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Isolation and characterization of Fusarium oxysporum, a wilt causing fungus, for its pathogenic and non-pathogenic nature in tomato (Solanum lycopersicum)

Collection of soil and plant samples from nine different geographical locations in Uttar Pradesh state of India was made. Composite soil was analyzed for its nutrient status. A total of sixty isolates of Fusarium species were recovered from the soil and plant samples. Among these, thirty nine isolates were identified as Fusarium oxysporum on the basis of their morphological and molecular identification. The pathogenicity test was conducted on tomato variety Pant T-3, disease incidence ranged from zero to 78.74%. Among F. oxysporum isolates, five were non pathogenic and three were found strongly pathogenic.Non-pathogenic isolates were tested for their antagonistic effect against most pathogenic isolates of F. oxysporum. The results showed that the Isolate no. 40 showed the highest antagonistic activity in inhibiting radial growth of pathogenic isolates.


INTRODUCTION
Fungi are an extremely versatile class of organisms comprised mostly of saprophytes, thriving on dead organic material. The soil-borne fungus, Fusarium oxysporum is the causal agent of vascular wilt, a disease that affects a large variety of economically important crops worldwide (Ortoneda et al., 2004) (Groenewald, 2006). Identification of Fusarium species by its morphology is notoriously difficult. Fusarium morphology, especially its conidiogenesis, can be easily changed by environment, particularly in the composition of the culture medium (Donnell, 2000). Generally, the appearance of a fungal culture, which results from its metabolism, is regulated by pH in association with the nitrogen source, in the medium (Kwaśna and Bateman, 2005). So the study of effect of abiotic factor, like pH and light on the growth of F.oxysporum is important. Tomato (Solanum lycopersicum) is one of the world's most cultivated vegetable crop, and amongst many, Fusarium wilt caused by F. oxysporum f. sp. lycopersici is one of the important disease (Snyder and Hansen, 1940) responsible for serious economic losses. Generally it occurs in midsummer when air and soil temperatures are high. Diseased plants develop yellowing of the older leaves (those near the ground). Often the yellowing is restricted to one side of the plant or to leaflets on one side of the petiole. At the nursery stage, plants infected by F. oxysporum may wilt and die soon after symptoms appear (Kennelly, 2007). The disease in field conditions appears late in the crop growth and therefore chemicals become quite ineffective. Moreover, the use of chemicals is not environmental friendly and economically viable, so, alternative methods of controlling the disease have been studied, with emphasis on biological control of Fusarium wilt. Naturally suppressive soils to Fusarium wilt have been reported in many regions of the world (Alabouvette, 1999;Silva and Wagner, 2005). Suppressive soils are characterized by a very low level of disease development even though a virulent pathogen and susceptible host are present. Biotic and abiotic elements of the soil environment contribute to suppressiveness of soil, however most defined systems have been identified as biological elements as primary factors in disease suppression. Many soils possess similarities with regard to microorganisms involved in disease suppression, while other attributes are unique to specific pathogen-suppressive soil systems. The organisms operative in pathogen suppression does so via diverse mechanisms including competition for nutrients, antibiosis and induction of host resistance (Mark 2002). Most of the biocontrol agents have been isolated from soils naturally suppressive to Fusarium wilt (Silva and Wagner 2005). This has led to the isolation and identification of antagonistic microorganisms from the soil. The use of biocontrol agents is a viable alternative for minimizing the yield loss. However, the survivability of a biocontrol agent, especially in tropical countries where the soils are in general, poor in organic matter has to address properly. Therefore, use of biocontrol agent of the same nature as pathogen could be an alternative. Non-pathogenic antagonistic Fusarium is a viable alternative (Minuto et al. 1995ab). These saprophytic species of non-pathogenic Fusarium have been found to be effective and play a critical role in reducing diseases caused by pathogenic F. oxysporum in cyclamen (Cyclamen persicum Mill.), gerbera (Gerbera jamesonii Hook.), basil (Ocimum basilicum L.), asparagus (Asparagus officinalis L.), eggplant (Solanum melongena L.), carnation (Dianthus canyophyllus L.), watermelon (Citrullus lanatus), tomato (Solanum lycopersicum L.), (Minuto et al., 1995ab;Larkin and Fravel, 1999;He et al., 2002;Reid et al., 2002;Silva and Wagner, 2005) and colonize the plant rhizosphere and roots without inducing any symptoms (Benhamou and Garand, 2001). The non-pathogenic antagonistic F. oxysporum have the same characteristics as pathogenic except that they are not disease causing and hence are important because these organisms can sustain up to the crop duration. A study was therefore undertaken to isolate the F. oxysporum from a large variable edapho-climatic condition, effect of abiotic factors on the growth under in-vitro, and characterize them as pathogenic/nonpathogenic in tomato.

