RHIZOBACTERIA AS ANTAGONIST AGAINST Fusarium oxysporum CAUSING TOMATO WILT

Tomatoes ( Lycopersicon esculentum L .) are the most extensively grown vegetable in the world. It belongs to the Solanaceae family and is commonly planted for its tasty fruits. Pests, weeds, diseases, and parasites are just a few of the numerous variables that significantly affect tomato growth and yield. The most common disease affecting tomatoes is fusarium wilt. Fifteen rhizobacterial strains were identified by morphological and biochemical analyses in this work, and they were employed as an antagonist against Fusarium oxysporum f.sp. lycopersici . Fifteen isolates were investigated for their antagonistic properties against Fusarium oxysporum f.sp. lycopersici using an invitro dual culture approach. The growth of Fusarium oxysporum f.sp. lycopersici was suppressed by each isolate. Out of the 15 rhizobacteria isolates, isolate RBS-5 exhibited the highest level of growth inhibition and strongly suppressed the development of Fusarium oxysporum f.sp. lycopersici , resulting in a 57.28 percent reduction in pathogen growth as compared to the control. The development of Fusarium oxysporum f.sp. lycopersici was suppressed by isolates RBS-12, RBS-6, and RBS-15, in decreasing order of merit, compared to the control by 53.7, 51.91, and 51.73 percent. Isolate RBS-13 showed the least amount of pathogen growth inhibition 20.83 percent. The data was statistically analyzed .


INTRODUCTION
Tomatoes are members of the nightshade family (Solanaceae).It is an extremely important and wellknown vegetable that is grown all over the world.It provides enough nutritional benefits and is competitively priced when compared to other vegetables.It is a fantastic source of vitamins A and C. Together with pigments like lycopene and βcarotene, it also contains minerals like phosphorus and iron.Tomatoes get their red colour from lycopene, which is essential for the production of βcarotene.(Chohan & Ahmad, 2008;Kumar et al., 2012).The tomato is a notable genetically identified diploid (2n) plant species with a relatively short life cycle, high fertility, and the capacity to procreate, which makes it an excellent subject for practical research.(Moyers et al., 20118).Tomatoes are a heavily farmed and eaten vegetable worldwide.It is a major profitable crop for small farmers in tropical Asia.(Saavedra et al., 2016).China is the global leader in tomato production.The tomato originated in America, and its original planting method was developed in Mexico.Tomatoes were brought to the subcontinent by British soldiers at the beginning of the 1800s.The first documented history of tomato cultivation in the Indo-Pak Subcontinent is recorded in William Roxburgh's 1832 book "Flora Indica."(Saavedra et al., 2016).Tomatoes are highly valued as a crop in Pakistan.It is the main ingredient in salads and is cooked with other vegetables to enhance their taste.Tomato cultivation has been greatly impacted by the surge in demand for tomato-based products like purees and ketchup brought on by the consumption of fast food.Tomato demand is growing, and this trend is likely to continue.Tomatoes provide 20% of the daily needed amount of vitamin A based on a 2000 calorie diet.A tomato supplies 26% of the recommended daily intake of vitamin C. (Ali et al., 2017).Among other limitations, diseases are the main factor affecting tomato yield and quality.(Pritesh & Subramanian, 2011).Anthracnose, bacterial canker, blights, fungal and bacterial wilts, and tomato spotted wilt are the most common tomato ailments.(Jones et al., 2014).One of the most common causes of losses in greenhouses and fields worldwide is Fusarium wilt.(Sheu et al., 2006;Abdel-Monaim et al., 2011).Crop losses from the disease can vary from 10% to 80% and perhaps 100% when it strikes tomato varieties that have not been modified.(Bharat & Sharma, 2014;Worku & Sahe, 2018).Tomato crops are susceptible to several diseases, including fungus, which can result in substantial damage, substantial financial losses, and low fruit production.(Lichtenzveig et al., 2006).F. oxysporum f. sp.lycopersici is an extremely dangerous soil-borne pathogen that may persist and proliferate in the soil for a long time.Fusarium wilt is a tomato disease that is widely dispersed and has a substantial commercial impact worldwide.(Abdesselem et al., 2016).Secondary metabolite production frequently happens concurrently with antagonistic PGPR action (Sliva et al., 2001).The most common antagonistic action method is direct physical contact with phytopathogens and the biocontrol agent (Mukerji & Chincholkar, 2007).

