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Keywords:

  • biological control;
  • fusarium wilt of banana;
  • Panama disease;
  • suppressive soils

Abstract

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

One of the most serious diseases of banana is fusarium wilt, caused by Fusarium oxysporum f.sp. cubense (Foc). The objectives of this study were to isolate and identify nonpathogenic F. oxysporum strains from soils suppressive to banana wilt, and to determine the diversity of these isolates. More than 100 Fusarium strains were isolated from the rhizosphere of banana plants and identified to species level. Pathogenicity testing was carried out to confirm that these isolates were nonpathogens of banana. A PCR-based RFLP analysis of the intergenic spacer region of the ribosomal RNA operon was used to characterize the nonpathogens. The isolates were also compared with isolates of Foc from South Africa and the known biological control isolate of F. oxysporum, Fo47. The species-specific primers FOF1 and FOR1, in addition to morphological features, were used to confirm the identity of F. oxysporum isolates included in the PCR-RFLP analysis. Twelve different genotypes could be distinguished, identified by a six-letter code allocated to each isolate following digestion with the restriction enzymes HaeIII, HhaI, HinfI, MspI, RsaI and ScrfI. Eleven of these included nonpathogenic F. oxysporum isolates, and these groups could all be distinguished from the genotype that included Foc. Fo47 was included in one of the genotype groups consisting of nonpathogenic F. oxysporum isolates from South Africa.


Introduction

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

Fusarium oxysporum f.sp. cubense (Foc) is a soilborne fungus responsible for fusarium wilt of banana (Stover, 1962). Fusarium wilt, commonly referred to as Panama disease, is one of the most serious and destructive diseases of banana (Ploetz & Pegg, 2000). The disease was discovered in Australia in 1876 and, by 1950, had spread rapidly to most of the banana-producing countries of the world (Ploetz et al., 1990). Fusarium wilt was first noticed in South Africa in 1946 (Ploetz et al., 1990), and today five of the six banana-producing areas are affected by the disease (A.V., unpublished data). No effective control measure for fusarium wilt has been found other than the use of resistant cultivars. Unfortunately, all Cavendish cultivars grown locally are highly susceptible to Foc‘subtropical’ race 4, the only variant of the fungus that occurs in South Africa. As only Cavendish banana cultivars are acceptable to the local market, and improvement of Cavendish bananas is difficult and time-consuming, it is important that strategies other than disease resistance be considered for management of fusarium wilt (Viljoen, 2002).

Biological control of fusarium wilt diseases has become an increasingly popular disease management consideration in recent years, given its environmentally friendly nature and the discovery of novel mechanisms of plant protection associated with certain microorganisms (Weller et al., 2002; Fravel et al., 2003). Fusarium wilt-suppressive soils have been reported in many regions of the world, and suppression has generally been shown to be due to biological factors (Scher & Baker, 1980; Alabouvette et al., 1993). This led to studies of antagonistic microorganisms in the soil, their identification, and the mechanisms involved in their disease suppression. Most of these studies have found that nonpathogenic strains of F. oxysporum are associated with the natural suppressiveness of soil to fusarium wilt diseases (Smith & Snyder, 1971; Alabouvette, 1990; Postma & Rattink, 1992; Larkin et al., 1996). Nonpathogenic isolates of F. oxysporum were also found effectively to colonize the plant rhizosphere and roots without inducing any symptoms (Elias et al., 1991; Olivain & Alabouvette, 1999).

Identification of Fusarium species from the soil is often challenging as it relies on minor differences in morphology, and different cultural conditions can cause the same species to vary (Doohan, 1998). The differentiation of F. oxysporum from several other Fusarium species that belong to the sections Elegans and Liseola can sometimes be especially difficult (Fravel et al., 2003). Molecular tools have therefore been developed to support morphological identifications. Edel et al. (1997a) developed a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method targeting a fragment of the ribosomal (r)DNA that includes the internal transcribed spacer (ITS) region for the identification of Fusarium species. Edel et al. (2000) also developed an rDNA-targeted oligonucleotide probe and PCR assay specific for F. oxysporum. Mishra et al. (2003) developed a PCR-based assay for rapid identification of some Fusarium species. This technique is based on the ITS region of the rDNA.