MATERIALS AND METHODS
Soil survey and sampling: Nine different geographical locations of Uttar Pradesh, a state of India were surveyed and samples of plants along with the soil were collected from tomato fields. Collection of plant and soil samples was done from ninety two fields i.e, of Bareilly (13 fields), Barabanki (32 fields), Kanpur (28 fields), Baheri (4 fields), Hardoi (3 fields), Pilibheet (6 fields), Furrukhabad (3 fields), Lakhimpur ( 2 fields) and Lucknow( 1 field)] Uttar Pradesh (Table 1). Each field represented five hundred square meters. Soil and root samples from every field (five samples were collected from each corner and the center of field) were, taken, pooled and mixed well into a single composite sample and kept within a plastic bag. Moist soil samples were immediately stored in sealed plastic bags at 4°C. Fungal isolation was done within one week of sampling. Soil nutrient analysis: All ninety two soil samples were used for soil nutrient analysis. Handling of large number of samples can make the of soil nutrient analysis difficult. Therefore, among these ninety two soil samples and the samples collected from nearby field were pooled and made twenty eight composite samples ( Table 2). Analysis of all twenty eight composite soil samples was done for oxidizable organic carbon (Walkey and Black, 1934), available nitrogen (Subbiah and Asija 1956), available phosphorus (Olsen et al., 1954) and potassium by Beckman DU flame photometric measurement. Electrical conductivity and pH was measured by COV 510 bench meter (Eutech Instrument Pte Ltd/Oakton Instruments) and MAC digital pH/mv meter MSW-552, respectively. Isolation of the fungi: Fusarium spp. was isolated from soil and root sample of tomato plants collected from the fields. Potato dextrose agar is a nutrient rich medium for growing a wide range of fungi therefore it is used10 -4 at a dilution for pour plating method of Ofunne (1999). For fungal isolation from plant, the roots were washed under tap water, chopped into 2 cm small pieces and surface sterilized in 0.5% NaOCl for two minutes then rinsed twice with triple distilled water and placed on PDA and finally kept in an incubator at 27°C under dark conditions. All the procedure was carried out into laminar hood under sterilize condition. After five days of incubation, small colonies of fungus appeared, which were picked with a sterilized tooth pick and transferred to fresh PDA plates. Morphological characterization of the isolated fungi: A total of sixty isolates of Fusarium were identified on the basis of colony morphology, morphological characteristic of macro-and micro-conidia and conidial measurement. Among these Fusarium isolates, F. oxysporum species was identified using manual of Booth (1971). Conidial measurement was carried out using Motic image plus 2.0 software at 400X magnification. Molecular identification of F. oxysporum using species specific primers: DNA of all sixty Fusarium isolates along with positive control (F. oxysporum, MTCC No. 4353) was isolated using the method given by Raeder and Broda (1985). Species specific primers, FOF1 (5'-ACA TAC CAC TTG TTG CCT CG-3') and FOR1 (5'-CGC CAA TCA ATT TGA GGA ACG-3') were used for the identification of Fusarium oxysporum (Nel et al., 2006). PCR Reactions were carried out in a 20µl reaction volume Sample no. at 27°C in continuous dark and alternate 12 hours dark and light conditions. The data for radial growth of F. oxysporum isolates were measured after 72 and 92 hours. Pathogenicity tests: All thirty nine isolates of F. oxysporum were grown in 250 ml volume conical flask, containing 100 ml Armstrong broth which is used for mass multiplication of inoculum of F. oxysporum (Booth, 1971). The flasks were incubated at 27°C, 120 rpm for six days into an incubator shaker. After six days, all cultures of F. oxysporum were filtered using four layers of muslin cloth for separating mycelium with spores. One gram mycelium of each isolate of F. oxysporum along with (12 ml) conidia (2x10 7 /ml) was inoculated in between sand layers into root trainers containing eighty grams sand. Tomato seedlings variety Pant T-3 was raised in steam sterilized soil. Twenty one days seedlings were transferred into the root trainers. The experiment was conducted in a glass house at 27°C and fourteen hours light period (500 lux). Plants were irrigated as and when required and supplied with Hoagland's solution weekly. Four replications were maintained for every isolate of F. oxysporum. The disease incidence was calculated after twenty days of transplantation.

