Differentiation between Tomato Fusarium Wilt Isolates by ISSR Markers and Virulence Analysis

Fusarium species cause various diseases in many host plants (Summerell et al., 2003; Booth, 1971). These soilborne fungi are economically crucial because many of their members cause root rot and vascular wilt diseases in cultivated crops worldwide (Woo et al., 1998). Because of its global distribution in soil, F. oxysporum has been dubbed the global mycoflora (Parkinson, 1981). Fusarium species are commonly identified using their macro and microscopic characteristics. However, these characteristics are generally reported to be unstable (Szecsi and Dobrovolsky, 1983; Nelson et al., 1985). The morphological, biochemical, and allozyme characteristics are the most commonly used methods for pathogens identification, which require professional experience and are still subject to error (Kheterpal, 2006). Currently, pathogens identification is based on nucleotide sequence and genetic markers analysis. DNA markers used in genetic variation analysis are characterized by high genetic divergence and the ability to detect multilocus data in the genome (Anne, 2006). Molecular markers such as Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), Cleaved Amplified Polymorphic Sequences (CAPs), and Inter Simple Sequence Repeats (ISSR) are widely used in constructing fungi genetic maps. ISSR markers amplify microsatellite DNA sequences, which are highly varied and widespread in the genome. ISSR is an efficient multilocus technique that enables scoring multiple polymorphic loci and does not need any previous sequence information; however, it is limited by repeatability issues. Furthermore, the ISSR technique has high stability, accuracy, and efficiency compared to morphological assessment, so it has been widely used in genetic diversity studies (Williams et al., 1990; Guo et al., 2001). Troncoso-Rojas et al. (2013) use ISSR and RAPD makers to evaluate the genetic diversity and genotyping of Alternaria solani isolates. DNA markers such as RFLP, AFLP, RAPD, ISSRs, simple sequence repeats (SSR), and expressed sequence tags (EST) were used in the analysis of the genetic variation in various F. oxysporum formae speciales (Bogale et al., 2005; Dubey and Singh 2008; Dubey et al., 2010; Sharma et al., 2014; Yuan et al., 2013). Singh and Kapoor (2018) used ISSR markers to develop sequence-characterized amplified region (SCAR) markers to identify and quantify Fusarium oxysporum f. sp. carthami. Molecular tools are the most accurate for identifying plant pathogens. Their use has increased in recent years due to the availability of specific equipment and chemicals and their ability to identify the types and aggressiveness of isolates (Adss et al., 2017). This study aimed to isolate, purify and identify F. oxysporum. f. sp. lycopersici isolates from six governorates of Egypt and differentiate these isolated using ISSR- PCR and virulence test.

MATERIALS AND METHODS

1. The causal fungal sample collections

Eight fungal isolates were isolated from infected tomato plants with typical wilt symptoms collected from six governorates in Egypt; El-Menofia (Talla and Shebeen Elkom), El-Menia, Assiut, Kafr El-Sheikh, Alexandria, El-Beheira (Abou-Homus and Kom Hamada).

2. Isolation and identification of the causal fungi

The collected tomato root rot samples were thoroughly washed in tap water and air-dried. Small pieces were taken from the internal tissues adjacent to the affected root parts, surface sterilized in 3 % sodium hypochlorite solution for 3 mins., rinsed in sterile distilled water, and dried between sterilized filter papers. After that, samples were transferred to PDA plates containing streptomycin sulphate (0.2g/L) and incubated at 28^oC for five days (Shaukat et al., 2005). The hyphal tip technique was used to purify the isolated fungi, as described by Brown (1924). Identifications of the causal fungi were made at the Department of   Plant Pathology, Faculty of Agriculture, Damanhour university, according to Gilman (1957), Singh (1982), and Summerell et al. (2003). The frequency of the fungal isolates recovered from the surveyed governorates was calculated

3. Pathogenicity test

The eight F. oxysporum f. sp. lycopersici isolates were tested for pathogenicity on the Carmen tomato cultivar. Tomato seeds were sterilized for five min. in 1% sodium hypochlorite, rinsed with sterile water, and sown in 20cm diameter plastic pots filled with 3Kg sterilized peat moss, clay, and sand (1:1:1). For Fungi inocula preparations, pathogens were cultured on Potato Dextrose Broth (PDB) medium in 250ml conical flasks and incubated at 25 ± 2°C for 10 days. The mycelial cultures were then filtered, and the spore suspensions were adjusted to 10⁶ conidia/ml using a haemacytometer slide. Plants were inoculated when they reached 15-20cm tall by adding 50 ml of the prepared conidial suspension to 1kg of soil (Elad and Baker,1985).

4. Disease assessments

The severity of wilting was rated 15-day post-inoculation (dpi) using a pathogenicity scale of 6 grades, according to Liu et al. (1995) (Table 1).