The ITS region can be used for the differentiation of species, although its variation at intraspecific level within F. oxysporum was found to be low (Edel et al., 1995). The intergenic spacer (IGS) region, which separates rDNA repeat units, appears to evolve more rapidly and is more variable than the ITS region (Hillis & Dixon, 1991). Edel et al. (1995) evaluated three different methods to determine the diversity within nonpathogenic isolates of F. oxysporum. They found that PCR-RFLP analysis of the IGS region was a rapid technique to determine the genetic relatedness among isolates of nonpathogenic F. oxysporum. Appel & Gordon (1995) also demonstrated that the diversity between pathogenic and nonpathogenic F. oxysporum isolates could be determined by RFLP analysis of the IGS region of rDNA.

The objectives of this study were to isolate and identify nonpathogenic F. oxysporum strains from the rhizosphere of healthy banana roots in fusarium wilt-suppressive soils; to use IGS sequence analysis to determine the genetic differences among these isolates; to determine the genetic relatedness of the nonpathogenic isolates; and to compare them with pathogenic isolates of F. oxysporum.

Materials and methods

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

Isolates used

Three sites with fusarium wilt-suppressive soil properties were identified in Kiepersol (Mpumalanga Province), one of the main banana-producing regions in South Africa. All these sites were planted to Williams, the most important Cavendish banana cultivar grown in the area. The three sites (referred to as sites 1–3) were geographically isolated from each other and occurred on three different farms in Kiepersol. Development of fusarium wilt of banana was slow or absent at all three sites, even though the plants in these sites were surrounded by diseased plants in severely affected banana fields. To isolate F. oxysporum strains from the rhizosphere of banana roots, five healthy banana plants were selected at each site, and five root pieces collected from each plant. Rhizosphere samples were collected twice from all three areas, once in October 2002 and once in March 2003, in order to counter any seasonal effect on the strains that were collected. The soil around the banana plants was tilled and roots were sampled about 15–30 cm deep. The samples were placed in 10-mL glass bottles containing sterile distilled water. Water and root suspensions were vigorously shaken to remove the adhering soil, after which the roots were removed from the glass bottles. Bottles were transported from the field to the laboratory in a cooler bag containing ice packs. In the laboratory, dilution series were made of each suspension and plated onto Komada medium (Komada, 1975) for isolation of F. oxysporum. Single-spore isolates were selected from each root sample collected. Each single-spore isolate was grown on filter paper overlaid on potato dextrose agar (PDA) (Difco). The colonized filter paper was removed from the agar plate, dried, and stored at 4°C at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

For comparative purposes, the nonpathogenic F. oxysporum isolate Fo47, from a wilt-suppressive soil of the Châteaurenard region of France, was supplied by Dr C. Steinberg (INRA, Dijon, France). The efficacy of Fo47 in reducing the severity of fusarium wilt diseases of other crops has been well demonstrated (Lemanceau & Alabouvette, 1991; Alabouvette et al., 1993; Fuchs et al., 1997). The Foc isolates (CAV 045 and CAV 129) used in this study were obtained from infected banana plant material in the KwaZulu-Natal area of South Africa.

Identification of Fusarium oxysporum from rhizosphere soil

Cultural and morphological identification

Isolates of F. oxysporum were identified according to their cultural and morphological characteristics as described by Nelson et al. (1983). The single-spore cultures were grown on PDA medium to determine their growth rate and colony pigmentation. Cultures were incubated at 25 and 30°C for 7–10 days in the dark, after which the colony diameter was measured and colony colour determined. Single-spore isolates were also placed on carnation leaf agar (CLA: 20 g Biolab agar, 1000 mL H2O), one or two 5 mm sterilized carnation leaves per Petri dish, and incubated for 14 days under cool-white and near-ultraviolet fluorescent lights to investigate the presence and shape of the macroconidia, microconidia and chlamydospores. Morphological characteristics were studied using light microscopy.

