Genetic affinities of Fusarim spp . and their correlation with origin and pathogenicity

Random amplified polymorphic DNA (RAPD) analyses was used in combination with pathogenicity assays to study the taxonomic kinships among five Fusarium species. A total of 46 isolates of Fusarium spp. obtained from diseased cotton seedlings showing typical root rot and dampping-off symptoms were characterized. Of 10 primers tested, four primers produced polymorphic amplification patterns with taxon-specific bands, in addition to individual-specific bands. Genetic analysis indicated into 2 main clusters, with the minor cluster included all F. moniliforme and F. solani at the genetic similarity of GS=57.82%. The major cluster consisted of all F. oxysporum, F. avenaceum and F. chlamydosporum clustered at 71% similarity. There was no clear-cut relationship between clustering in the RAPD dendrogram, pathogenicity test and geographic origin of tested isolates. The results suggest that RAPD-PCR is a useful method for analysing genetic variation within and between Fusarium spp.


INTRODUCTION
Since Fusarium species are among the most common fungi associated with seedling roots of cotton, it is important to determine their effects on seedling growth.These fungal pathogens are frequently isolated from diseased roots of cotton seedlings (Johnson et al., 1978;Roy and Bourland, 1982;Colyer, 1988;Zhang et al., 1995;Aly et al., 1996;El-Samawaty 1999).Pathogenicity tests are cumbersome, time-consuming (50-60 days), require extensive facilities, and are influenced by variability inherent in the experimental system.Furthermore, pathogenicity data alone provide no information about genetic diversity within, or relatedness among, races of the pathogen.Knowledge of genetic diversity is needed for resistance deployment to be effective and to identify shifts in race or population structure that might occur (McDonald, 1997).
In recent years numerous DNA-based fingerprinting methods that reveal the genetic diversity of similar organisms have arisen.Random amplified polymorphic DNA (RAPD) analysis is a fast, PCR-based method of genetic typing based on genomic polymorphisms.A technique widely used to assess inter-and intra-specific *Corresponding author; phone: (49 431) 880 2993, fax: (49 431) 880 1583, e-mail: kaabdelsalam@msn.comAbbreviations: RAPD, random amplified polymorphic DNA; GS, genetic similarity; UPGMA, unweighted pair group method using arithmetic.genetic variation on a nuclear level is based on the use of random amplified polymorphic DNA (RAPD) markers, produced by the polymerase chain reaction (PCR) (Williams et al., 1990;Welsh and McClelland, 1990).RAPD analysis has been applied widely in the detection and genetic characterization of phytopathogenic fungi (Lanfranco et al., 1995;Miller, 1996;Brown, 1998), including race differentiation in several formae speciales of F. oxysporum, such as cubense (Bentley et al., 1994), dianthi (Manulis et al., 1994;Migheli et al., 1998), pisi (Grajal-Martin et al., 1993), and vasinfectum (Assigbetse et al., 1994).Abd-Elsalam et al., (2003) used RAPD markers to study inter-and intra-specific variation of twelve Fusarium species isolated from cotton-growing areas in Egypt.In this study, we examined the possibility of identifying and differentiating Fusarium spp.isolates using the RAPD method and their association with origin and pathogenicity.

Fungal isolates
Forty-six monosporic isolates of Fusarium species, reported to be associated with seedlings disease of cotton, were obtained from different geographic locations in Egypt (Table 2).

Pathogenicity test of Fusarium spp. on Giza 89
The growth medium consists of 100 g of sorghum grains and 80 ml of tap water in 500 ml glass bottles which were autoclaved for 30 minutes.Isolated inoculum, taken from one-weak-old culture on potato dextrose agar (PDA), was aseptically introduced into the bottle and allowed to colonize the sorghum for 3 weeks.The present test was carried out by using autoclaved clay loam soil.Batches of soil were infested separately with inoculum of each isolate at the rate of 50 g/kg of soil.Infested soil was dispensed in 10-cm-diameter clay pots and these were planted with 10 seeds per pot of cotton cultivar Giza 89.In the control treatment, sterilized sorghum grains were mixed thoroughly with soil at the rate of 50 g/kg of soil.Pots were randomly distributed on greenhouse bench under temperature regime ranging from 25 to 35 °C.Preemergence damping-off was recorded 15 days after planting.Postemeergence damping damping-off, survival, plant height (cm), and dry weight (mg/plant) were recorded 45 days after planting.

