• Gibberella zeae;
  • head blight ;
  • maize;
  • rice;
  • wheat


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

A collection of group II Fusarium graminearum isolates obtained from maize, wheat and rice from different locations in Nepal were identified using a combination of morphological and molecular criteria. The variation within this collection was analysed using RAPD markers, intergenic spacer (IGS) RFLP and PCR polymorphisms. The isolates were divided into two groups, A and B, by RAPD analysis. Isolates in group A yielded four different PCR polymorphic markers, but all the isolates in group B yielded a single polymorphic marker. The IGS RFLP analysis was consistent with division of the isolates into two groups. Isolates from wheat and rice were more frequently placed in group A, with isolates from maize more evenly distributed between the groups. Results indicate that host preference might be a factor in the division of isolates, although the year of isolation may also have had an influence. No geographical factors or agricultural practices could be identified to account for the observed variation.


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

Fusarium graminearum (teleomorph Gibberella zeae) is a pathogen of a wide range of graminaceous crops including the three main food crops in Nepal: maize, rice and wheat. Among the diseases caused by F. graminearum are ear blight (also known as scab or head blight), stem and root infections of wheat ( Parry et al., 1995 ), ear blight of rice ( Ou, 1985), and ear and stalk rot of maize ( Francis & Burgess, 1975; Vigier et al., 1997 ). In addition to causing yield losses, infection by F. graminearum can lead to contamination of grain by mycotoxins ( Joffe, 1986).

The identification and classification of Fusarium isolates based on morphological and physiological criteria have proved problematic ( Nelson, 1991). The study of Fusarium spp. has been greatly advanced by the adoption of molecular techniques including random amplified polymorphic DNA (RAPD) analysis ( Ouellet & Seifert, 1993; Voigt et al., 1995 ), specific diagnostic PCR primers ( Schilling et al., 1996 ; Nicholson et al., 1998 ), the analysis of PCR products by either restriction fragment length polymorphisms ( Nicholson et al., 1993 ; Appel & Gordon, 1995; Bateman et al., 1996 ; Edel et al., 1996 ) or DNA sequencing ( Appel & Gordon, 1996; O'Donnell et al., 1998 ) and amplified fragment length polymorphisms ( Leissner et al., 1997 ). A molecular systematics approach based on the DNA sequences of several loci detected considerable variation within the Gibberella fujikuroi complex, and has resulted in an increase in the number of proposed species within this complex ( O'Donnell et al., 1998 ). Similarly, RAPD analysis of group I and group II isolates of F. graminearum ( Purss, 1971; Francis & Burgess, 1977) has shown the two groups to be as different from each other as F. graminearum is from Fusarium culmorum and Fusarium crookwellense ( Schilling et al., 1994 ). Subsequent analysis based on both morphological and DNA sequence analysis has led to the reclassification of group I F. graminearum to Fusarium pseudograminearum ( Aoki & O'Donnell, 1999). In addition to clarifying the taxonomic status and relationships among Fusarium isolates and species, these techniques have enabled the detection and quantification of coinfecting Fusarium species ( Nicholson et al., 1998 ), studies on genetic variation ( Nicholson et al., 1993 ; Voigt et al., 1995 ; Schilling et al., 1997 ; Dusabenyagasani et al., 1999 ), and epidemiology of a characterized, introduced strain of F. graminearum ( Miller et al., 1998 ).

Inoculum for ear rot and blight may come from a number of different sources, including crop debris ( Sturz & Johnston, 1985; Fernandez, 1991; Miller et al., 1998 ), F. graminearum infections on other parts of the plant ( Parry et al., 1995 ), infected seed ( Duthie & Hall, 1987), and spread from adjacent fields ( Suty et al., 1996 ). In addition to graminaceous crops, F. graminearum has also been recovered from nongraminaceous crops ( Fernandez, 1991; Harrington et al., 2000 ), wild grasses ( Nyvall et al., 1999 ) and common broad-leaved weeds ( Jenkinson & Parry, 1994), although the contributions of these hosts to inoculum levels have not been determined. Crop rotation and tillage practices have been shown to affect the severity of disease ( Sturz & Johnston, 1985; Smiley et al., 1996 ) and the composition of the F. graminearum population ( Miller et al., 1998 ). Changes within the F. graminearum population may be important as individual isolates have shown different levels of aggressiveness when tested for pathogenicity to rye seedlings ( Miedaner & Schilling, 1996). The relative importance to the infection of graminaceous crops of different inoculum sources, agricultural practices and the genetic variation present within the F graminearum population remains unclear. A fuller understanding of the genetic variation and the relationship between this variation and the ecology of F. graminearum will help in the design of agricultural management practices and in the development of resistant crop cultivars.