Molecular identification

For DNA extractions, all isolates of F. oxysporum were grown on half-strength PDA medium for 7 days at 25°C under cool-white and near-ultraviolet fluorescent lights. Mycelium was then scraped directly from agar plates and used for DNA isolation. Total genomic DNA was isolated using the method described by Raeder & Broda (1985), with minor modifications. Cultures were homogenized with a pestle in 300 µL DNA extraction buffer in an Eppendorf tube, freeze-dried in liquid nitrogen and boiled in water for 5 min. After adding 700 µL phenol–chloroform (1 : 1), samples were vortexed and centrifuged for 7 min at 20 817 g. The upper aqueous layer was transferred to a new tube and the phenol–chloroform step repeated until the white interface disappeared. The rest of the procedure was performed similarly to that described by Raeder & Broda (1985), with the only exception that tubes were centrifuged for 10 min after the precipitation step. DNA was dried under vacuum, after which the resulting pellet was resuspended in 100–200 µL sterile distilled water (SABAX water). RnaseA (10 µµL−1) was added to the DNA samples, and incubated at 37°C for 3–4 h to digest any residual protein or RNA. The DNA was visualized on a 1% agarose gel (wt/v) (Boehringer Mannheim) stained with ethidium bromide and viewed under ultra-violet light. DNA concentrations were estimated by comparing the intensity of ethidium bromide fluorescence of the DNA sample to a known concentration of lambda DNA marker (marker III, Roche Diagnostics).

Isolated DNA (50–90 ng) was used as template for the PCR reaction. Two primers, designed specifically to the ITS region of the rDNA operon of F. oxysporum (Mishra et al., 2003), were used for the molecular identification of F. oxysporum isolates from the banana root rhizosphere. The primer pair of FOF1 (5′-ACA TAC CAC TTG TTG CCT CG-3′) and FOR1 (5′-CGC CAA TCA ATT TGA GGA ACG-3′) was synthesized by Inqaba Biotec, Pretoria, South Africa. Amplification conditions were similar to those described by Mishra et al. (2003). Reactions were carried out in a 20-µL reaction volume containing PCR buffer (10 mm Tris–HCl, 1·5 mm MgCl2, 50 mm KCl pH 8·3) (Roche), 0·2 mm each dNTP (Roche), 0·3 µm of each primer FOF1 and FOR1, and 1 U Taq DNA polymerase (Roche). SABAX water was used to achieve the final volume of 20 µL.

DNA amplifications were performed in a Mastercycler gradient PCR machine (Eppendorf Scientific) using an initial denaturation temperature of 94°C for 60 s, followed by 25 cycles of template denaturation for 60 s at 94°C, primer annealing for 30 s at 58°C and chain elongation for 60 s at 72°C, with a final extension of 7 min at 72°C. Negative and positive controls were included in each reaction, containing SABAX water and no template, and DNA of a known F. oxysporum isolate, respectively. The amplified products were verified using 2% agarose gel electrophoresis in 1 × Tris acetic acid EDTA (TAE, pH 8·0) buffer, stained with ethidium bromide and visualized under ultra-violet light. A 100-bp molecular weight marker XIV (Roche) was used to determine the size of the PCR products.

Pathogenicity testing

A subsample of 60 isolates identified as F. oxysporum, and the biological control agent Fo47, were evaluated for their ability to cause disease in small banana plantlets. Isolates were grown for 7 days on half-strength PDA in Petri dishes at 25°C under cool-white and near-UV fluorescent lights, after which mycelia of these cultures were transferred to Armstrong Fusarium sporulation media (Booth, 1977) in 500-mL Erlenmeyer flasks. The flasks were placed on a rotary shaker operating at 170 r.p.m. at 25°C for 5 days, after which the different suspensions were passed through cheesecloth to separate the mycelia from the spores. The spore concentration in the liquid medium was determined using a haemocytometer, and diluted with sterile distilled water to a concentration of 5 × 106 spores mL−1.

