ANTIFUNGAL EXPLOITATION OF FUNGICIDES AND PLANT EXTRACTS AGAINST FUSARIUM OXYSPORUM F.SP. MELONGENAE CAUSING FUSARIUM WILT OF EGGPLANT

Eggplant/Brinjal ( Solanum melongena L.) is the most significant and widely consumed as nutritious vegetable. Fusarium wilt is the major destructive disease in most of the vegetable growing regions of Pakistan. The research was conducted to assess fungicides against Fusarium oxysporum f. sp. melongenae from eggplant in field and in-vitro efficacy of numerous plant extracts. In current research, three treatments (T) i.e; shincar, flumax, Topsin-M, along with control were used at recommended dose with three consecutive sprays, on comparison of disease incidence with control, the Shincar showed 21.03% disease incidence, Topsin-M showed 31.94% disease incidence and the Flumax was close to control as it showed the least effect against the Fusarium wilt by showing 35.85% disease incidence. Furthermore, for plant extracts, poisoned food technique was used in the lab. The results were significant for all treatments (T), concentrations of (C), days (D) and their interactions (T x D), (T x C) and (C x D). Zingiber officinale showed less growth of colony (1.28cm) following Allium sativum (1.62cm), Datura stramonium (2.17cm), Eucalyptus globulus (2.50cm), Azadirachta indica (2.76cm) compared to control, respectively. Results of the present study showed a significant decrease in fungal growth of tested fungicides such as Shincar (Carbendazim) and Zingiber officinale (Ginger).


INTRODUCTION
Brinjal/Eggplant (Solanum melongena L.) is the most significant and widely consumed as nutritious vegetable. It has great status/importance as vegetable in diet of human because of its worth in vitamins, proteins and fundamental minerals. On facing malnutrition problems, vegetables are important producers in developing nations (Rahman et al., 2011, Perrone et al., 2012. In Pakistan, brinjal is used as a common vegetable in cooking due to its availability at reasonable price round the year, area under cultivation is 8,427 hectares with a total annual production of 84,255 tons (Anonymous, 2019). Pakistan stood at 22 nd position in world ranking for Eggplant cultivation (FAO, 2021). Successful eggplant production is subject to numerous fungal, viral and bacterial disorders due to the inability to obtain disease resistant seeds (Abdel-Monaim, 2012). Among those constraints, Fusarium wilt caused by Fusarium oxysporum has major destructive impact. F. oxysporum decreases level ofquality and quantity of eggplant's fruit in vegetable growing areas- (Fayzan, 2012). Thermophilic, soilborne and systemic pathogen, which leads to the deterioration of the vascular system, nutrient inhibition and water transfer, yield and quality by disrupt physiological procedures (Morid et al., 2012). The Fusarium is a pathogen which invades into the roots of the host eggplant-through the feeding of nematode pinholes, natural-and other-wounds. Afterward its invasion, it penetrates in the xylem vessels of plant which-transfer water and nutrients-from roots towardscrown and fungal mycelium-makes toxins,-as-itproliferate -through-plant. Suddenly, death of plant occurs owing to blockage of the xylem vessel (Pietro et al., 2003). Fusarium wilt characterized by yellowing of leaves and drooping of-shoots which results in expiry of entire-plant. Soil inhabiting fungi which colonized in senescing tissues of infected plant and could persist in soil-for decades (Joseph et-al., 2008). A sequence of wilt and death may occur in mature-leaves. Xylem vessel of infected tissue show brown discoloration. The most pronounced symptom is epinasty of leaves and partial stunting of eggplant by Fusarium wilt (Altinok, H.H 2005). Numerous management strategies are widely adopted to reduce disease incidences such as cultural operations, biological controls, resistant varities, soil solarization, crop rotation & less availability of soil moisture (Kamal et al., 2009), but for all these techniques it is necessary to demonstrate their activities against new prolonged virulently pathogen races as well as highly skilled precise measurements (Amini and Sidovich 2010). Production can be increased by cultivating diseased and pest free crops. The healthy fruit with the good appearance takes hold of high-prices at market level. In the emerging countries, there are some vegetables and fruits are banned to grow because of their defenselessness to sever disease and insect/pest attacks (Sain, 2018). Researchers are-finding low-priced and-more-ecofriendly compounds-to control plant infectious diseases due-to environmental and economic issues. Someanalysis has shown that extracts of some plants and bacteria could be-applied as biocontrol agents for managing soil-inhabiting-pathogens like Fusarium oxysporum (Pegg-and-Brady, 2002). Conversely, using systemic fungicides in order to prevent the occurrence, gravity and growth of Fusarium wilt has been easily handled, alternatives are developed and drained into the soil, directly and quickly . Pakistani farmers are aware rather than adverse effects of these fungicides that are increasing their output, yield and financial return (Sitara and Hasan, 2011). A wide range of fungicide classes have also achieved a reputation in Pakistan because of irrelatively low costs and efficiency (Diaz et al., 2005) and are widely used worldwide. The protection of plants and curative interventions depend on the use of suitable systemic fungicides gradually. In addition, successfully managing wilting problems is crucial to ensuring that the brinjal farming is economical. However, it is an essential component of modern research to prevent the incidence of this thermophilic soil-borne disease through substances. Furthermore, structural and ecological fungicide recognition is the requirement of the present era. Therefore, it is very necessary to implement such fungicides that are quick to act and less phytotoxicity effect against Fusarium oxysporum. Therefore, in the current studies, the most efficient fungicides have been used to avoid disease for brinjal crop. Current study was performed with the aim of identifying best fungicide against Fusarium wilt commercially suited for treatment in Pakistan induced in field and in laboratory conditions for Fusarium f. sp. melongenae by plants extracts. In the eggplant world, this disease causes huge losses.