Disease severity (%) = {Σ (Rating no x no. plants in rating category) / (Total no. plants x highest rating value)} × 100

Table 1. Assessment scale of disease severity of F. oxysporum of tomato according to Liu et al. (1995).

5. Differentiation between F. oxysporum isolates using ISSR analysis

5.1. Fungal DNA extraction

Fungal genomic DNA was isolated by Wizard Genomic DNA purification kit (Promega, Germany) according to the manufacturer's procedures.

5.2. ISSR reactions

PCR amplification was performed using nine ISSR primers (Table 2) to differentiate and fingerprint the F. oxysporum isolates. PCR reaction (25µl) contained 10x PCR buffer (2.5µl), 4mM dNTPs (2.5µl), 50mM MgCl₂ (2.5µl), 50pmol primer (7µl), 50ng of fungal genomic DNA (1µl), and 5 units/µl Taq DNA polymerase (0.2µl) (Promega, Germany). The PCR program was: initial denaturation at 95ºC for 5 min, then 40 cycles of 95ºC for 1 min, annealing at a temperature depending on primer for 1 min (Table 2), extension at 72ºC for 1 min, and a final extension at 72ºC for 10 min (Istock et al., 2001). The amplified DNA products were electrophoresed on 1.5% (w/v) agarose gel in 0.5 x TBE buffer, stained with ethidium bromide (0.5 µg/cm3, w/v), and photographed using a gel documentation system.

Table 2. ISSR primers sequences and annealing temperature used in this study.

5.3. Construction of dendrogram based on ISSR-PCR DNA banding patterns.

ISSR-PCR banding patterns for each primer were recorded depending on the presence and absence of the amplified bands. A band is scored as (1) or (0), depending on whether it is present or absent. Multi-Variate Statistical Package (MVSP) Version 3.1 was used to compare the F. oxysporum isolates and the unweighted pair group method with arithmetic averages (UPGMA) used to compute the similarity coefficients and to conduct cluster analysis.

6. Statistical analysis

Disease severity data were analyzed with SAS ver. 9.4 (SAS Institute Inc., Cary, NC, USA), and the least significant difference (LSD) test was used to differentiate means at a p ≤ 0.05.

RESULTS AND DISCUSSIONS

F. oxysporum. f. sp. lycopersici is the disease agent of tomato fusarium wilt and one of the most critical tomato pathogens, and it causes significant damage and economically severe crop losses, particularly at high temperatures and humidity in the indoor environment(Jones et al., 1982; Smith et al., 1988; Agrios, 2005).

1. Isolation and Identification of F. oxysporumisolates

Eight isolates of F. oxysporum (FOT) were obtained and identified from tomato-infected plants (Table 3) collected from El-Menofia (Talla (FOT1) and Shebeen Elkom (FOT2)), El-Menia (FOT3), Assiut (FOT4), Kafr El-Sheikh (FOT5), Alexandria (FOT6), El-Beheira governorate (Abou-Homus (FOT7) and Kom Hamada (FOT8)).

2. Pathogenicity test

The eight F. oxysporum isolates were tested for pathogenicity on the highly susceptible tomato cultivar Carmen. Data presented in Table (3) and Fig (1) showed that all eight isolates were virulent. Isolates FOT4 from Assiut, FOT2 from El-Menofia (Shebeen Elkom), andFOT5 from Kafr El-Sheikh were highly virulent. In comparison, isolates FOT6 from Alexandria and FOT8 from El-Beheira (Kom Hamada)were moderately virulent. While isolates FOT7 from Abou Homus, FOT3 from El-Menia and  FOT1 from El-Menofia (Talla) were weakly virulent. These results agreed with those of Abou-Zeid et al. (2016). They obtained six F. oxysporum isolates from diseased pepper collected from Gharbya and Dakahlya governorates in Egypt. The F3 isolate from Dakahlya was more aggressive (96.67% wilt severity) than Tanta F4 isolate (71.67% severity).

Table 3. The pathogenicity test of eight F. oxysporum. f. sp.  lycopersici isolates on Carmen tomato cultivar

Fig. 1. Reaction of Carmen tomato cultivar to the infection with eight F. oxysporum. f. sp.  lycopersici isolates

Fig.  2. ISSR-PCR using primer A: HB-09, B: HB-12, C: HB-14, D: UBC814, E: UBC840, F: UBC868, G: UBC835, H: HB-11 and I: HB-13.  M: DNA marker. Lanes 1: FOT1, Lanes 2: FOT7, Lanes 3: FOT3, Lanes 4: FOT5, Lanes 5: FOT6, Lanes 6: FOT2, Lanes 7: FOT8 and Lanes 8: FOT4.

Fig. 3. The dendrogram shows the phylogenetic relationship among the eight F. oxysporiumbased on ISSRs analysis.

Table 4. A similarity matrix between the tested F. oxysporum isolates based on ISSR band pattern analysis and Jaccard index.