Pathogen-free tissue-culture banana plantlets (Williams cultivar) obtained from Du Roi Laboratories, Letsitele, South Africa, were used for pathogenicity testing. The 5-cm plants were planted in an aquaculture system. After a 4-week period of adjustment during which the plants received nutrition, three plants were inoculated with each of the F. oxysporum isolates. Each 250-mL plastic cup was inoculated with 5 mL of a spore concentration of 5 × 106 spores mL−1, which gave a final inoculum concentration of 105 spores mL−1. The roots of the plants were slightly bruised by manually squeezing the rootball to ensure infection by the pathogen. The pathogenic Foc isolates CAV 045 and CAV 129 were included as positive controls. Healthy banana plants inoculated with sterile distilled water were used as negative controls. Plants were placed in phytotrons with a photoperiod of 12 h and at a day/night temperature regime of 28/20°C. After 4 weeks the plants were removed from the cups and evaluated for the presence of the disease. The standard disease rating scale (Carlier et al., 2002) for fusarium wilt of banana was used to record severity of disease.

Genotypic characterization of nonpathogenic Fusarium oxysporum isolates

PCR amplification of the IGS region

To determine genetic relatedness among nonpathogenic isolates from the banana root rhizosphere, isolates identified as F. oxysporum were analysed by means of PCR-RFLPs. The IGS region of the rDNA of each isolate was amplified using the oligonucleotide primers PNFo (5′-CCC GCC TGG CTG CGT CCG ACT C-3′) and PN22 (5′-CAA GCA TAT GAC TAC TGG C-3′). The forward primer PNFo, which anneals to the nucleotides 636–657 in the IGS sequence, was designed according to the IGS sequence of F. oxysporum f.sp. melonis Fom24 (Edel et al., 1995), while the reverse primer PN22 was taken from a conserved region at the 5′ end of the Saccharomyces cerevisiae 18S rRNA gene. Primers were synthesized by Inqaba Biotec. The PCR conditions were similar to those described by Edel et al. (1995). Amplifications were performed in volumes of 50 µL containing the following: DNA template (50–90 ng), PCR buffer (10 mm Tris–HCL, 1·5 mm MgCl2, 50 mm KCl pH 8·3), 0·25 mm each dNTP, 0·2 µm of each primer PNFo and PN22, and 2 U Taq DNA polymerase. SABAX water was used to achieve the final volume of 50 µL.

PCR reactions were performed in an Eppendorf Mastercycler gradient PCR machine. Conditions consisted of 30 cycles of denaturation at 95°C for 90 s, followed by primer annealing at 50°C for 60 s and extension at 72°C for 90 s. A sample containing SABAX water and no DNA template was included as a negative control, while DNA of a known F. oxysporum isolate was included as positive control. The PCR products were run on a 0·8% agarose gel stained with ethidium bromide and visualized under ultra-violet light. A lambda DNA marker (marker III) (Roche) was used to determine the size of the PCR products.

DNA restriction digests and electrophoresis

Restriction enzymes selected were similar to those used by Edel et al. (1995). Restriction enzymes HaeIII, HinfI, MspI, RsaI, ScrFI (Roche) and HhaI (Promega) are all four-base-cutting enzymes. The restriction enzyme (2 U) was added directly to 10 µL unpurified PCR amplification products, 1× restriction buffer and SABAX water to achieve an end-reaction volume of 20 µL, which was incubated at 37°C for 3–4 h. Digested fragments were run on an ethidium bromide-stained gel consisting of 3–4% agarose at 60 V for 2 h. Fragments were run against a 100-bp molecular marker for size estimation. Gels were visualized under UV light.