MATERIALS AND METHODS:
Collection, isolation, purification and identification of F. oxysporum f. sp. Melongenae: Brinjal plants which were already infected with Fusarium wilt were collected from different fields of Faisalabad, the collected diseased samples were processed in lab for isolation of pathogen Fusarium oxysporum f.sp. melongenae. Thoroughly cleaned and sliced the infected roots into tiny parts and sterilized with 1% of HgCl2. Roots were dried on sterilized filter paper and placed on watch glass. Atleast, two portions of diseased roots were placed in petri-dish which consists of potato dextrose agar (PDA) medium. The plates were incubated for fungal growth at 25 °C ± 2 °C (Sarwar et al., 2005) for 48 to 72 hours. Then the pathogen colonies have been purified. Fusarium oxysporum f.sp. melongenae were identified by morphological features under stereomicroscope such as white and violet mycelium and brief microconidia on short conidiophores (Soesanto et al., 2011). Pathogenicity test through sick field: The pathogenicity test was conducted through a nursery in which the pure Fusarium oxysporum f. sp. melongenae cultures were mixed into the soils before transplant. Establishment of sick field: Two consecutive pure culture sprays F. oxysporum f.sp. melongenae were performed in experimental areas at an interval of 10 days before the most susceptible eggplant varieties were replanted like desi on the ground. Susceptible varieties with a fungal suspension of 1 x 10 6 spores per ml of H2O were again inoculated. This suspension was done by inserting 3-4 ml of fluid in petri-plates containing F. oxysporum 7-10 days old, and after shaking water was poured into 250 ml beaker. 1ml of this suspension in a haemocytometer was retained and no. of spores was evaluated three times, stereo microscopically. Spores in the beaker were adjusted by incorporating sterilized water in the beaker to 1 x 10 6 spores/ml of H2O (Sarwar et al., 2005). Inoculation of this spore suspension was done close to plant's root area for the peak production of inoculum in field. The plants were brought into the laboratory for an evaluation on presence of F. oxysporum f. sp. melongenae after symptoms observation. After testing, the affected crops were ploughed in soil and irrigated to improve decomposition of crop waste and to create circumstances for peak fungal development (Naik et al., 2008). Artificially diseased crops have been reisolated to fulfill postulates of Koch (Ignjatov et al., 2012).

