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IN VITRO TOXICITY EVALUATION OF CULTURE FILTRATES OF FUSARIUM OXYSPORUM F. SP. LYCOPERSICI ON GROWTH AND PHYSIOLOGY OF TOMATO UNDER CHROMIUM (VI) STRESS.

Byline: S. Khurshid A. Shoaib and A. Javaid

: ABSTRACT

In vitro toxicological influence of culture filtrates of Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder and Hans was investigated on seed germination seedling growth and physiology of tomato (Lycopersicum esculentum Mill.) under chromium(VI) stress. Original culture filtrates of the fungus and three concentrations of Cr(VI) viz. 50 75 and 100 mg L- 1 were used in laboratory bioassays either alone or in different combinations. Experiment was carried out at 25 2 C in triplicate using completely randomized design and different growth and physiological assays were recorded in 15-days old tomato seedlings. Germination growth and biomass was declined significantly up to 40% 85% and 70% due to original culture filtrate of the F. oxysporum and by 40-60% 10-50% 50-80% under combine stress of original culture filtrate of the fungus along with increasing concentration of Cr(VI) and by 10-20% 0-25% and 30-60% due to increasing concentration of Cr(VI) alone. Chlorophyll contents was

significantly declined due to original culture filtrate of the F. oxysporum f. sp. lycopersici alone or combined with Cr(VI) while non-significantly different due to solitary effect of Cr(VI) over control treatments. Whereas catalase and peroxidase activities increased significantly in treatments supplemented with Cr(VI) alone or combination with fungus and decreased due to culture filtrate of fungus alone as compared to control. The present study concluded that culture filtrates of F. oxysporum f. sp. lycopersici are hazardous to tomato seedlings either alone or in combination with Cr(VI).

Key words: Chromium fungal culture filtrates Fusasium oxysporum Lycopersicum esculentum tomato.

INTRODUCTION

Toxicogenic fungi mostly belong to genus Aspergillus Penicillium and Fusarium with ability to utilize variety of substrates to produce their toxins as low molecular weight secondary metabolites during metabolic processes (Jalonder and Gachonde 2011). The metabolites are products of some cyclic peptides phenols and plant growth regulators (Madhosing 1995). Fusarium oxysporum f. sp. lycopersici the most common soil-borne ascomycetous fungus is notorious for causing wilt in tomatoes (Park et al. 2013). The fungus provoke devastating losses in agriculture and lead to food contamination by producing well-known biologically active mycotoxins like fusaric acids fumonisins beauvericin enniatin moniliformin and trichothecenes. These fungal toxins are known to cause destruction of plants by causing necrosis chlorosis wilting and sometimes by inhibiting seed germination (Idris et al. 2003). Mycotoxins have been recognized as environmental pollutants that are present

virtually in all parts of the world and their toxicology in food arising life-threatening infections in humans have been documented globally (Lopez-Berges et al. 2013). Due to extensive industrialization chromium (Cr) contamination has become a setback in Pakistani agriculture (Rizwan et al. 2009). Cr(VI) is considered as the second most common contaminant in groundwater soil and sediments (Kar et al. 2008). The water quality of major cities of Pakistan is deteriorating because of unchecked disposal of Cr loaded untreated tannery effluent that left residual effects in food chain (Qadir et al. 2008). Discharge rate of effluent only from tanneries is approximately 1.1 million liters per day. In fact there is no safe level of Cr and even a very dilute content can cause adverse health effects (Sen and Ghosh 2010). Plants the primary producers are the first ones that are being exposed to the contaminants present in the soil. This phtotoxicity causes chlorosis

tissue necrosis reduced enzyme activity damage to membrane and diminished photosynthesis (Srivastava and Thakur 2006; Scoccianti et al. 2008). Tomato (family Solanaceae) is the most intensively studied model species for plant-pathogenic interaction as well as for biomechanical studies (Zamir and Giuliano 2012). It is the most ubiquitous vegetable of Pakistan with the annual production of 529.6 thousand tones cultivated on an area of 52.3 thousand hectares (Anonymous 2011). Cultivation of tomato has been limited by a variety of biotic (fungal bacterial viral nematodes diseases) and abiotic (environmental factors) deteriorating factors (Saeed and Khan 2011). Earlier studies were focused on the negative response of tomato to either Cr or F. oxysporum f. sp. lycopersici stress alone (Liu et al. 2008; Maia et al. 2011). However the growth and physiological response under simultaneous stress of toxin of fungal pathogen and metal needs to be addressed.