Collection of Samples for Fusarium oxysporum
Isolation: The diseased tomato root, stem and soil sample was collected from chashma achozai, Quetta, Balochistan.These samples were brought into Plant Pathology laboratory of Balochistan Agriculture College and stored at room temperature for observation of different pathological aspects.

Collection of samples for Rhizobacteria Isolation:
For isolation of Rhizobacteria soil samples were collected from maize field in chashma achozai, Quetta, Balochistan.The samples were brought into Plant Pathology laboratory of Balochistan Agriculture College and stored at room temperature for observation of different Rhizobacteria isolates.

Preparation of Media
Preparation of PDA from commercial powder: For preparation of 1 litter of media added 39g commercial powder in 600ml of water and mix it on hot starer plate, add 400ml of water to make 1000ml/ 1 litter of media.For sterilization of media, autoclave was used for 15 minutes at 121°C.Preparation of NA Media: For 1 liter of media add 28g commercial powder in 600ml of water and mix it on hot starer plate, add 400ml of water to make 1000ml/ 1 litter of media.For sterilization of media, autoclave was used for 15 minutes at 121°C.

Isolation and Purification of Fusarium oxysporum:
The wilt disease-causing Fusarium oxysporum f.sp.lycopersici was isolated from tomato plants that were afflicted and exhibiting wilt symptoms.Samples that were infected were gathered from Quetta's tomatogrowing fields.After chopping the infected roots into tiny pieces and surface sterilising them for 20 to 30 seconds with a 1% sodium hypochlorite (NaOCl) solution, the infected roots were thoroughly cleaned with sterilised distilled water.After plating three pieces on Potato Dextrose Agar (PDA) medium, they were cultured for five to seven days at 28 ºC ± 2 ºC.The fungal material was isolated using the single hyphal tip method (Rangaswami, 1972), stored at 25 ºC, and utilised for subsequent experimental investigations and identification.Refrigerated at 4ºC were the pure culture tubes.To identify the culture, employed morphological and microscopic investigation.Morphological characterization of Fusarium: oxysporum: The initial step in the morphological characterisation method was to use a microscope and human eye to examine the characteristics of culture plates.Using PDA plates, the morphological properties of fungal colonies were investigated.The characteristics of the colony were studied using a stereoscope.The morphological features of chlamydospores, macroconidia, and microconidia were investigated under a compound microscope.The fungal isolate's morphological identity was determined by using the procedure outlined by Leslie et al. (2006).The microscopic features that were looked at in order to identify the fungal pathogen were the colony's size, color, and appearance; the forms of the macro-and microconidia; and the chlamydospores.

Isolation and Purification of Rhizobacteria:
Rhizospheric soil that was taken from the root zone was used to isolate rhizobacteria.In this investigation, a soil sample was obtained from Quetta's field regions where maize is grown, and rhizobacteria were identified using the serial dilution method.Serial Dilution Method: The rhizobacteria were recovered from rhizospheric soil by serial dilution method (Wollum, 1982).To remove the rhizosphere soil from the root zone, the roots were gently shaken and then submerged in sterile water in the laboratory.Soil samples were put in a test tube with 9 mL of distilled water, and the mixture was vortexed to homogenize it.Take 1 mL of the first dilution (10-1 ) and transfer it to a fresh clean tube along with 9 mL of diluent to create a second dilution.As a result, the solution was diluted up to 10-8 .Each time, the solution was thoroughly mixed using a vortex mixer.After a thorough shaking, 10-6 , 10-7 , and 10-8 dilutions were evenly distributed on a Petri plate filled with solidified nutritional agar (NA) medium.Petri plates that were taped were incubated at 26 ± 2 °C.