Results

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

Identification of Fusarium oxysporum from rhizosphere soil

Cultural and morphological identification

Fusarium oxysporum produced abundant oval- to kidney-shaped microconidia in false heads and abundant macroconidia, slightly sickle-shaped, thin-walled and delicate, with an attenuated apical cell and a foot-shaped basal cell (Fig. 1). Short monophialides could be seen, and chlamydospores were present and formed singly or in pairs (Fig. 1). Culture growth was rapid on PDA, producing white aerial mycelium, some tinged with purple. The under-surface in some cultures was dark purple, while in others it varied from light purple to pink and even peach. Fusarium solani and Fusarium semitectum, as well as a number of unknown Fusarium spp., were also isolated from the soil. However, these were not included in the PCR-RFLP analysis.

image

Figure 1. Morphological characteristics of assorted nonpathogenic Fusarium oxysporum isolates from suppressive soils in Kiepersol, South Africa. (a) Oval- to kidney-shaped microconidia; (b) microconidia produced in false heads on short monophialides; (c) macroconidia are slightly sickle-shaped, thin-walled and delicate, with an attenuated apical cell and a foot-shaped basal cell; (d) chlamydospores formed singly or in pairs. Scale bar = 10 µm.

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Molecular identification

High concentrations of DNA (50–90 ng µL−1) were obtained for all isolates. The primer set FOF1 and FOR1 permitted the amplification of a single DNA fragment c. 340 bp in size (Fig. 2). All isolates identified morphologically as F. oxysporum were amplified by this primer set.

image

Figure 2. PCR amplification products of the internal transcribed spacer region of the ribosomal DNA of Fusarium isolates from suppressive soils in Kiepersol, South Africa. PCR products visualized on a 2% agarose gel stained with ethidium bromide. Lane 1, 100 bp molecular weight marker; lane 2, water control; lane 3, Fusarium semitectum; lanes 4–7 and 9–11, Fusarium oxysporum isolates; lanes 8 and 12, Fusarium solani isolates.

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Pathogenicity testing

Only one of the 60 isolates of F. oxysporum (CAV 278) from the rhizosphere soil, and the two pathogenic Foc isolates, caused wilt of banana plantlets. All other isolates, including the biocontrol agent Fo47, were nonpathogenic and were therefore considered as nonpathogens of banana.

Genotypic characterization of nonpathogenic Fusarium oxysporum isolates

The primer set PNFo and PN22 resulted in amplification of a single DNA fragment of the IGS region of c. 1700 bp for each of the 60 isolates of F. oxysporum from the rhizosphere soil, as well as for the Foc isolates included for comparison (Fig. 3). No size variation was observed between any of the PCR products. The six restriction enzymes each produced unique patterns. Patterns that displayed fragments with similar sizes were grouped together and a letter was awarded to each specific pattern (A–H) (Figs 4 and 5). Enzyme HaeIII produced five; HhaI three; HinfI four; MspI four; RsaI eight; and ScrfI two different restriction patterns (Fig. 5). Restriction fragments <50 bp were excluded as it was not possible to determine their correct size by means of electrophoresis.

image

Figure 3. PCR amplification products of the intergenic spacer region of the ribosomal DNA of Fusarium oxysporum isolates. PCR products visualized on a 0.8% agarose gel stained with ethidium bromide. Lane 1, λ molecular weight marker; lanes 2–13, Fusarium oxysporum isolates; lane 14, water control.

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image

Figure 4. Restriction fragments of amplified intergenic spacer products of various Fusarium oxysporum isolates digested with RsaI (top) and HinfI (below). Isolates of F. oxysporum with similar restriction patterns were grouped together and assigned the same letter. Lane M, 100 bp molecular weight marker. Restriction fragments were visualized on a 3–4% agarose gel stained with ethidium bromide.

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image

Figure 5. RFLP patterns obtained for Fusarium oxysporum isolates from the root rhizosphere of banana plants. Each illustration represents the RFLP pattern produced when the intergenic spacer region of the ribosomal DNA was digested with restriction enzymes HaeIII; HhaI; HinfI; MspI; RsaI; and ScrFI.

