Genetic variability among Fusarium oxysporum isolates from melon ( Cucumis melo ) in Qazvin province , Iran

Fusarium wilt is a soilborne disease which is the result of Fusarium oxysporum Schl. This fungi is one of the most important pathogens, which is causes wilting in some crops such as cantaloupe, cucumber, wax gourd, muskmelon, watermelon, and other types of melon. Melon (Cucumis melo) of the Cucurbitaceae family is one of the most important agricultural products in Qazvin Province, Iran. Fusarium is a soilborne fungus that is found in all soils and has global distribution. All isolates are able to survive in soil and growth in the rhizosphere area of many plant species. In the previous reports, more than 120 different formae speciales of F. oxysporum have been described based on host specificity (Armstrong and Armstrong, 1981). Wilting symptoms are the result of fungal spores and mycelium that block the xylem. Infected plants looking healthy at the beginning, but the vascular tissue is brown and discolored. To support morphological identification of Fusarium species, we can use of diversity in DNA sequences of genes. Furthermore, to estimate the genetic relationship for F. oxysporum we can use of phylogenetic analysis on DNA sequences (Hsuan et al., 2011; Harrow et al., 2010). Genetic diversity of a particular “forma specialis” revealed by using of multitude ways. Those include restriction fragment length polymorphism (Koenig et al., 1997), random amplified polymorphic DNA (RAPD) (Windels, 1992), amplified fragment length polymorphism )Leslie et al., 2006). There aren’t any affective fungicides for controlling this pathogen on melon. Furthermore, crop rotation is not effective because isolates on the melon plant can live on the other crops (Gordon et al., 1989). Hence, host resistance is the best way for disease management. To host plant resistance breeding, we must know if exists the variability within the pathogen population. Therefore, the present research study was performed to explain variation among F. oxysporum isolated from melon using DNA sequencing of translation elongation factor 1α (TEF1α). The goal of this study was to determine phylogenetic relationship among F. oxysporum isolates, gathered from melon plants, and determine diversity correlates with location origin and pathogenicity. Genetic variability among Fusarium oxysporum isolates from melon (Cucumis melo) in Qazvin province, Iran


INTRODUCTION
Fusarium wilt is a soilborne disease which is the result of Fusarium oxysporum Schl.This fungi is one of the most important pathogens, which is causes wilting in some crops such as cantaloupe, cucumber, wax gourd, muskmelon, watermelon, and other types of melon.Melon (Cucumis melo) of the Cucurbitaceae family is one of the most important agricultural products in Qazvin Province, Iran.Fusarium is a soilborne fungus that is found in all soils and has global distribution.All isolates are able to survive in soil and growth in the rhizosphere area of many plant species.In the previous reports, more than 120 different formae speciales of F. oxysporum have been described based on host specificity (Armstrong and Armstrong, 1981).Wilting symptoms are the result of fungal spores and mycelium that block the xylem.Infected plants looking healthy at the beginning, but the vascular tissue is brown and discolored.To support morphological identification of Fusarium species, we can use of diversity in DNA sequences of genes.Furthermore, to estimate the genetic relationship for F. oxysporum we can use of phylogenetic analysis on DNA sequences (Hsuan et al., 2011;Harrow et al., 2010).Genetic diversity of a particular "forma specialis" revealed by using of multitude ways.Those include restriction fragment length polymorphism (Koenig et al., 1997), random amplified polymorphic DNA (RAPD) (Windels, 1992), amplified fragment length polymorphism ) Leslie et al., 2006).There aren't any affective fungicides for controlling this pathogen on melon.Furthermore, crop rotation is not effective because isolates on the melon plant can live on the other crops (Gordon et al., 1989).Hence, host resistance is the best way for disease management.To host plant resistance breeding, we must know if exists the variability within the pathogen population.Therefore, the present research study was performed to explain variation among F. oxysporum isolated from melon using DNA sequencing of translation elongation factor 1α (TEF1α).The goal of this study was to determine phylogenetic relationship among F. oxysporum isolates, gathered from melon plants, and determine diversity correlates with location origin and pathogenicity.

MATERIALS AND METHODS
In recent years, prevalence a disease had caused a severe decreasing in melon yield in Qazvin Province, Iran (Middle of Iran, 36°15'N -50°0'E).Disease symptoms in melon farmlands were similar to Fusarium wilt.F. oxysporum consists of pathogenic and non-pathogenic strains that are morphologically indistinguishable.

Sampling, Identifi cation, and Storage
Melon plants symptoms of Fusarium wilt were elected from melon farmland of Qazvin province from 2013 to 2015.Each sample was rinsed under running water for 20 min.Six pieces from root, crown, and stem (5 mm) were surface sterilized with 2% NaCl 2 for 2 min.These pieces were cultured onto potato dextrose agar (PDA; 200 g potato, 17 g glucose and 17 g agar/L) supplemented with 1 drop lactic acid.Petri dishes were incubated at 25°C for 10 days under fluorescent light (12 h photoperiod) (Nirenberg, 1976).Morphological identification was done on PDA and carnation leaf agar medium according to the morphological criteria keys of Nelson et al. (1983).For long-time storage, mycelium from F. oxysporum was stored desiccated on sterile glass fiber filter paper at −18°C (Hanson and Hill, 2004).