Biochemical Characterization of Rhizobacteria:
The biochemical characterization of each isolate was mostly finished in compliance with the procedure outlined in (Joseph, Patra, & Lawrence, 2007).Gram staining: Procedure performed as prescribed by Vincent & Humphrey (1970).Catalase Test: Catalase is an enzyme that breaks down hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O) was performed as Ninama et al. (2012).Starch Hydrolysis Test: Starch agar medium is used in this experiment.Pure bacterial cultures were streaked on Petri plates filled with solidified starch agar medium, and the plates were incubated for 24 hours as instructed by Cappuccino & Sherman (1983).Potassium Hydroxide (KOH) Solubility Test: A loopful of bacterial culture was collected on a clear, dry glass slide, and then mixed with a drop of 3% potassium hydroxide until an even solution was formed for the KOH test, as per Kirsop & Doyle (1991).Ammonia production: Ammonia was measured in rhizobacterial strains using peptone water, as per Cappuccino & Sherman's (1992) methodology.Fresh rhizobacterial cultures were placed in test tubes with 10 mL of peptone water, and they were incubated for two days at 28°C.The test tubes turned brown to orange, indicating that the appropriate rhizobacteria had started to create ammonia when 0.5 ml of Nessler's reagent was added to each one.Salt and PH Tolerance : The ability of the isolated rhizobcterial isolates to grow in different concentration of salt was tested by streaking them on NA medium containing 1.0%, 3.0% and 5% (wt/v) NaCl.Differences in pH tolerance was tested by adjusting the pH to 6.5, 7.5, and 8.0.All the plates incubated at 28°C for 72 hours and NA medium plates was used as controls.Dual Culture Method (Dennis & Webster.,1971):A nine-millimeter culture disc was removed from the periphery of a seven-day-old F. oxysporum f.sp.lycopersici culture and positioned about 75 millimetres away from the edge of the Petri dish containing 15 millilitres of sterile, solidified PDA media.The two-day-old Rhizobacterial strains and the pathogenic culture were evenly spaced apart on the medium and faintly streaked with it.The zone of inhibition (mm) and the mycelial growth of F. oxysporum f.sp.lycopersici were measured.The pathogen's growth was impeded during the process of choosing the most potent antagonists.The percentage of mycelia growth inhibition was computed according to Vincent (1947).
By using Formula  = Microscopically, Fusarium oxysporum f.sp.lycopersici has been identified based on characteristics of the colony and spore shape as described by Leslie et al. (2006).The mycelium that developed on PDA plates had cottony growth and was hyaline.Microscopic examination revealed septate and branching hyphae.Branched conidiophores hosted microconidia that ranged in shape from straight to bent.Morphological characterization of Rhizobactria: It is one the phenotypic studies of cell to evaluate and identify the colony of various species of bacteria which is help full to identify the physical properties of cell colonies such as the size, the shape, elevation, edges and color.For the colony morphology, the size, the shape, elevation, edges, surface and color were considered for fixing the standard for this parameter.

Salt and pH Tolerance Test:
The ability of the isolated rhizobcterial isolates to grow in different concentration of salt was tested by streaking them on NA medium containing 1%, 3% and 5% (wt/v) NaCl.
All the rhizobacterial strains show positive result at 1%, 3% and 5% NaCl concentrations, except RBS-4 and RBS-10 at 5% NaCl concentration.Differences in pH tolerance was tested by adjusting the pH to 6, 7, and 8.All the rhizobacterial isolate shows positive growth at pH 6, 7 and 8 except strain RBS-4, RBS-7 and RBS-10 which shows negative growth at pH 8.

In Vitro antagonistic activity against FOL:
Antagonistic activities of 15 isolates were tested against Fusarium oxysporum f.sp.lycopersici by dual culture in laboratory.All the isolates were found to inhibit the growth of Fusarium oxysporum f.sp.lycopersici.The percent inhibition was measured by the formula Percent inhibition (%) = (C-T)/CX100.T  In vitro efficacy of rhizobacterial strains against Fusarium oxysporum f.sp.lycopersici: Antagonistic activities of 15 isolates were tested against Fusarium oxysporum f.sp.lycopersici by dual culture in laboratory.All the isolates inhibited the growth of Fusarium oxysporum f.sp.lycopersici.In 15 isolates of rhizobacteria the isolate RBS-5 show maximum growth inhibition and significantly inhibited the growth of Fusarium f.sp.lycopersici, which is 57.28 percent reduction on the growth of pathogen when compared to control.This is followed by isolate RBS-12, RBS-6, and RBS-15 in decreasing order of merit, which inhibited the growth of fusarium oxysporum f.sp.lycopersici by 53.7, 51.91 and 51.73 percent over control.The least growth inhibition of pathogen was exhibited by isolate RBS-13 which is 20.83 percent.