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Twelve IGS genotypes could be distinguished by a six-letter code designated to each isolate (Table 1). IGS genotype DBBACB (group 6), was the most common and consisted of 18 nonpathogenic F. oxysporum isolates. The two pathogenic Foc isolates grouped with the single pathogenic F. oxysporum isolate in IGS genotype ABAACA (group 2), while the well known biological control isolate Fo47 grouped with three nonpathogenic F. oxysporum isolates within the IGS genotype DBBADB (group 7). Several of the genotypes were found at all three sites (Table 2). All IGS genotypes found in this study were present in site 1, while only eight and six of the genotypes were present in sites 2 and 3, respectively (Table 2). The latter sites, however, also yielded the least number of isolates. The isolates sampled in October 2002 group in a similar way to those sampled in March 2003 (Table 1), with the exception of three genotypes. However, these exceptions contained only a few isolates.

Table 1.  Intergenic spacer (IGS) region genotype groups obtained with RFLP analysis of Fusarium oxysporum isolates collected from the rhizosphere soil of banana plants in fusarium wilt-suppressive soils, and their pathogenicity status
GroupIsolateCollection siteDate isolatedPathogenicityIGS genotypea
HaeIIIHhaIHinfIMspIRsaIScrFI
  • a

    RFLP analysis of the PCR amplified intergenic spacer region of the ribosomal (r)DNA following endonuclease digestion. Each letter represents the same PCR-RFLP pattern.

  • b

    Pathogenic isolates of Fusarium oxysporum f.sp. cubense from KwaZulu-Natal, South African.

  • c

    Known nonpathogenic Fusarium oxysporum isolates Fo47 from fusarium wilt-suppressive soils in France.

1CAV 251Kiepersol, site 1, SA03/03NonpathogenABBAAB
CAV 257Kiepersol, site 1, SA03/03NonpathogenABBAAB
CAV 259Kiepersol, site 1, SA03/03NonpathogenABBAAB
CAV 272Kiepersol, site 2, SA03/03NonpathogenABBAAB
CAV 275Kiepersol, site 2, SA03/03NonpathogenABBAAB
CAV 276Kiepersol, site 2, SA03/03NonpathogenABBAAB
2CAV 045bPort Edward, SA PathogenABAACA
CAV 129bPort Edward, SA PathogenABAACA
CAV 278Kiepersol, site 1, SA03/03PathogenABAACA
3CAV 221Kiepersol, site 1, SA10/02NonpathogenABAAGA
CAV 227Kiepersol, site 1, SA10/02NonpathogenABAAGA
4CAV 239Kiepersol, site 1, SA10/02NonpathogenBBBACB
CAV 279Kiepersol, site 1, SA03/03NonpathogenBBBACB
CAV 212Kiepersol, site 2, SA10/02NonpathogenBBBACB
CAV 280Kiepersol, site 2, SA03/03NonpathogenBBBACB
CAV 281Kiepersol, site 2, SA03/03NonpathogenBBBACB
CAV 282Kiepersol, site 3, SA03/03NonpathogenBBBACB
5CAV 236Kiepersol, site 1, SA10/02NonpathogenCACCEB
CAV 261Kiepersol, site 1, SA03/03NonpathogenCACCEB
CAV 271Kiepersol, site 2, SA03/03NonpathogenCACCEB
CAV 249Kiepersol, site 3, SA10/02NonpathogenCACCEB
6CAV 224Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 225Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 226Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 229Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 230Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 242Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 243Kiepersol, site 1, SA10/02NonpathogenDBBACB
CAV 252Kiepersol, site 1, SA03/03NonpathogenDBBACB
CAV 256Kiepersol, site 1, SA03/03NonpathogenDBBACB
CAV 258Kiepersol, site 1, SA03/03NonpathogenDBBACB
CAV 264Kiepersol, site 1, SA03/03NonpathogenDBBACB
CAV 210Kiepersol, site 2, SA10/02NonpathogenDBBACB
CAV 217Kiepersol, site 2, SA10/02NonpathogenDBBACB
CAV 220Kiepersol, site 2, SA10/02NonpathogenDBBACB
CAV 269Kiepersol, site 2, SA03/03NonpathogenDBBACB
CAV 201Kiepersol, site 3, SA10/02NonpathogenDBBACB
CAV 205Kiepersol, site 3, SA10/02NonpathogenDBBACB
CAV 247Kiepersol, site 3, SA10/02NonpathogenDBBACB
7Fo47cFrance NonpathogenDBBADB
CAV 254Kiepersol, site 1, SA03/03NonpathogenDBBADB
CAV 211Kiepersol, site 2, SA10/02NonpathogenDBBADB
CAV 277Kiepersol site, 2, SA03/03NonpathogenDBBADB
8CAV 240Kiepersol, site 1, SA10/02NonpathogenDBBAHB
CAV 202Kiepersol, site 3, SA10/02NonpathogenDBBAHB
9CAV 262Kiepersol, site 1, SA03/03NonpathogenDBBBBB
CAV 263Kiepersol, site 1, SA03/03NonpathogenDBBBBB
CAV 274Kiepersol, site 2, SA03/03NonpathogenDBBBBB
10CAV 231Kiepersol, site 1, SA10/02NonpathogenDBBDCB
11CAV 241Kiepersol, site 1, SA10/02NonpathogenDCDAFB
CAV 255Kiepersol, site 1, SA10/02NonpathogenDCDAFB
CAV 265Kiepersol, site 1, SA03/03NonpathogenDCDAFB
CAV 219Kiepersol, site 2, SA10/02NonpathogenDCDAFB
CAV 270Kiepersol, site 2, SA03/03NonpathogenDCDAFB
CAV 273Kiepersol, site 2, SA03/03NonpathogenDCDAFB
CAV 200Kiepersol, site 3, SA10/02NonpathogenDCDAFB
CAV 244Kiepersol, site 3, SA10/02NonpathogenDCDAFB
CAV 248Kiepersol, site 3, SA10/02NonpathogenDCDAFB
CAV 253Kiepersol, site 3, SA03/03NonpathogenDCDAFB
12CAV 233Kiepersol, site 1, SA10/02NonpathogenEBBACB
CAV 209Kiepersol, site 2, SA10/02NonpathogenEBBACB
CAV 245Kiepersol, site 3, SA10/02NonpathogenEBBACB
CAV 246Kiepersol, site 3, SA10/02NonpathogenEBBACB
Table 2.  Number of Fusarium oxysporum isolates obtained from fusarium wilt-suppressive soils in banana fields in Kiepersol, South Africa
IGS groupaNumber of isolates from site
123
  1. aIsolates were grouped according to PCR-RFLPs of the intergenic spacer (IGS) region.

