Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants
Version of Record online: 17 JAN 2006
Volume 55, Issue 2, pages 207–216, April 2006
How to Cite
Nel, B., Steinberg, C., Labuschagne, N. and Viljoen, A. (2006), Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants. Plant Pathology, 55: 207–216. doi: 10.1111/j.1365-3059.2006.01343.x
- Issue online: 14 MAR 2006
- Version of Record online: 17 JAN 2006
- Accepted 29 August 2005
- biological control;
- fusarium wilt of banana;
- Panama disease;
- suppressive soils
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.
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
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.
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 µg µL−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.
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.
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.
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.
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.
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.
|Group||Isolate||Collection site||Date isolated||Pathogenicity||IGS genotypea|
|1||CAV 251||Kiepersol, site 1, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|CAV 257||Kiepersol, site 1, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|CAV 259||Kiepersol, site 1, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|CAV 272||Kiepersol, site 2, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|CAV 275||Kiepersol, site 2, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|CAV 276||Kiepersol, site 2, SA||03/03||Nonpathogen||A||B||B||A||A||B|
|2||CAV 045b||Port Edward, SA||Pathogen||A||B||A||A||C||A|
|CAV 129b||Port Edward, SA||Pathogen||A||B||A||A||C||A|
|CAV 278||Kiepersol, site 1, SA||03/03||Pathogen||A||B||A||A||C||A|
|3||CAV 221||Kiepersol, site 1, SA||10/02||Nonpathogen||A||B||A||A||G||A|
|CAV 227||Kiepersol, site 1, SA||10/02||Nonpathogen||A||B||A||A||G||A|
|4||CAV 239||Kiepersol, site 1, SA||10/02||Nonpathogen||B||B||B||A||C||B|
|CAV 279||Kiepersol, site 1, SA||03/03||Nonpathogen||B||B||B||A||C||B|
|CAV 212||Kiepersol, site 2, SA||10/02||Nonpathogen||B||B||B||A||C||B|
|CAV 280||Kiepersol, site 2, SA||03/03||Nonpathogen||B||B||B||A||C||B|
|CAV 281||Kiepersol, site 2, SA||03/03||Nonpathogen||B||B||B||A||C||B|
|CAV 282||Kiepersol, site 3, SA||03/03||Nonpathogen||B||B||B||A||C||B|
|5||CAV 236||Kiepersol, site 1, SA||10/02||Nonpathogen||C||A||C||C||E||B|
|CAV 261||Kiepersol, site 1, SA||03/03||Nonpathogen||C||A||C||C||E||B|
|CAV 271||Kiepersol, site 2, SA||03/03||Nonpathogen||C||A||C||C||E||B|
|CAV 249||Kiepersol, site 3, SA||10/02||Nonpathogen||C||A||C||C||E||B|
|6||CAV 224||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 225||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 226||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 229||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 230||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 242||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 243||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 252||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||A||C||B|
|CAV 256||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||A||C||B|
|CAV 258||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||A||C||B|
|CAV 264||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||A||C||B|
|CAV 210||Kiepersol, site 2, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 217||Kiepersol, site 2, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 220||Kiepersol, site 2, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 269||Kiepersol, site 2, SA||03/03||Nonpathogen||D||B||B||A||C||B|
|CAV 201||Kiepersol, site 3, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 205||Kiepersol, site 3, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 247||Kiepersol, site 3, SA||10/02||Nonpathogen||D||B||B||A||C||B|
|CAV 254||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||A||D||B|