The present study was therefore accomplished to investigate the solo and simultaneous influence of culture filtrates of F. oxysporum and Cr(VI) on tomato seedling growth and physiology.

MATERIALS AND METHODS

Preparation of fungal culture filtrates: F. oxysporum f. sp. lycopersici was isolated from infected tomato plants collected from tomato fields. The fungus was cultured on 2 % malt extract agar medium and identified on the basis of morphological characters (El-Kazaz et al. 2008). Fungal culture filtrate was prepared by inoculating 100 mL autoclaved malt extract broth with 2 mm F. oxysporum f. sp. lycopersici disc in 250-mL flasks incubated for 10 days under constant shaking at 100 rpm (25 3 C; pH 5.5). Fungal culture filtrate was collected by filtration of the fungal mat through Whatmann filter paper No.1.

Preparation of metal solutions: A stock solution of 1000 mg L-1 of potassium dichromate (K2Cr2O7) (Merk Germany) was prepared by dissolving 2.825 g of salt in 100 mL of water and final volume of 1000 mL was made by addition of double distilled water. Further dilutions of 50 75 and 100 mg L-1 were prepared by adding appropriate quantity of sterilized double distilled water.

A similar set of metal concentrations was also prepared in fungal culture filtrates instead of distilled water.

Plant growth bioassays: Seeds of tomato variety LA- 2662 were procured from Vegetable Research Center Ayub Agriculture Research Institute Faisalabad Pakistan. Healthy seeds were separated and surface sterilized using 0.1% sodium hypochlorite solution for 3 minutes and rinsed 4 times with sterile distilled water. Sterilized seeds were placed in sterilized Petri plates (9- cm) lined with double layer of filter papers moistened with 3 mL of each of different concentrations of Cr(VI) solutions. In second set Petri plates were supplied with 3 mL of culture filtrate amended with each of different doses of Cr(VI). Two control treatments were devised; one received 3 mL of sterilized distilled water and second received 3 mL of culture filtrates of F. oxysporum f. sp. lycopersici. Each treatment was replicated three times with 25 seeds each. The Petri plates were arranged in a completely randomized design and incubated at 25 2 C and 10 hours daily light period. Data regarding germination length and

fresh and dry weight of shoot and roots were recorded 15-days after seed sowing. Germination rate Germination index (GI) and relative germination rate (RGR) were calculated for each treatment (Li 2008). Formula

Physiological assays: Chlorophyll extraction was performed by homogenizing 500 mg fresh leaf material with 10 mL of chilled 80% acetone. Resultant homogenate was centrifuged at 800 rpm for 15 minutes and supernatant was analyzed for chlorophyll content (Li 2008). For antioxidant enzymes extraction 0.5 g leaves were ground in chilled mortar using 5 mL phosphate buffer. Homogenate was centrifuged at 13000 rpm for 20 minutes at 4 oC and supernatant was further employed for enzymes activities assays. For catalase (CAT) activity

1.0 mL supernatant was added to reaction mixture containing 3.0 mL of 0.1 M phosphate buffer (pH 6.8) and l mL of H2O2 (0.01M). Ten mililitres of 2% H2SO4 was added after l minutes at 20 C. The reaction mixture was titrated against 0.005 N KMnO4 to determine the quantity of H2O2 utilized by the enzyme. Catalase activity was expressed as number of moles of H2O2 utilized min-1 mg-1 protein (Machly and Chance 1967). Peroxidase activity was determined by taking enzyme extract (0.5 mL) in 2 mL of 0.1 M phosphate buffer (pH 6.8) and 1 mL of 0.01M pyrogallol. This solution was filled with 0.05 M H2O2 (5:5 in H2O2 and distilled water) incubated at 25 C and reaction was stopped by adding 2.5 N H2SO4 (24.5 mL of H2SO4 + 100 mL of distilled water). Absorbance was recorded at 420 nm to determine the amount of purpurogalline formed against blank. One unit of enzyme activity denoted the quantity of the enzyme that inhibited 50% of the auto-oxidation rate of pyrogallol at 25 C.