ABBAAB 3 3 0
ABAACA 1 0 0
ABAAGA 2 0 0
BBBACB 2 3 1
CACCEB 2 1 1
DBBACB11 4 3
DBBADB 1 2 0
DBBAHB 1 0 1
DBBBBB 2 1 0
DBBDCB 1 0 0
DCDAFB 3 3 4
EBBACB 1 1 2
Total301812

Discussion

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

A diverse population of F. oxysporum isolates nonpathogenic or saprophytic to banana was found in the rhizosphere of healthy banana plants in fusarium wilt-suppressive soils in three different fields in South Africa. At one site, a pathogenic isolate of Foc was obtained from healthy roots, suggesting that some soils can be suppressive. Previous studies on other fusarium wilt diseases have found that populations of saprophytic Fusarium spp. are more diverse and reach higher levels in suppressive than in conducive soils (Wensley & McKeen, 1963; Louvet et al., 1981). Nash & Snyder (1965) and Smith & Snyder (1971, 1972) observed that saprophytic F. oxysporum established easily in suppressive soils in a variety of clonal types, while pathogenic F. oxysporum appeared to establish with difficulty in such soils. Consequently, saprophytic Fusarium clones have the ability to utilize substrates better, and compete more effectively against the pathogen for the ecological sites, in suppressive than in conducive soils (Louvet et al., 1981; Gordon et al., 1989).