Genomic DNA extraction
DNA was isolated from mycelial mats of each single-spore isolate grown for 6 days at 28°C in 5ml of potato dextrose broth (PDB) (Difco) in 15-ml Falcon tubes.The mycelial mats were collected by filtration, mats were ground with a mortar and pestle in liquid nitrogen and stored immediately at -20°C.DNA was extracted from these preparations according to the method of Liu et al. (2000).

RAPD using arbitrary primers
For the development of the RAPD method for Fusarium isolates, the 10 different 10-mer primers (Kit A, Roth, Germany) (Table 1) were screened for the amplification of template DNA from five isolates of Fusarium spp.four primers were selected for final RAPD typing.Since PCR in the RAPD method is affected by the experimental parameters (Tyler et al., 1997).All of the screening steps in this study were performed under the conditions described below.DNA amplifications for the screening of primers or final typing were carried out in buffer (50 mM KCl, 1.5 mM MgCl2 , 10 mM Tris HCl, pH 8.8) containing 200 mM (each) dATP, dCTP, dGTP, and dTTP; 0.50 mM primer; 2.0 U of Taq DNA polymerase (Promega); and 50 ng of template DNA.The temperature cycling program used with a thermocycler (Primus 69 HPL, MWG Biotech, Germany) was as follows: 2 initial cycles consisting of 94°C for 4 min, 35°C for 2 min, and 72°C for 2 min, followed by 35 cycles consisting of 94°C for 30 s, 35°C for 1 min, and 72°C for 2 min and a final extension step consisting of 72°C for 5 min.All manipulations were carried out with dedicated DNA-free pipettes in a sterile hood to minimize the risk of contamination.

Gel electrophoresis
The amplified products were electrophoresed at 75 V in a horizontal 10-by 15-cm 1.5% agarose gel in Tris-borate buffer for about 4.5 h.The amplified DNA bands were visualized after ethidium bromide staining and photographed under UV light.A 100-bp ladder (Promega, Germany) was used as a marker in determining the sizes of the amplification products.

Computer comparisons of RAPD patterns
Digitized RAPD fingerprints were analyzed by using onedimensional software (Advanced American Biotechnology and Imaging, Fullerton CA 92831, USA).After normalization and background subtraction with mathematical algorithms, the levels of genetic similarity between RAPD patterns were calculated with the Pearson product-moment correlation coefficient (r) (Pot et al., 1994).For cluster analysis of RAPD banding patterns, the unweighted pair group method using average linkages (UPGMA) was used (Vauterin and Vauterin, 1992)

Pathogenicity test
Of the 46 isolates of Fusarium spp.tested under greenhouse conditions 38 isolates (82.4%) were pathogenic to seedlings of Giza 89.They included twenty three isolates of F. oxysporum, seven isolates of F. moniliforme, and five isolates of F. solani.Also, two isolates of F. avenaceum and one isolate of F. chlamydosporum were pathogenic (Table 2).Only one isolates of F. moniliforme was non-pathogenic during preemergence stage.Isolate no.24 of F. oxysporum showed peculiar behavior because it significantly reduced preemergence damping-off, that is, it significantly improved germination.Thirty-seven isolates were induced postemergence damping-off cotton seedlings.They included twenty one isolates of F. oxysporum, four isolates of F. solani, two isolates of F. avenaceum and seven F. moniliforme isolates.More than 75% F. oxysporum, F. moniliforme and F. solani isolates were pathogenic.

Identification of informative primers
To identify primers that generate informative arrays of PCR products, five Fusarium isolates were selected from the entire panel of isolates.They had been isolated from different geographic sites and belonged to different species.Ten oligonucleotides, each 10 nucleotides long, with a G/C content of 60 to 70%, and containing no palindromic sequences, were tested (Table 1).The choice of selected primers was based on the number of bands generated (with as few low-intensity bands as possible) as well as the quantity of different and reproducible patterns yielded.Four primers (2, 3, 7, and 9) were selected because they satisfied the characteristics described above.A set of reproducible bands produced for a particular primer was defined as a "pattern."

