands III. Monographella nivalis and Fusarium spp. in winter wheat fields and seed lots. Neth J Plant Pathol 97, 105114.
  • Doohan, F.M., Parry, D.W. and Nicholson, P. (1999) Fusarium ear blight of wheat: the use of quantitative PCR and visual disease assessment in studies of disease control. Plant Pathol 48, 209217.
  • Doohan, F.M., Brennan, J. and Cooke, B.M. (2003) Influence of climatic factors on Fusarium species pathogenic to cereals. Eur J Plant Pathol 109, 755768.
  • Edwards, S.G., Pirgozliev, S.R., Hare, M.C. and Jenkinson, P. (2001) Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine the efficacy of fungicides against Fusarium head blight of winter wheat. Appl Environ Microbiol 67, 15751580.
  • Fernandez, M.R. and Chen, Y. (2005) Pathogenicity of Fusarium species on different plant parts of spring wheat. Plant Dis 89, 164169.
  • Glynn, N.C. (2002) Studies on the epidemiology and chemical control of Fusarium seedling blight of wheat using molecular techniques. PhD Thesis, Harper Adams University College, Shropshire, UK.
  • Glynn, N.C., Hare, M.C., Parry, D.W. and Edwards, S.G. (2005) Phylogenetic analysis of EF-1 alpha gene sequences from isolates of Microdochium nivale leads to elevation of varieties majus and nivale to species status. Mycol Res 109, 872880.
  • Hare, M.C. (1997) Epidemiology and control of Fusarium seedling blight of winter wheat (Triticum aestivum L.). PhD Thesis, Harper Adams University College, Shropshire, UK.
  • Hilton, A.J., Jenkinson, P., Hollins, T.W. and Parry, D.W. (1999) Relationship between cultivar height and severity of Fusarium ear blight in wheat. Plant Pathol 48, 202208.
  • Humphreys, J., Cooke, B.M. and Storey, T. (1995) Effects of seed-borne Microdochium nivale on establishment and grain yield of winter-sown wheat. Plant Var Seeds 8, 107117.
  • Jones, R.K. (1999) Seedling blight development and control in spring wheat damaged by Fusarium graminearum Group 2. Plant Dis 83, 10131018.
  • Kane, R.T. and Smiley, R.W. (1987) Relative pathogenicity of selected Fusarium species and Microdochium bolleyi to winter wheat in New York. Plant Dis 71, 177181.
  • Lee, S.B. and Taylor, J.W. (1990) Isolation of DNA from fungal mycelia and single spores. In PCR Protocols: A Guide to Methods and Applications ed. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. pp. 282287. San Diego, CA: Academic Press.
  • Millar, C.S. and Colhoun, J. (1969) Fusarium diseases of cereals VI. Epidemiology of Fusarium nivale on wheat. Trans Br Mycol Soc 52, 195204.
  • Nicholson, P., Lees, A.K., Maurin, N., Parry, D.W. and Rezanoor, H.N. (1996) Development of a PCR assay to identify and quantify Microdochium nivale var. nivale and Microdochium nivale var. majus in wheat. Physiol Mol Plant Pathol 48, 257271.
  • Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W. and Joyce, D. (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol 53, 1737.
  • Nicholson, P., Turner, A.S., Edwards, S.G., Bateman, G.L., Morgan, L.W., Parry, D.W., Marshall, J. and Nuttall, M. (2002) Development of stem-base pathogens on different cultivars of winter wheat determined by quantitative PCR. Eur J Plant Pathol 108, 163177.
  • Parry, D.W. and Nicholson, P. (1996) Development of a PCR assay to detect F. poae in wheat. Plant Pathol 45, 383391.
  • Parry, D.W., Pettitt, T.R., Jenkinson, P. and Lees, A.K. (1994) The cereal Fusarium complex. In Ecology of Plant Pathogens ed. Blakeman, P. and Williamson, B. pp. 301320. Wallingford: CAB International.
  • Parry, D.W., Jenkinson, P. and McLeod, L. (1995) Fusarium ear blight in small grain cereals – a review. Plant Pathol 44, 207238.
  • Paveley, N.D. and Davies, J.M.L. (1994) Cereal seed treatment – risks, costs and benefits. In Seed Treatment: Progress and Prospects, British Crop Protection Council Monograph No. 57 ed. Martin, T. pp. 2735. Farnham: British Crop Protection Council Publication.
  • Pettitt, T.R., Parry, D.W. and Polley, R.W. (1993) Improved estimation of the incidence of Microdochium nivale in winter-wheat stems in England and Wales, during 1992 by use of benomyl agar. Mycol Res 97, 11721174.
  • Rossi, V., Ravanetti, A., Pattori, E. and Giosue, S. (2001) Influence of temperature and humidity on the infection of wheat spikes by some fungi causing Fusarium head blight. J Plant Pathol 83, 189198.
  • Schilling, A.G., Moller, E.M. and Geiger, H.H. (1996) Polymerase chain reaction-based assays for species-specific detection of Fusarium culmorum, F. graminearum, and F. avenaceum. Phytopathology 36, 515522.
  • Simpson, D.R., Weston, G.E., Turner, J.A., Jennings, P. and Nicholson, P. (2001) Differential control of head blight pathogens of wheat by fungicides and consequences for mycotoxin contamination of grain. Eur J Plant Pathol 107, 421431.
  • Waalwijk, C., De Koning, J.R.A., Baayen, R.P. and Gams, W. (1996) Discordant groupings of Fusarium spp. from sections Elegans, Liseola and Dlaminia based on ribosomal ITS1 and ITS2 sequences. Mycologia 88, 361368.
  • Waalwijk, C., Kastelein, P., Vries, I., Kerenyi, Z., Lee, T., Hesselink, T., Kohl, J. and Kema, G. (2003) Major changes in Fusarium spp. in wheat in the Netherlands. Eur J Plant Pathol 109, 743754.
  • Waalwijk, C., Van De Heide, R., De Vries, I., Van Der Lee, T., Schoen, C., Corainville, G.C-d., Häuser-Hahn, I., Kastelein, P. et al. (2004) Quantitative detection of Fusarium species in wheat using TaqMan. Eur J Plant Pathol 110, 481494.
  • White, T.J., Bruns, S., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications ed. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. pp. 315322. San Diego, CA: Academic Press.
  • Wiese, M.V. (1987) Compendium of Wheat Diseases. St Paul, MN: American Phytopathological Society.
  • Zadoks, J.C., Chang, T.T. and Konzak, C.F. (1974) A decimal code for the growth stages of cereals. Weed Res 14, 415421.