In-vitro antagonistic assay between pathogenic and non pathogenic Fusarium:
Based on the pathogenicity test of tomato crop, the most virulent strains of F. oxysporum Isolate no. 21, 22 and 27 were used against most promising five non-pathogenic F. oxysporum (Isolates no. 16, 29, 37, 40 and 50) of F. oxysporum, which did not give any symptom of wilt disease. To check the antagonistic property of non-pathoginic F. oxysporum isolates against pathogenic strains, 5mm disc of fully grown PDA plate of the pathogenic as well as non pathogenic F. oxysporum were cut and both the culture disc of pathogenic and non pathogenic F. oxysporum inoculated at the opposite ends of the PDA plates. Both pathogenic and non pathogenic F. oxysporum were also inoculated on petri plates separately which served as control. The antagonistic potential of different isolates of F. oxysporum was observed by measuring the radial growth of pathogenic F. oxysporum isolates and percent inhibition in the growth of the pathogenic F. oxysporum was recorded comparing with control. The percent inhibition in growth was calculated according to the following formula Percent inhibition= (C-T) X 100/C Where C, indicated as the radial growth of the test pathogen in control plate; T= Radial growth of the test pathogen in treatment Statistical analysis: Statistical analysis of pathogenicity test was done using one way ANOVA after angular transformation. Results were compared using least significant difference (LSD) test at P 5%.

Soil nutrient analysis:
Twenty eight composite samples were used for soil nutrient analysis. In general, the soil samples were poor in phosphorus though organic carbon was comparatively better, which is probably because of the use of compost in the vegetable fields ( Table 2). Identification of fusarium: On the basis of colony morphology and characteristics of macro and micro conidia, sixty fungal isolates were identified as Fusarium.
On further microscopic study, thirty nine isolates were identified as F. oxysporum on the basis of macro conidia characteristics which were thin walled generally 3-5 septate, fusoid falcate macro conidia with somewhat hooked apex and pedicillate base (Booth, 1971). The macro conidia of F. oxysporum were found in the rage of 5.3 to 28.4 μm and the micro conidia were found in the rage of 2.3 to 11.8 μm (Table 3). Molecular identification of F. oxysporum: Thirty nine isolates were confirmed as F. oxysporum using species specific primers amplifying the DNA fragment of 340bp.

Effect of abiotic factors on the growth of F. oxysporum:
The acidic as well as alkaline conditions affected the radial growth of the Fusarium. The maximum radial growth of most of the F. oxysporum isolates was found under slightly alkaline conditions. Isolate no. 13,16,21,29,27 and 47, showed better growth under slightly acidic conditions (Fig. 1). Isolates of F. oxysporum were quite variable in their growth rates as well as sporulation in light and dark conditions. All the isolates showed increased growth rate under light conditions except Isolate no. 15, 47, 53, 54 and 58 (Fig. 2). All the thirty nine F. oxysporum isolates showed better sporulation in dark to light alteration. Measurement of disease incidence: Percent disease incidence was recorded by counting the dead plants after twenty days of transplantation. Disease incidence was found within the range of 0 %-78.7%. The isolates having maximum disease incidence (78.7%) were further used in the experiment. Three isolates 21, 22 and 27were the most aggressive pathogens. Isolate no.16, 29, 37, 40 and 50 were non-pathogenic (   4.0 f 3.5 e 2.86 d 2.0 a 2.2 b 2.7 c P = Pathogenic isolate, NP = Non-pathogenic isolate. Each value represents the mean of three replicates. In every row, any two values without common latter in their superscript are significantly different. and compared using least significant difference at P 5%. Table 6. Percent inhibition of pathogenic F. oxysporum by different antagonistic F. oxysporum. Each value represents the mean of three replicates. In every column, any two values without common latter in their superscript are significantly different. which was compared using least significant difference at P 5%. Radial growth (cm) 5.5 pH 6.5 pH 7.5pH