|CAV 211||Kiepersol, site 2, SA||10/02||Nonpathogen||D||B||B||A||D||B|
|CAV 277||Kiepersol site, 2, SA||03/03||Nonpathogen||D||B||B||A||D||B|
|8||CAV 240||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||A||H||B|
|CAV 202||Kiepersol, site 3, SA||10/02||Nonpathogen||D||B||B||A||H||B|
|9||CAV 262||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||B||B||B|
|CAV 263||Kiepersol, site 1, SA||03/03||Nonpathogen||D||B||B||B||B||B|
|CAV 274||Kiepersol, site 2, SA||03/03||Nonpathogen||D||B||B||B||B||B|
|10||CAV 231||Kiepersol, site 1, SA||10/02||Nonpathogen||D||B||B||D||C||B|
|11||CAV 241||Kiepersol, site 1, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 255||Kiepersol, site 1, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 265||Kiepersol, site 1, SA||03/03||Nonpathogen||D||C||D||A||F||B|
|CAV 219||Kiepersol, site 2, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 270||Kiepersol, site 2, SA||03/03||Nonpathogen||D||C||D||A||F||B|
|CAV 273||Kiepersol, site 2, SA||03/03||Nonpathogen||D||C||D||A||F||B|
|CAV 200||Kiepersol, site 3, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 244||Kiepersol, site 3, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 248||Kiepersol, site 3, SA||10/02||Nonpathogen||D||C||D||A||F||B|
|CAV 253||Kiepersol, site 3, SA||03/03||Nonpathogen||D||C||D||A||F||B|
|12||CAV 233||Kiepersol, site 1, SA||10/02||Nonpathogen||E||B||B||A||C||B|
|CAV 209||Kiepersol, site 2, SA||10/02||Nonpathogen||E||B||B||A||C||B|
|CAV 245||Kiepersol, site 3, SA||10/02||Nonpathogen||E||B||B||A||C||B|
|CAV 246||Kiepersol, site 3, SA||10/02||Nonpathogen||E||B||B||A||C||B|
|IGS groupa||Number of isolates from site|
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).
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.
- 1990. Biological control of Fusarium wilt pathogens in suppressive soils. In : HornbyD , ed . Biological Control of Soil-Borne Plant Pathogens. Wallingford, UK: CABI Publishing, 27–43. ,
- 1993. Recent advances in the biological control of Fusarium wilts. Pesticide Science 37, 363–73. , , ,
- 1995. Intraspecific variation within populations of Fusarium oxysporum based on RFLP analysis of the intergenic spacer region of the rDNA. Experimental Mycology 19, 120–8. , ,
- 1996. Relationships among pathogenic and nonpathogenic isolates of Fusarium oxysporum based on the partial sequence of the intergenic spacer region of the ribosomal DNA. Molecular Plant–Microbe Interactions 9, 125–38. , ,
- 1999. Genetic characterisation and detection of Fusarium wilt. In : MolinaAB , MasdekNHN , LiewKW , eds . Banana Fusarium Wilt Management: Towards Sustainable Cultivation. Proceedings of the International Workshop on Banana Fusarium Wilt Disease, Los Banos. Laguna, Philippines: INIBAP Publications, 143–51. , , , , ,
- 1977. Fusarium: Laboratory Guide to the Identification of the Major Species. Kew, UK: Commonwealth Mycological Institute. ,
- 2002. Global evaluation of Musa germplasm for resistance to Fusarium wilt, Mycosphaerella leaf spot diseases and nematodes. INIBAP Technical Guidelines No 6 . Montpellier, France: INIBAP Publications. , , ,
- 1998. The use of species-specific PCR-based assays to analyse Fusarium ear blight of wheat. Plant Pathology 47, 197–205. ,
- 1995. Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathology 85, 579–85. , , , , ,
- 1997a. Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species. Mycological Research 101, 179–87. , , , ,
- 1997b. Populations of nonpathogenic Fusarium oxysporum associated with roots of four plant species compared to soilborne populations. Phytopathology 87, 693–7. , , , ,
- 2000. Ribosomal DNA-targeted oligonucleotide probe and PCR assay specific for Fusarium oxysporum. Mycological Research 104, 518–26. , , , ,
- 2001. Genetic diversity of Fusarium oxysporum populations isolated from different soils in France. FEMS Microbiology Ecology 36, 61–71. , , , , ,
- 1991. Analysis of vegetative compatibility groups in non-pathogenic populations of Fusarium oxysporum isolated from symptomless tomato roots. Canadian Journal of Botany 69, 2089–94. , , ,