In the current study a combination of traditional and molecular techniques was used to characterize and compare the F. graminearum populations isolated from three hosts, maize, wheat and rice, from two areas of Nepal, and an attempt made to identify and exclude factors that could contribute to the observed genetic variation.

Materials and methods

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

Origin and maintenance of fungal strains

Fungi were isolated from surface-sterilized rice, maize and wheat seed as described by Desjardins et al. (2000a, 2000b). Surface-sterilized seeds were placed on a Fusarium-selective agar containing pentachloronitrobenzene ( Nelson et al., 1983 ), and one Fusarium isolate from each seed was purified by single-spore isolation. A total of 67 isolates of Fusarium collected from three areas of Nepal were used in this study ( Table 1). From the first sample area, the mid-west districts of Lamjung and Kaski ( Fig. 1), 12 isolates from rice (RL1–12), 12 from wheat (WL1–12), and 15 from maize (ML1–15) were selected and termed the Lamjung isolates. From the second sample area, in the Central region including the Kathmandu valley ( Fig. 1), 12 isolates from rice (RK1–12), seven from wheat (WK1–7) and eight from maize (MK1–7 and 9) were selected and termed the Kathmandu isolates. One isolate, MK8, from a third area, the Dhankuta District in the Eastern Hills ( Fig. 1), was also included and was grouped with the Kathmandu isolates.

Table 1.  Origin and Fusarium graminearum-specific PCR products of isolates
   F. graminearum-specific PCR
Code aOriginal codeYear isolatedF16N F/R bF16F/R c
  • a

    The first letter in the code denotes the host from which the isolate was obtained: R, rice; W, wheat; M, maize. The second letter denotes the sample area from which the isolate was collected: L, Lamjung; K, Kathmandu.

  • b

    Primer pair F16NF/R yields a single fragment of 0·28 kb with all isolates of F. graminearum tested (scored as +) and no amplification product with other species (scored as –).

  • c

    Primer pair F16F/R yields polymorphic fragments with isolates of F. graminearum (scored as type 1, 0·42 kb; type 2, 0·51 kb; type 3, 0·54 kb; type 4, 0·58 kb; type 5, 0·52 kb) and no amplification products with other species (scored as –).


Figure 1. Map of Nepal indicating the eight districts where Fusarium graminearum was isolated. Upper and lower dashed lines indicate elevations of approximately 2000 and 700 m, respectively. Districts are numbered as: 1, Kaski; 2, Lamjung; 3, Nuwakot; 4, Bhaktapur; 5, Kathmandu; 6, Lalitpur; 7, Kavre; 8, Dhankuta. Isolates from districts 1 and 2 were termed Lamjung isolates; those from districts 3–8, Kathmandu isolates.

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Cultures were maintained at 15°C on potato dextrose agar (PDA) (Difco, West Molesey, UK) to which streptomycin sulphate (50 mg L−1) and penicillin-G (50 mg L−1) had been added. For long-term preservation, mycelium was stored in vapour-phase liquid nitrogen vessels.

Fungal isolate morphology

Isolates were identified as F. graminearum on morphological criteria according to the procedure of Nelson et al. (1983) . Each isolate was cultured on PDA and carnation leaf agar (CLA). On PDA the isolates were scored for the density and colour of aerial mycelium, and the characteristic carmine red undersurface of the colony. On CLA the isolates were scored for the presence of cylindrical macroconidia, the absence of microconidia, and later for the presence of chlamydospores and perithecia.

Extraction of DNA

DNA was extracted from the mycelium of 4- to 5-day-old cultures grown in potato dextrose broth. The mycelium was freeze-dried and ground to a fine powder, and the DNA extracted using the CTAB method of Nicholson et al. (1997) .