The enzyme activity was expressed as unit mg-1 protein (Arnon 1949). All the data were analyzed through analysis of variance technique and means were compared by Duncan's Multiple Range Test (P = 0.05) to separate mean differences (Steel et al. 1997).

RESULTS AND DISCUSSION

In general all the fungal culture filtrates and Cr(VI) concentrations either alone or in combinations suppressed plant growth biomass and physiology over control treatment. The highest decline in plant growth and alteration in physiology was recorded in fungal culture filtrates treatments. Original culture filtrates of F. oxysporum f. sp. lycopersici did not exhibit significant reduction in seed germination and relative germination rate whereas germination index was significantly declined by 38% over control. Shoot and root lengths were significantly decreased by 50% and 85% respectively over control. Similarly seedlings fresh biomass was significantly reduced by 86% and dry biomass by 70% over control. Total chlorophyll (chlorophyll a + b) content and catalase activity was decreased by 20% and 36% respectively while peroxidase activity was increased 2-folds over control (Table 1 and 2). Reduction in growth and alteration in physiological parameters was recorded due to the effect of

culture filtrates of F. oxysporum may be attributed to inhibitory action of variety of mycotoxins and enzymes present in the culture filtrate (Karaman and Matavuly 2005). Fusarium toxins in culture filtrate have been well-known for their properties related to virulence of pathogenic strain (Xu et al. 1993). Therefore negative influence of mycotoxins may be well-correlated with hypersensitive response in plant tissue that results in liberation of reactive oxygen species which induce high level of lipid peroxidation mediating damage to DNA or protein in tomato tissues (El-Khallal 2007). Therefore it could be speculated that pathogen toxin disturb normal plant physiology by facilitating nutrients leakage from the macerated tissues (Nafie 2003) thus consequences with overall reduction in plant growth and biomass (El- Khallal 2007; Houssien et al. 2010; Maia et al. 2011).

When tomato seeds were exposed to different concentrations of Cr(VI) solution germination rate germination index and relative germination rate were significantly dropped by 10-20% with increase in metal concentration from 50 to 100 mg L-1. Root and shoot lengths were reduced by 0-25% and both fresh and dry weight were declined by 30-60% at metal concentration of 50-100 mg L-1 over control (Table 1). Total chlorophyll content was declined up to 6% and there was non-significant difference among the treatments for this parameter as compared to control. Catalase as well as peroxidase activities significantly increased with increase in metal concentration as compared to control (Table 2).

The toxicology of Cr(VI) might be owing to its adverse effect on seed germination and early seedling growth that could results in functional abnormality in hydrolytic enzymes auxin synthesis and osmotic regulation (Noggle and Fritz 1991; Barton et al. 2000). Metals are also reported to depress the uptake of O2 hence inhibit normal physiological process (Sharma and Sharma 2003). The decline in chlorophyll content of chromium treated seedlings might be due to either inhibition of chlorophyll biosynthesis or correlated with the increasing Cr concentrations (Panda and Choudhury 2005; Scoccianti et al. 2006; Liu et al. 2008). Cr(VI) is thought to be involved in termination of enzymes responsible for chlorophyll biosynthesis by degrading d-aminolevulinic acid dehydratase (ALA) thus results in upsurge of ALA and decline in the chlorophyll content (Vajpayee et al. 2000). Chromium-induced oxidative stress resulted in elevated production of reactive oxygen species producing oxidative damage and