Pathogenicity Tests
A total of 120 Fusarium isolates cultured from diseased melon, tomato and watermelon were surveyed for pathogenicity on a Fusarium wilt susceptible melon plant Minoo 095P ergon (Ergon International N.V.The Netherlands).All isolates of Fusarium were grown in 250 ml volume conical flask, containing 100 ml Armstrong broth which is used for mass multiplication of inoculum of Fusarium (Booth, 1971).The flasks were incubated at 27°C, 120 rpm for 6 days into an incubator shaker.After 6 days, all cultures of Fusarium isolates were filtered through a muslin cloth before adjusting the spore density with distilled water to 2 × 10 7 spores/g of soil mixture in pots.Melon seedlings variety Minoo 095P was raised in steam sterilized soil.21 days seedlings were transferred into the root trainers.The experiment was carried out in a greenhouse at 27°C and 13 h light period (600 lux).Plants were irrigated as and when required and supplied with Agrimel's solution weekly.Four replications were maintained for every isolate of F. oxysporum.The disease incidence was measured after 4 weeks of transplantation (Joshi et al., 2013).All tests were conducted at least twice.Disease severity was recorded using a scale containing 4 grades suggested by Matsumoto et al. (2011): Grade: 0 = No symptoms, 1 = Small lesions on leaves, 2 = Leaves strongly affected, 3 = Plant death.After 4 weeks, roots and crown were removed to reisolate and confirmed the presence of Fusarium species.Disease index (DI) values were analyzed using PROC MIXED (SAS) with t-test (Dunnett, 1955), to determine if there were significant differences among our isolates or not.Half (50%) of the isolates from different locations and pathogenicity were selected for phylogenetic analysis (Hill et al., 2011) (Table 1).

DNA Extraction from Fungal Cultures
Each fungal culture was inoculated in liquid potato dextrose broth and grown on a rotary shaker at 24°C for 5 days 100 rpm with 10 h of light per day and aeration.The fungal mycelium was harvested, ground to fine powder in liquid nitrogen.Genomic DNA was extracted from each culture by cetyltrimethylammonium bromide method.DNA samples were dried under vacuum and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), followed by incubation with RNase A at a concentration of 100 μg/mL at 37°C for 1 h (Gurjar et al., 2009).

DNA Amplifi cation
We used an intron region of TEF1α, to describe in F. oxysporum isolated from diseased melon plants.The primer used was described initially by O'Donnell et al.

Sequencing and Phylogenetic Analysis
The PCR products were purified using a Gene Elute PCR clean-up kit (Sigma, USA) pursuant to the manufacturer's protocol.The purified PCR products sent for sequencing to a service provider.The EF1α sequences were aligned with clustalx-2.1 (Thompson et al., 1997).Then, default gap penalties were used in the pair-wise alignment.A neighbor-joining tree was delineated with the Jukes-Cantor model.The reliability of neighbor-joining trees was calculated by bootstrap method with 1000 random stepwise along with replicates.An isolate of Fusarium culmorum was selected as the outgroup.

Species Identifi cation
Based on the morphological characteristics of isolates from farmlands, we identified F. oxysporum, Fusarium acuminatum, Fusarium graminearum, Fusarium proliferatum, and Fusarium solani.Identification based on morphological characteristics of our isolates was corroborated by comparison with sequences of their isolates in the GenBank.

Pathogenicity Testing and Determination
In the field and greenhouse, symptoms were include combinations of wilting, yellowing and chlorosis.Severely infected plants were wilted and died, but plants affected to a lesser degree became unproductive.In some cases, signs of decay on roots were observed.The most prominent internal symptom was vascular browning in plants.Symptoms of Fusarium wilt occurred after 37 days from the start.Reisolation was done from all the plants.Any signs of disease observed in the control plants.
All isolates were considered nonpathogenic (NP) in the experiment, when the F-test indicated no significant differences among our isolates and also control (Table 1).When our isolates indicated significant differences, t-test was used to compare all isolates with the control treatment, with P = 0.05.Those isolates (at least two times of experiments) which were significantly higher DI than the control treatment were calculated pathogenic (P).Those isolates which had a significant differences from the control treatment in few experiments but not all of the experiments, were calculated, low pathogenic (LP).26 were identified as F. oxysporum and surveyed for pathogenicity on melon seedlings among of 50 Fusarium species that were gathered from melon and tomato in 2013.All of these were pathogenic in pathogenicity test on melon seedlings.In 2014, 40 Fusarium cultures were isolated from melon that 12 were identified as F. oxysporum and surveyed for pathogenicity.Six isolates of these isolates were pathogenic on melon plants.Furthermore, one isolate of F. solani and F. proliferatum identified that were NP.In 2015, 30 Fusarium cultures were isolated from melon that 9 isolates of F. oxysporum were collected from melon and watermelon were tested, and of these isolates, only three were pathogenic on melon plants.Seven F. oxysporum cultures isolated from watermelon in 2014 and 2015 (S4, S6, S9, A16, A17, AV7, AV9) were distinguished as non-pathogenic on melon seedlings.Moreover, three F. acuminatum were identified that all of them were LP.We had three F. graminearum cultures isolates from melon that all of them were non-pathogenic on melon seedlings (Table 1).