- 2003. Fusarium oxysporum and its biocontrol. New Phytologist 157, 493–502. , , ,
- 1997. Non-pathogenic Fusarium oxysporum strain Fo47 induces resistance to Fusarium wilt of tomato. Plant Disease 81, 492–6. , , ,
- 1989. Colonization of muskmelon and non-susceptible crops by Fusarium oxysporum f.sp. melonis and other species of Fusarium. Phytopathology 79, 1095–100. , , ,
- 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology 66, 411–53. , ,
- 1991. Repetitive genomic sequences for determining relatedness among strains of Fusarium oxysporum. Phytopathology 81, 331–6. , , ,
- 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Review of Plant Protection Research 8, 115–25. ,
- 1996. Suppression of Fusarium wilt of watermelon by non-pathogenic Fusarium oxysporum and other microorganisms recovered from a disease-suppressive soil. Phytopathology 86, 812–9. , , ,
- 1991. Biological control of Fusarium diseases by fluorescent pseudomonas and non-pathogenic Fusarium. Crop Protection 10, 279–86. , ,
- 1981. Microbiological suppressiveness of some soils to Fusarium wilts. In : NelsonPE , ToussounTA , CookRJ , eds . Fusarium: Disease, Biology and Taxonomy. University Park, PA, USA: Pennsylvania State University Press, 261–75. , , ,
- 2003. Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria. FEMS Microbiology Letters 218, 329–32. , , ,
- 1965. Quantitative and qualitative comparison of Fusarium populations in cultivated fields and non-cultivated parent soils. Canadian Journal of Botany 43, 939–45. , ,
- 2006. The potential of nonpathogenic Fusarium oxysporum and other biological control organisms for suppressing fusarium wilt of banana. Plant Pathology, doi 10.1111/j.1365-3059.2006.01344.x . , , , ,
- 1983. Fusarium Species: An Illustrated Manual for Identification. University Park, PA, USA: Pennsylvania State University Press. , , ,
- 1999. Process of tomato root colonization by a pathogenic strain of Fusarium oxysporum f.sp. lycopersici in comparison with a non-pathogenic strain. New Phytologist 141, 497–510. , ,
- 2000. Fungal diseases of root, corm and pseudostem. In : JonesD , ed . Diseases of Banana, Abaca and Enset. Wallingford, UK: CABI Publishing, 143–59. , ,
- 1990. Importance of Fusarium wilt in different banana-growing regions. In : PloetzRC , ed . Fusarium Wilt of Bananas. St Paul, MN, USA: APS Press, 9–26. , , , , , , ,
- 1992. Biological control of Fusarium wilt of carnation with a non-pathogenic isolate of Fusarium oxysporum. Canadian Journal of Botany 70, 1199–205. , ,
- 1985. Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17–20. , ,
- 1980. Mechanism of biological control in a Fusarium-suppressive soil. Phytopathology 70, 412–7. , ,
- 1971. Relationship of inoculum density and soil types to severity of Fusarium wilt of sweet potato. Phytopathology 61, 1049–51. , ,
- 1972. Germination of Fusarium oxysporum chlamydospores in soils favourable and unfavourable to wilt establishment. Phytopathology 62, 273–7. , ,
- 1997. Phenotypic characterization of natural populations of Fusarium oxysporum in relation to genotypic characterization. FEMS Microbiology Ecology 24, 73–85. , , , , , ,
- 1962. Fusarial Wilt (Panama Disease) of Bananas and Other Musa Species. Kew, UK: Commonwealth Mycological Institute. ,
- 1961. Studies on Fusarium wilt of bananas. VII. Field control. Canadian Journal of Botany 39, 197–206. , , ,
- 2002. The status of Fusarium wilt (Panama disease) of banana in South Africa. South African Journal of Science 98, 341–4. ,
- 2003. Molecular biological studies of the Fusarium wilt pathogen of banana in South Africa. Pretoria, South Africa: University of Pretoria. PhD Thesis. ,
- 2002. Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annual Review of Phytopathology 40, 309–48. , , , ,
- 1963. Populations of Fusarium oxysporum f.sp. melonis and their relation to the wilt potential of two soils. Canadian Journal of Microbiology 9, 237–49. , ,