disturb normal cell functioning. Catalase and peroxidase are antioxidant defense mechanisms generated in plants against danger posed by the presence of reactive oxygen species (Meloni et al. 2003). The activities of antioxidant enzymes assures their role in antioxidant defense so various employed Cr(VI) concentrations results in alteration of their enzyme activities (Sinha et al. 2002). Combined effect of culture filtrates of F. oxysporum f. sp. lycopersici and Cr(VI) exhibited a significant inhibition of 10-20% in germination and relative germination rate and 40-60% in germination index with increase in metal concentration. Root and shoot lengths were declined by 10-50% and 20-50% respectively over control. Likewise fresh and dry biomasses of the seedlings were reduced by 50-80% in a mixture prepared by adding various concentration of Cr (VI) in fungal culture filtrates (Table 1). Total chlorophyll contents were decreased by 30-40% while catalase activity

was increased up to 14%. However peroxidase activity showed non-significant response towards simultaneous influence of Cr(VI) concentrations and fungal culture filtrates (Table 2). Negative growth of plants under simultaneous action of culture filtrate of pathogen and Cr(VI) could be related

Table 1. Effect of culture filtrates of F. oxysporum f. sp. lycopersici (FO) alone and in combination with various Cr(VI)

###concentrations on growth of tomato.

###Treatments###Germination###Germination###Relative###Shoot###Root###Seedling###Seedling

###(%)###index###germination###length###length###fresh weight dry weight

###rate###(cm)###(cm)###(mg)###(mg)

Control###1000a###1.10a###10.01a###50.01a###2 0.01a###8700.01a###83 0.01a

FO culture filtrate###1000a###0.680.01b###10.02a###2.50.02cd###0.30.0c###100 0.01f###25 0.02e

Cr(VI) 50 mg L-1###890.01b###0.980.07a###0.890.01bc###5 0.02a###2 0.01a###4800.01c###49 0.02 c

Cr(VI) 75 mg L-1###820.02bc###0.910.03a###0.820.04cd###4 0.01b###2 0.02a###390 0.01d###340.01d

Cr(VI) 100 mg L###-1###79 0.01 c###0.870.02 b###0.790.06 cd###4 0.01 b 1.50.03 ab###3310.0 d###320.02 d

FO + Cr(VI) 50 mg L-1###90 0b###0.550.06c###0.90.01abc###4 0.01b###1.40.01ab###320 0.01d###39 0.01d

FO + Cr(VI) 75 mg L###-1###86 0.02 b###0.510.02 c###0.860.03 c###3 00.02 c 10.02 b###240 0.02 e###26 0.01e

FO + Cr(VI) 100 mg L-1###79 0c###0.480.04d###0.790.04cd###2.50.01cd###1 0.01b###2100.01e###25 0.02e

with negative outcome generated by synergistic action of both. It might be possible that Cr toxicity weaken roots and provide more absorption side to fungus to get inside roots during early germination stages and therefore weaken plant while oxygen depletion generated by Cr prompted that negative effect on overall growth biomass chlorophyll and enzyme assays.

Table 2. Effect of culture filtrates of F. oxysporum f. sp. lycopersici (FO) and Cr(VI) on chlorophyll content

###peroxidase and catalase activity of tomato.

###Chlorophyll content###Peroxidase activity###Catalase activity

###Treatments

###(mg g-1)###(Unit-1 min-1 mg-1 protein) (Unit-1 min-1 mg-1 protein)

Control###0.890a###0.2540.01d###5.50.02b

###b###c

FO culture filtrate###0.740.01###0.430.02###3.50.03d

Cr(VI) 50 mg L-1###0.870.02a###0.4540.03c###6.30.04a

Cr(VI) 75 mg L-1###0.850.03a###0.770.02b###6.50.02a

Cr(VI) 100 mg L-1###0.840.01a###0.960.04a###6.90.03a

###-1###c###c

FO + Cr(VI) 50 mg L###0.620.04###0.350.02###5.50.02b

###-1###c###c

FO + Cr(VI) 75 mg L###0.610.03###0.330.02###60.03b

###-1###d###d

FO + Cr(VI) 100 mg L###0.570.02###0.310.01###6.50.02a

Conclusions: The present study concluded that F. oxysporum f. sp. lycopersici culture filtrates and Cr(VI) either present alone or in combination have detrimental influence on seed germination seedlings growth and physiology of tomatoes.

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