The large number of IGS genotypes found within nonpathogenic isolates of the clonally reproducing fungus F. oxysporum on banana was highly significant. Similarly, 11 different genotypes among 60 strains of nonpathogenic F. oxysporum were isolated from the rhizoplane of four plant species (flax, melon, tomato and wheat) (Edel et al., 1995). These polymorphisms observed among isolates of F. oxysporum are probably due to sequence differences, as demonstrated by Appel & Gordon (1995, 1996); Edel et al. (1997b, 2001) and Steinberg et al. (1997), who also observed substantial differences among nonpathogenic isolates of F. oxysporum. Their data provided further evidence that isolates morphologically identifiable as F. oxysporum are genetically diverse. Intraspecific variation in the IGS region may reflect a slow rate of concerted evolution in a species characterized by infrequent sexual reproduction, or a predominantly clonal mode of reproduction by selective mutation (Appel & Gordon, 1996). Vegetative compatibility group (VCG) studies can be used to help determine clonality among nonpathogenic populations of F. oxysporum. It was shown that one IGS type might include one or several VCGs, most of them being a single member in the case of nonpathogenic populations, but one VCG never includes isolates from different IGS types. It has also been observed that one VCG may be present among the pathogenic isolates, while a number of VCGs can be found among isolates from soil (Steinberg et al., 1997). Similarly, few DNA polymorphisms have been detected among isolates from the same VCG in Foc (Kistler et al., 1991). Bentley et al. (1999) observed that PCR-RFLP analysis of 14 South African Foc isolates representing VCG 0120 all belonged to the same IGS type.

The single isolate identified as F. oxysporum (CAV 278) virulent to banana plantlets shared the same IGS genotype (IGS group 2) as the two pathogenic isolates of Foc included in this study. This was a significant finding, considering that the isolates were obtained from two widely separated banana-growing regions in South Africa. While it has been speculated that Foc was introduced into the Kiepersol area from KwaZulu-Natal (Viljoen, 2002), the same RFLP fingerprinting pattern again indicated the highly conserved nature of this clonal pathogen that was introduced into a different area several decades ago. This isolate also produced a characteristic aroma in culture known to be associated with some wilt-inducing isolates (Stover, 1962). Only three pathogenic isolates were used in this study, but the fact that the Foc pathogen does not have a great diversity in South Africa (Visser, 2003), while the natural populations of F. oxysporum in banana soils are highly diverse, raises even more questions concerning the reproductive mode and evolution of this important fungal species.

Similar genotypes of the nonpathogenic F. oxysporum isolates were obtained from all three collection sites in Kiepersol, more being obtained from site 1 than from sites 2 and 3. This could be due to physical and chemical soil composition, temperature, sampling procedure and, most likely, the cropping history of the various sites. Using IGS-RFLP analysis, Edel et al. (1997b) compared the F. oxysporum population structure of uncultivated soil with populations isolated from the roots of four plant species. Considerable diversity within the populations of F. oxysporum was observed, and genotypic population structure of uncultivated soil differed from the populations associated with the roots of wheat and tomato. Certain IGS genotypes were detected more commonly on tomato, whereas others were found more commonly on wheat, suggesting that the roots of wheat and tomato plants had a selective effect on the population structure of F. oxysporum (Edel et al., 1997b). In the current study, it is possible that isolates from sites 1–3 are genetically similar because banana root exudates from the Williams banana cultivar may favour their selectiveness, as shown previously by Stover et al. (1961).

In the current study the known biological control agent Fo47, isolated in France, grouped with three of the nonpathogenic isolates of F. oxysporum from two collection sites in Kiepersol: more discriminating molecular analysis is required to compare relatedness. Comparison of indigenous isolates to suppress fusarium wilt of banana with that of Fo47 is discussed by Nel et al. (2006).

Acknowledgements

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

We thank the Banana Growers Association of South Africa (BGASA), the Technology and Human Resources of Industry Programme (THRIP) and the National Research Foundation (NRF) for financial assistance, the banana growers in Kiepersol for their assistance during field collections, and Dr Ben Eisenberg for assistance with statistical analysis of data.

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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