Fig.1. Effect of pH on radial growth (cm) of F. oxysporum
was inhibited by all the nonpathogenic isolate whereas the maximum percentage of inhibition was 50% by Isolate no. 37 was obtained.

DISCUSSION
The present study recovered sixty Fusarium isolates from the rhizosphere of tomato plants. Thirty nine Fusarium oxysporum isolates were identified to species level, morphologically. On further study, all the thirty nine isolates were found positive by PCR amplification of ITS region using species specific primers of F. oxysporum.
The amplification of ITS region of all isolates of F. oxysporum strains showed that both pathogenic and non pathogenic forms of this fungus may have evolved from single ancestors. Molecular identification of F oxysporum in this study further demonstrates that PCR analysis is an effective and fast way to detect F. oxysporum strains using species specific primer sets FOF1and FOR1. Identification of Fusarium species from the soil has always been difficult as it relies on minor differences in morphology, and different cultural conditions can cause the same species to vary (Doohan, 1998;Edel et al., 2000). A diverse population of non-pathogenic and pathogenic F. oxysporum isolates was found in rhizosphere and endorhizosphere of tomato plants. It reflects that isolates of F. oxysporum show the morphological and culture differences. Diversity of nonpathogenic and pathogenic populations of F. oxysporum was compared by variation in conidial morphology; large variability appeared but it had no relation with pathogenicity. All the isolates of F. oxysporum were quite variable in their growth rate in different abiotic stress conditions. Isolates did not show much variation in their growth rate at different light and dark conditions but the sporulation in fugal culture was more in light treatment. Most of the isolates favored the pH of 7.5, slightly alkaline conditions. In a previous study by Srobar (1978), it was found that micelia of F. oxysporum which grew within the range of pH 2-12, pH 0-6 was the most suitable for the growth of all Fusarium species while a highly acidic medium was unsuitable for the sporulation of all Fusarium species. Chuang (1991) reported that the germination of chlamydospores of F. oxysporum was inversely co-related with pH from 4-8, but that the pathogen survives longer in alkaline condition (pH 8, 9 and 10) than in very acidic condition (pH 2, 3 and 4). In pathogenicity test, results indicated that most of F. oxysporum were pathogenic. These isolates caused wilt symptoms on at least one plant while on inoculation of non pathogenic isolates on the tomato plant, wilt did not occur at all. There are reports of nonpathogenic F. oxysporum demonstrating competing for infection sites and for nutrients, and by induction of resistance (Larkin and Fravel 1999;Benhamou and Garand 2001 Rest of the four non pathogenic isolate of F.oxysporum reduced the colony growth of pathogenic F. oxysporum varied to reduce growth from 0% to 50%. It has been observed that antagonistic fungi are specific in their antagonistic activity against specific fungi (Saleem et al. 2000).
The results of the confrontation assay showed that Isolate no. 21 is most aggressive pathogen and results of the pathogenicity test provided evidence for the ability of selected non-pathogenic F. oxysporum to reduce the fusarium wilt of tomato. Similarly in the previous study, it has been reported that the F. oxysporum Fo47 increased disease suppression (Park et al.,1988). On the basis of present study we could see that the different nonpathogenic F. oxysporum were able to inhibit the growth of different pathogenic F. oxysporum to different extent. Their capability of inhibiting the growth of pathogenic behavior varies from isolate to isolate. One effective means of controlling Fusarium wilt is nonpathogenic F. oxysporum which appears to be practical and economically efficient control measure for the management of Fusarium wilt. It was concluded that among various isolates of F. oxysporium, nonpathogenic Isolate no. 40 has the potential to suppress growth of pathogenic F. oxysporum f. sp lycopersici, and could be used as a biocontrol agent against the management of wilt disease in tomato.