F. graminearum species-specific PCR

F. graminearum-specific PCR was performed using primer pair Fg16NF/R, which produces a monomorphic product of 0·28 kb with DNA from F. graminearum and no products with DNA from any other fungi tested ( Nicholson et al., 1998 ). Amplification reactions were carried out in volumes of 50 µL containing 10–20 ng fungal DNA. The reaction buffer consisted of 100 µm each of dATP, dCTP, dGTP and dTTP, 100 n m each of forward and reverse primer, and 0·8 units of Taq polymerase (Roche Diagnostics Ltd, Lewes, UK) in 10 m m Tris HCl (pH 8·3), 1·5 m m MgCl2, 50 m m KCl, 100 µg mL−1 gelatine, 0·05% Tween 20 and 0·05% Nonidet P-40. Reaction mixtures were overlaid with mineral oil prior to PCR in a Perkin-Elmer Cetus 480 DNA thermal cycler. DNA was amplified using touchdown PCR ( Don et al., 1991 ). The annealing temperature was 66°C for the first five cycles and 64°C for the next five cycles, followed by 15 cycles at 62°C. The temperature cycle consisted of denaturation (95°C) for 30 s, annealing (as described above) for 20 s, and extension (72°C) for 45 s, with maximum transition rates between temperatures. A final extension of 72°C for 5 min was incorporated into the programme, followed by cooling to 5°C until recovery of the samples. PCR products were separated by electrophoresis through 2% agarose gels. Gels were stained with ethidium bromide and photographed under UV light with Polaroid 665 positive/negative film.

rDNA sequence analysis

A region containing the 3′ end of the 18S rRNA gene, the 5·8S rRNA gene, and the internal transcribed spacers (ITS1 and ITS2) was amplified and sequenced as described by Carter et al. (1999) . The sequences for the ITS1 and ITS2 regions were used individually to search the EMBL and GenBank databases using blast ( Altschul et al., 1990 ).

Polymorphic PCR

PCR was performed using primer pair Fg16F/R which produces polymorphic products with DNA from F. graminearum, but no products with DNA from any other fungi tested ( Nicholson et al., 1998 ). The amplification conditions and analysis of reaction products were identical to those described for the monomorphic, species-specific primer pair Fg16NF/R.

Random amplified polymorphic DNA assays

The PCR cycle for RAPD analysis consisted of 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C. The reaction buffer used was identical to that used for the species-specific PCR. Transition times were 114 s from denaturing to annealing, and 72 s from annealing to extension. RAPDs were produced using 200 n m of seven oligonucleotide primers selected from a preliminary screen of kits OPT and OPU (Operon Technologies Inc, Alameda, CA, USA). These were OP-T01 (GGGCCACTCA); OP-T04 (CACAGAGGGA); OP-T06 (CAAGGGCAGA); OP-T16 (GGTGAACGCT); OP-U13 (GGCTGGTTCC); OP-U15 (ACGGGCCAGT); and OP-U19 (GTCAGTGCGG).

The RAPD products were separated on 2% agarose gels which were photographed under UV light after staining with ethidium bromide, and the presence or absence of bands recorded. The data were converted to a distance matrix using algorithm number 2 (Jaccard) of rapdist version 1·04 ( Armstrong et al., 1994 ). The distance matrices were then used to construct dendrograms by the unweighted pair-group method with arithmetic mean (UPGMA) using the neighbor program contained in phylip version 3·572 ( Felsenstein, 1995). A program to produce bootstrapped data sets was kindly supplied by John Armstrong (ANU, Canberra, Australia), and these data sets were analysed using the neighbor and consense programs contained in phylip.

IGS amplification and restriction fragment analysis

The intergenic spacer (IGS) region of the nuclear ribosomal RNA gene sequence was amplified using primers CNS1 and CNL12 ( Anderson & Stasovski, 1992). The PCR cycle consisted of 35 cycles of 30 s at 95°C, 20 s at 60°C, and 45 s at 72°C, with maximum transition rates between temperatures. A final extension of 72°C for 5 min was incorporated into the programme, followed by cooling to 5°C until recovery of the samples. Aliquots (10 µL) of the amplification products were digested in PCR buffer with 5 units of either AluI, CfoI, HpaI, HinfI or HaeIII at 37°C for 18 h. Digests were size-fractionated by electrophoresis through 1·3% MetaPhor agarose gel (Flowgen, Lichfield, UK) and viewed and photographed as above.