Interpretation of RAPD fingerprints performed with selected primers
To investigate the degree of genetic diversity among and within collections of Fusarium species, a total 46 isolates comprised of one isolate of F. chlamydosporum, two isolates of F. avenaceum, six isolates of F. solani, nine isolates of F. moniliforme and twenty-eight isolates of F.
oxysporum were analyzed.RAPD patterns amplified with primers 2, 3, 7, and 9 (Figure 1) were clearly differentiated within and between species.The PCR bands were more polymorphic between the species and less variable within the species.Genetic similarity (GS) values obtained among isolates of F. oxysporum, F. moniliforme and F. solani revealed remarkable differences for inter-and intraspecific comparisons.A UPGMA dendrogram is shown in Figure 1.The tree obtained indicated that genetic similarity between these isolates was high (57%).Nine F. moniliforme isolates were clustered into the first main cluster, the genetic similarity values between populations of this cluster ranged from 62 to 98%.Isolates Fm7 and Fm8 showed high genetic relatedness (98.91%).Isolates Fs1, Fs2, Fs3, Fs4 and Fs5 were grouped into a separate subcluster in the first main cluster.The second major cluster consist of all F. oxysporum isolates and F. avenaceum isolates at the genetic similarity of 71.40%, Isolates Fo1, Fo2, Fo3 and Fo4 showed very high genetic similarity of 98.50.Between Fusarium spp., similarities ranged from 57 to 71% for inter-specific comparisons.The genetic similarities ranged from 71 to 99% for intraspecific comparisons.High intra-specific variability was observed in F. oxysporum, F. solani, F. moniliforme and F. avenaceum.No tendency was detected between geographical origin and pathogenicity of isolates and genetic diversity.

DISCUSSION
The advent of DNA-based molecular methods has provided useful tools with which to study the phylogeny of Fusarium and to differentiate species, formae speciales, races, and strains.RAPD markers have been previously used to study inter-and intra-specific variation of twelve Fusarium species isolated from cotton-growing areas in Egypt (Abd-Elsalam et al., 2003).Donaldson et al. (1995) examined the usefulness of primer sets designed to amplify introns within conserved genes in filamentous ascomycetes to differentiate 35 isolates representing six different species of Fusarium commonly found in association with conifer seedlings.
The genetic similarity values of 57% between F. solani and F. oxysporum isolates in this study are in contrast with that of 20% between F. solani and F. oxysporum found by Szecsi and Dobrovolsky (1985).We can draw no conclusions for F. chlamydosporum because of the limited number of isolates tested.The polymorphisms observed for RAPD markers revealed a high degree of genetic diversity in Fusarium spp at the inter-specific level.Donaldson et al. (1995) found that the degree of inter-specific polymorphism observed in the PCR products from the six Fusarium species allowed differentiation by a limited number of amplifications and restriction endonuclease digestions.Our findings indicate no clear relationship between the RAPD profiles and pathogenicity tests/geographic origin for Fusarium spp., and the conclusions are in accord with these obtained by Abdel-Sattar et al. (2003) and Kiprop et al. (2002).Analyzing the association of specific RAPD markers and aggressiveness did not reveal a clear relation in a worldwide collection of 41 F. culmorum isolates (Miedaner et al., 1997).There were also no relationships among pathogenicity and RAPD profiles of F. avenaceum (Satyaprasad et al., 2000).Yli-Mattila et al. (1996) also found no correlation between isozymes, RAPD-PCR and UP-PCR patterns on the one hand, and either geographic origin or particular host plants on the other.Nevertheless, in a RAPD analysis of F. oxysporum, Assigbetse et al. (1994) reported a correlation between genetic similarity and geographic origin.RAPD analysis is extremely powerful and can separate individuals having intra-and inter-specific variability.It gives more comprehensive information regarding the genetic variability among the pathogen populations as it is based on the entire genome of an organism (Achenback et al., 1997).Our study showed that, RAPD technique is influential tool for discriminating different Fusarium species as well as Fusarium isolates within species.

Figure 1 .
Figure 1.Dendrogram obtained by genomic DNA fingerprinting with the random amplification of polymorphic DNA (RAPD) method.The percentage of genetic homology between banding patterns is indicated.Patterns of Fusarium spp.were combined with one-dimensional analysis software (Advanced American Biotechnology and Imaging, Fullerton CA 92831, USA) and grouped with the unweighted pair group algorithm with arithmetic averages (UPGMA).

Table 1 .
Code and nucleotide sequence of primers used in the random amplified polymorphic DNA (RAPD) reactions, G+C content, and number of polymorphic bands produced in the Fusarium spp.isolates studied.

Table 2 .
List of Fusarium isolates indicating location and effect of Fusarium spp.isolates on Giza 89 cotton seedlings growing in artificially infested soil under greenhouse conditions (1998).Percentage data were transformed into arsine angles before carrying analysis of virulence.bValues of a column followed by the same letter(s) are not significantly (P£0.05) according to Duncan´s multiple range test.* an asterisk denotes a significant difference from the control. a