DISCUSSION
The tomato, or Solanum lycopersicum L., is a member of the Solanaceae family and is grown worldwide because of its short growing season and excellent yield.Carotenoids, antioxidants, vitamins C and E, and other nutrients found in tomatoes can assist improve the nutritional state of the general public, especially the under privileged.(Rai et al., 2012).
Healthy plants are essential for the welfare of both humans and animals.Pathogenic bacteria negatively impact the nutritional content, yield, and general well-being of plants (Fletcher et al., 2010).The infamous tomato pathogen Fusarium oxysporum f. sp.Lycopersici (FOL) is the cause of wilt disease and causes considerable crop losses.(Ahmed, 2011).
In this study, 15 Rhizobacterial isolates were isolated and tentatively identified.The isolates were identified as genus; Bacillus, Pseudomonas and Streptomyces.As an antagonist against Fusarium oxysporum f.sp.lycopersici, the isolates were used.In dual culture, every bacterial isolate showed reduction of Fusarium oxysporum mycelial growth.Numerous investigators have also seen similar results with a range of fungi, such as Fusarium species.(Sivamani & Gnanamanickam, 1988;Khan & Zaidi, 2002).
Seven Bacillus isolates, six Pseudomonas isolates, and two Streptomyces isolates were found among the fifteen rhizobacterial isolates.Isolate RBS-13 was shown to be least efficient against Fusarium oxysporum mycelial growth, whereas isolate RBS-5 demonstrated the best growth inhibition at 57.28 percent.The biocontrol capability of seven Bacillus isolates obtained from the rhizosphere was assessed in vitro against FOL.The findings showed that every Bacillus isolate inhibited F. oxysporum's mycelial growth to a different extent, with isolate RBS-2 suppressing it by 27.32% and isolate RBS-5 suppressing it by 57.28%.Several investigations have used Bacillus species as biological control agents and biofertilizers.(Cao et al., 2011;Li et al., 2012;Chen et al., 2013It has been observed that B. subtilis produces a volatile material with antifungal qualities against soil-borne infections.(Fiddaman & Rossales, 1993).
Six isolates of Pseudomonas and two isolates of Streptomyces were evaluated for their biocontrol potential aginst FOL in vitro.The result revealed that Pseudomonas and Streptomyces isolates inhibit the mycelial growth of Fusarium oxysporum ranging from 20.83% to 51.91%.A review of earlier research has demonstrated Pseudomonas species' potential as biological control agents for harmful plant diseases.(Pastor et al., 2010;Khalimi & Surpata, 2011;Karimi et al., 2012;Saravanan et al., 2013).
The bioefficacy of thirty P. fluorescens isolates against Fusarium sp. was reported by Shahzaman et al. (2016).Pf3 was shown to be the most effective antagonist, showing an inhibiting percentage of 93.33 percent.Similar findings were reported by Vethavalli et al. (2012) The PV2 isolates exhibited the highest percentage of inhibition, with 77.22 percent suppressing F. oxysporum f.sp.lycopersici growth over control.The efficiency of three isolates' in vitro antagonistic interactions was assessed in this work (PV2, BVE1, and SM1).
Peudomonas flourescens, Putida, Chloropyrus, Bacillus subtilis, Streptomyces pulcher, S. corchorusii, and S. mutabilis are bacterial bio control organisms that may be able to control Fusarium oxysporum f. sp.lycopersici, according to Monda (2002).Rhizobacteria can work as direct or indirect biological fertilisers and biostimulants by generating plant growth hormones that include indole acetic acid, gibberelin, cytokinin, ethylene, and dissolved minerals.Because they produce antibiotics and siderophore, they can additionally effectively impede the development of harmful microbes (Sarma et al., 2009).

CONCLUSION
It is concluded that the Rhizobacteria inhibit the mycelial growth of Fusarium oxysporum f.sp.Lycopersici.Among the Rhizobacteria, Bacillus show highest mycelial growth inhibition, followed by Pseudomonas and Streptomyces

Figure 1
Figure 1 Colony morphology of Fusarium oxysporum f.sp.lycopersici Characterization of Rhizobacteria: Through serial dilution method 15 rhizobacterial strains were isolated and characterized on the basis of morphological characters like shape, size, elevation, edges, surface and colour

Figure 3
Figure 3 Catalase activity test Starch Hydrolysis test: One important test to assess a bacteria's capacity to produce starch is the hydrolysis of starch test.For starch hydrolysis test the strain RBS-1, RBS-2, RBS-4, RBS-5, RBS-8 RBS-12, RBS-13 and RBS-15 show positive result while strain

Figure 6
Figure 6 Ammonia production test

Figure 7
Figure 7 (A) Inhibition by Bascillus, (B) Inhibition by Pseudomonas (C) Inhibition by Streptomyces (D) Mycllium growth in control

Table 1
Colony Morphology of Rhizobacteria

Table 4
Salt and pH Tolerance Test of Rhizobacterial Strains