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

Identification of fungal isolates

All of the isolates in Table 1 were similar to F. graminearum for all of the morphological criteria, except for one strain, ML10, which failed to produce perithecia. All of the isolates except WL6 and MK4 yielded a PCR product of 0·28 kb when amplified using the F. graminearum-specific primer pair Fg16NF/R. Isolates RL1, ML4, WL9, RK5, WL1, WL6 and MK4 were selected for rDNA sequence analysis. Of these isolates, RL1, ML4, WL9, RK5 and WL1 had ITS1 and ITS2 sequences identical to those reported for G. zeae (EMBL accession number U34578), although these sequences have also been shown to be shared with F. culmorum (EMBL accession number AF006342). In contrast, the ITS1and ITS2sequences obtained from isolates WL6and MK4were different both to the other Nepalese isolates and to each other, and were identical to those of F. fujikuroi ( U34557) and F. napiforme ( U34570), respectively.

Polymorphic PCR

Isolates WL6 and MK4 failed to yield any products when tested with primer pair Fg16F/R. The remaining isolates yielded products of five apparently different sizes ( Table 1). Only three isolates, all obtained from rice at the same village in the Lamjung region, yielded a 0·42 kb product. Six Kathmandu isolates, three each from wheat and rice, yielded 0·58 kb products. The remaining 0·51, 0·54 and 0·52 kb products were yielded by both Lamjung and Kathmandu isolates. The majority of isolates from maize (16 out of 23) yielded a 0·51 kb product, with the remaining isolates yielding a 0·54 kb product. Of the 24 isolates obtained from rice, three yielded 0·42, five 0·51, 11 0·54, three 0·58 and two 0·52 kb products. Of the 18 isolates from wheat, 11 yielded a 0·54 kb, three a 0·51 kb, three a 0·58 kb and one a 0·52 kb product.

RAPD analysis

Following their failure to amplify with the F. graminearum-specific PCR primer pair Fg16NF/R and the polymorphic product PCR primer pair Fg16F/R, and their ITS1 and 2 sequence identity to F. fujikuroi and F. napiforme, respectively, WL6 and MK4 were excluded from the RAPD analysis. Isolates WL1, WL12 and ML11 were also excluded due to difficulty in accurately scoring the RAPD bands obtained for them. For the remaining 62 isolates the data obtained from the seven primers were pooled, and a total of 134 bands scored. Only six of the isolates had profiles identical to another isolate. One pair of isolates with identical RAPD profiles, WL10 and WK3, was obtained from different sample areas, while the remaining pairs of isolates sharing RAPD profiles (ML1 and ML2; ML6 and ML7) were isolated from the same farms. No common RAPD profiles were detected between isolates obtained from different crops.

A dendrogram constructed using the RAPD data ( Fig. 2a) shows the isolates to be divided into two groups. The larger group, group A, supported by a bootstrap value of 73%, contains 39 isolates of which only six were obtained from maize. All of the isolates that yielded 0·42, 0·52, 0·54 and 0·58 kb Fg16 products were in group A, with the majority of isolates in this group (28 of 39) yielding 0·54 kb products. Within group A all three of the isolates with 0·42 kb Fg16 products were grouped together with bootstrap support of 100%. Isolates with the 0·58 kb Fg16 product grouped together, but without bootstrap support. Group B, supported by a bootstrap value of 77%, contained 23 isolates of which 16 had been obtained from maize. Within group B, all of the isolates yielded 0·51 kb Fg16 products.


Figure 2. Relationship between the Nepalese Fusarium graminearum isolates based on (a) RAPD and (b) IGS RFLP analysis. Numbers after isolate names refer to the Fg16 polymorphic PCR product yielded (in kb): 1, 0·42; 2, 0·51; 3, 0·54; 4, 0·58; 5, 0·52. Dendrograms were produced after data were converted to a distance matrix using algorithm number 2 (Jaccard) for RAPD or algorithm number 1 (Nei) of rapdist version 1·04 ( Armstrong et al., 1994 ). The distance matrices were used to construct the dendrograms by the unweighted pair-group method with arithmetic mean (UPGMA) using the neighbor program contained in phylip version 3·572 ( Felsenstein, 1995). Bootstrapped data sets (500 replicates) were produced using a program supplied by John Armstrong (ANU, Australia) and analysed using the neighbor and consense programs contained in phylip; bootstrap values >50% are shown on the dendrograms.