Phylogenetic Analysis
Phylogenetic tree from neighbor-joining showed progressively increasing resolution.Datasets were including five small groups of isolates (III-VIII) and two large groups (I, II) (Figure 1).Group (I) is resolved into smaller groups and groups (V-VIII) were found at the base of the tree (Figure 1).In clade (I), F. oxysporum from melon and watermelon grouped with NP (F14, F16, F22, F23, S4, S6, S9, AV7, AV9) from melon.

DISCUSSION
F. oxysporum is due to vascular wilt diseases in a large variety of important crops (Beckman, 1987).Fusarium can attack to vascular system of the plants and secrete its toxins, which cause the vascular wilting and then death within a few days or weeks (Champaco et al., 1993).Phylogenetic relationships in F. oxysporum have been studied by many researchers to determine evolutionary orientation among Fusarium species and to distinguish the dynamism in the different formae speciales (Gordon and Martyn, 1997).Using conserved gene regions in some of studies have showed that formae speciales of F. oxysporum are monophyletic, but some of other studies have distinguished polyphyletic groups (Koenig et al., 1997;Jimenez Gasco et al., 2002).
In this study, the phylogeny and the pathogenicity of F. oxysporum isolates were analyzed.et al., 1997).However, genetic resistance may enable management in some locations, but some of the farmers have announced that managing of Fusarium wilt is decreased in different conditions.Although, they are ubiquitous but, the variability and pathogenicity of these isolates are rarely reported (Gordon and Okamoto, 1992).
A lot of studies of genetic diversity have been presented the possible correlation between genotypes and pathotypes (Nelson et al., 1997;Vakalounaki and Fragkiadakis, 1999).Variability in resistance in different locations shows races within F. oxysporum f. sp.melonis (Fom) (Jimenez-Gasco et al., 2002).It can propose races in Fom has been firmly-fixed on cross-pathogenicity among of isolates of melon, tomato and watermelon.However, in this study genetic variability and geographical locations had no obvious correlation for the F. oxysporum isolates.This is similar to a lack of correlation between the UP-PCR (by EF1α) and geographic location that is reported for F. oxysporum (Zhao et al., 2014).This is the opposite of a correlation observed between the RAPD pattern and geographic locations in a previous research study (Assigbetse et al., 1994).
Both in the field and greenhouse, the environmental conditions may participate diversity in pathogenicity response.Many of researchers found that inoculation quantity, plant variety, inoculation method, plant stage, and temperature can effect on the pathogenicity of Fom isolates.The cross pathogenicity of F. oxysporum isolates and their polyphyletic nature can be get the resistance breeding attempts much tough than other fungi.These findings would be required to assess tactics of resistance breeding in field or greenhouse conditions.
This research study indicates the first molecular phylogenetic studies of F. oxysporum from melon in Qazvin province, Iran as compared to Zhao et al. (2014), which compared some species using EF1α in Liaoning Province, China.This work supports the importance of expanding genetic markers to achieve a better perception of the evolutionary structure of fungi especially Fom.Furthermore, This study was include more geographical locations and similar numbers of isolates from each geographical location, so showed a more precise picture of aggregations between location origin, pathogenicity and phylogeny than other research studies.

Figure 1 :
Figure 1: Neighbor-joining tree of 60 isolates of Fusarium oxysporum inferred from translation elongation factor 1α using Jukes-Cantor method.An isolate of F. culmorum was used as the out group

Table 1 : Geographic origin and pathogenicity on melon seedling (variety Minoo 095P) of Fusarium oxysporum, Fusarium acuminatum, Fusarium graminearum, Fusarium proliferatum and Fusarium solani cultured from melon, tomato and watermelon
The results indicated the genetic variability in Fusarium spp.from different locations in Qazvin Province.They indicated the genetic similarities and differences among the tested Fusarium spp.In this study, F. oxysporum strains from melon being placed in clades next and close to F. oxysporum strains from watermelon and tomato that show probably F. oxysporum from melon plants is polyphyletic.Clade (I) contained of pathogenic and NP isolates from Takestan, Buein zahra, Sagez abad, and Avaj.The isolates of F. solani, F. proliferatum, F. acuminatum, and F. graminearum evaluated form four clades separated each other which situate out of the four major clades.The identification isolates of F. oxysporum, F. solani, F. proliferatum, F. acuminatum, and F. graminearum by morphological methods supported with the results of the sequence of BLAST analysis.