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A further set of dendrograms, constructed from the RAPD data for isolates from each host crop separately ( Fig. 3), were consistent with the division of the isolates into the groups evident in Fig. 2a, with the division again supported by strong bootstrap values. The numbers of isolates from each of the three hosts that were placed in the two RAPD groups is shown in Table 2. A χ2 test showed that isolates from wheat and rice were more likely to be classified in group A (P < 0·05). There was no significant (P > 0·05) division of isolates from maize between the two groups, although 16 of the 22 isolates were placed in group B.


Figure 3. Dendrograms based on RAPD analysis of Nepalese Fusarium graminearum isolates obtained from (a) wheat; (b) rice; (c) maize. Numbers after isolate names refer to the Fg16 polymorphic PCR marker type yielded. Dendrograms were produced as described in Figure 2.

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Table 2.  Number of isolates of F. graminearum in each of the two RAPD groups from each of the hosts sampled
 Number of isolates
Host of origin Total RAPD group A RAPD group B Expected aχ2 calculated P value
  • a

    The expected number of isolates in a RAPD group if isolates were equally likely to be placed in either group.


IGS RFLP analysis

The same 62 isolates used in the RAPD analysis were also used for the IGS RFLP analysis ( Fig. 2b). Of the 30 different profiles produced, 13 were shared by more than one isolate, and one by 14 isolates. The isolates that had been placed together in group B on the basis of the RAPD analysis ( Fig. 2a), all of which yielded a 0·51 kb Fg16 product ( Table 1), formed three groups each with 100% bootstrap support. None of the isolates placed in group A on the basis of the RAPD analysis (none of which yielded a 0·51 kb Fg16 product) was present in these three groups.


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

The fungal isolates were identified using a combination of morphological and molecular techniques. Each isolate had to show the correct morphology on both PDA and CLA, and yield a 0·28 kb PCR product with the species-specific Fg16NF/R primers, before it was identified as F. graminearum and included in the RFLP and IGS RFLP analysis. Other monomorphic primer pairs have also been reported that are specific for F. graminearum ( Schilling et al., 1996 ), and this method has been shown to have the potential to differentiate F. graminearum from the closely related F. culmorum and F. crookwellense ( Schilling et al., 1996 ; Nicholson et al., 1998 ). The production of perithecia was used as supporting evidence for the identification, but was not relied upon due to its variability under laboratory conditions. To confirm the identification, isolates RL1, ML4, WL9, RK5 and WL1, which represented each of the Fg16 PCR polymorphic product sizes, and isolates MK4 and WL6, which failed to yield PCR products with either Fg16NF/R or Fg16F/R primer pairs, were subjected to ITS sequence analysis. The ITS sequences obtained were consistent with isolates RL1, ML4, WL9, RK5 and WL1 being classified as F. graminearum, although this sequence analysis on its own could not distinguish F. graminearum and F. culmorum. The ITS sequences of isolates MK4 and WL6 suggest their classification as F. napiforme and F. fujikuroi, respectively, rather than F. graminearum. The combination of morphological and molecular techniques used allows identification of the isolates used in the RAPD and IGS RFLP analysis as F. graminearum with a greater degree of certainty than the use of either approach on its own.

The RAPD analysis, IGS RFLP analysis and the PCR polymorphic markers suggest that the Nepalese F. graminearum isolate collection was highly genetically varied. Isolates of G. zeae are able to reproduce sexually in the laboratory, although the extent of sexual recombination in the field is not known ( Bowden & Leslie, 1999). A comparison of the RAPD analysis and the polymorphic Fg16 PCR marker shows that the two assays produced similar groupings of isolates ( Fig. 2a) and the Nepalese F. graminearum isolates could be divided into two groups, A and B, with all of the isolates yielding a 0·42, 0·52, 0·54 or 0·58 kb product being classified in group A, and all of the isolates yielding a 0·51 kb product being classified in group B. The dendrogram produced from the IGS RFLP analysis did not agree with the Fg16 PCR polymorphic marker data as closely as the dendrogram drawn from the RAPD data, but the grouping of all the isolates that yielded a 0·51 kb Fg16 product was consistent with group B derived from the RAPD data analysis.

A number of different explanations could be proposed for the division of the Nepalese F. graminearum isolates into two groups, including host preference, year of isolation, management practices and geographic location.

Isolates from different hosts were not evenly distributed between groups A and B, indicating that an element of host preference may be involved. Although isolates from each group were obtained from all three hosts, group A isolates were recovered with similar frequency from all three hosts, whereas group B isolates were recovered less frequently from wheat and rice than from maize. This might imply that group B isolates are less aggressive to wheat and rice than they are to maize. In a previous study the only F. graminearum isolate that yielded a 0·51 kb Fg16 product, characteristic of group B, was from maize in Honduras ( Nicholson et al., 1998 ). Although variation in aggressiveness has been reported for isolates of F. graminearum on both maize ( Cullen et al., 1982 ) and rye ( Miedaner & Schilling, 1996), it has not been reported for different hosts.

The explanation of the division of isolates into two groups as being due to host preference is, however, complicated by the year of isolation. The majority of the isolates obtained from maize were collected in 1993, and no isolates from other hosts were collected in this year. Group A and B isolates were obtained in both sample years. Although the contribution of different Fusarium species to levels of head blight has been examined ( Sturz & Johnston, 1985; Schilling et al., 1997 ; Vigier et al., 1997 ; Nyvall et al., 1999 ), there are fewer studies on the intraspecific variation in F. graminearum in different years. RAPD analysis of F. graminearum populations from Ontario and Quebec, Canada, isolated 4 years apart, detected no correlation between the populations' genetic make-up and the year of sampling ( Dusabenyagasani et al., 1999 ).

It is unlikely that the variation observed was related to the geographic source of the isolates. The same percentage, 63%, of the Lamjung and Kathmandu isolates were placed in group A on the basis of their RAPD profiles, and isolates that yielded 0·51 or 0·54 kb Fg16 PCR products were obtained from both sample areas (34 and 53% of the Lamjung isolates and 37 and 37% of the Kathmandu isolates, respectively). The only minor differences detected between the two sample areas were with the 0·42 and 0·58 kb Fg16 PCR products which were obtained only from Lamjung or Kathmandu isolates, respectively.

There are no clear agronomic practices that would lead to the development of separate F. graminearum populations on maize compared with rice and wheat. A wide range of different agricultural practices are used in the hill regions of Nepal, but the growing seasons of all three hosts overlap, with this overlap being greatest between maize and wheat (A.E. Desjardins, personal observation). One factor that may affect the spread of disease to subsequent crops is the practice of removing crop debris after harvest, either by burning or for use as animal feed. The removal or ploughing in of infected crop debris have been implicated in the reduction of soil inoculum levels and in changes of Fusarium species composition ( Warren & Kommedahl, 1973; Miller et al., 1998 ). Although F. graminearum is largely associated with organic debris in soil ( Wearing & Burgess, 1977; Sturz & Johnston, 1985), the relative importance of this as a source of inoculum compared with aerial spread from adjacent fields or seedborne infection remains unclear. The ability of RAPD analysis and the Fg16 polymorphic PCR marker to distinguish between different F. graminearum isolates from Nepal will provide a tool for further investigations into this pathogen. By being able to distinguish different isolates, and by combining these diagnostic techniques with quantitative approaches ( Nicholson et al., 1998 ), it will be possible to investigate the relative competitive ability of isolates on different hosts to allow any host preferences to be determined, and to allow the roles of different sources of inoculum to be identified. In addition to contributing to the understanding of ear rot and head blight and improving crop productivity in Nepal, this information will improve our basic understanding of the ecology of this pathogen and will be of importance to many other agricultural systems.


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

This work was supported by the Ministry of Agriculture, Fisheries and Food, UK, and the USDA, USA. J.P. Carter was supported by ELL grant ERBIC18-CT98-0312. The collection of fungi in Nepal by A.E. Desjardins was supported by the United States Fulbright Foreign Scholarship Program and by The Nepalese Agricultural Research Council. We thank Stephanie Fulmar for technical support.


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