Exploitation of commercial fungicides against Fusarium wilts of Eggplant: Most effective concentrations of fungicides Topsin-M, Flumax and
Shincar with control (water) were evaluated-against Fusarium wilt of eggplant in field environments. In diseased fields, 15 day old seedlings of susceptible varieties were planted and row-to-row and plant-toplant distance was maintained under RCBD. Every treatment was applied with three replicates. Spray was done with 2 weeks interval and influence of spray-ondisease was observed. Data related to disease incidence (%) has been collected after one week interval-up tocrop maturity. T1=Topsin-M (Thiophanate-Methyl) @ 3gm/L, T2= Flumax (Fluazinam Metalaxal-M) @ 2ml/L, T3= Shincar (Carbendazim) @ 3ml/L, T4= Control (water). In-vitro evaluation and their potential against Fusarium oxysporum f.sp. Melongena of several plant extracts: Extracts-of 5 -plants namely Neem (Azadirachta indica), Garlic (Allium sativum), Eucalyptus (Eucalyptus globulus), Datura (Datura stramonium) and (Zingiber officinale) with one control (water) were examined-against-Fusarium oxysporum f.sp. melongenae by using poisoned food-technique. Several parts of these plants like leaves and roots @30g/plant were collected and brought to lab for processing and extraction. These were suspended in 100 ml sterilized water and grand in blender apparatus (Yelmame et al., 2010) and then 1 gram washing powder was added to the solution and then filtered with muslin cloth in beaker and then left for 24 hours. Then, 50 ml distilled water was added and mixed in a beaker. 10% and 15% concentration were made by adding prepared plant extract in sterilized water, and the extract was added in semicooled-PDA media in-vessel and mixed well-by using-rotatory shaker-and 15ml medium was poured-into 9 cm petri dish-in laminar-flow (Yelmame et al., 2010). In cooled medium PDA center and a pure medium of culture of the previously incubated pathogen, a sterilized cork borer was used for making of the 6 mm hole with one diameter in hole. Every treatment with control (water) has been replicated 3 times and then incubation was done at ± 25C for 2 to 3 days under-CRD. Data collection wascarried out-after 4, 7 and 10 days-based upon-colonygrowth. T1= Neem, T2= Ginger, T3= Garlic, T4= Eucalyptus, T5= Datura, T6= Control (water). Data-Analysis: SAS/STAT-statistical softwares were used to perform all statistical tests (SAS Institure, 1990). Means-of all treatments were separated-via-Fisher's protected-least-significant-difference (ANOVA) and SAS/STAT software-packages were used to develop interaction-among different treatmentsand their-combinations.

RESULTS
ANOVA indicated that all the treatments (T), sprays, varieties and their interactions showed significant results as shown in The above (Table. 2) describe the results of the field evaluation of fungicides, on comparison of disease incidence with control the Shincar fungicide was the best against the Fusarium wilt disease as it showed 21.03% disease incidence and then the Topsin-M showed 31.94% disease incidence and the Flumax was close to control as it showed the least effect against the Fusarium wilt disease by showing 35.85% disease incidence. Mean values in rows and column sharing similar small and capital letters do not differ significantly P>0.05.
The comparison of the three sprays with the fungicidal effects is discussed in the (Table 3). It shows that diseaseincidence recorded after first sprays of-Topsin-M was 8.70% and that time control was at 9.31%, therefore, Flumax (8.90%) and Shincar (9.15%) respectively. After 2 nd spray, the Shincar showed (25.26%), Topsin-M showed 30.01% and Flumax showed 35.51%. After the third spray, there was significant difference between Shincar effect and control as it showed 28.83% incidence and then Topsin-M showed 57.11% following Flumax (57.36%). Mean values in rows and column sharing similar small and capital letters do not differ significantly P>0.05.

Fig:1 Evaluation of different plants extracts against Fusarium oxysporum f.sp. melongenae
The above graph showed the response of five plant extracts (neem, garlic, datura, ginger and garlic against the Fusarium oxysporum. As comparison with the control the plant extract of Ginger shows the best response (by showing minimum growth of 1.28 cm) against Fusarium oxysporum and followed by Garlic (1.62 cm), Datura (2.17 cm), Eucalyptus (2.5 cm) and neem (2.76 cm) respectively (Fig 1). Mean values in rows and column sharing similar small and capital letters do not differ significantly P>0.05

Fig: 2 Evaluation of various plant extracts concentrations against Fusarium oxysporum f.sp.
The above graph showed the response of five plants extracts conc. @ 10% blue bars and 15% red bars (neem, garlic, datura, ginger and garlic) against the Fusarium oxysporum. As comparison with the control the plant extract of Ginger shows the best response at 15% (by showing reduced growth of 0.99 cm against Fusarium oxysporum and followed by Garlic (1.28 cm), Datura (1.98 cm), Eucalyptus (2.31 cm) and neem (2.6 cm) respectively. And at 10% concentration the fungus growth was ginger (1.56cm), Garlic (1.96 cm), Datura (2.37 cm), Eucalyptus (2.65 cm) and neem (2.9 cm) respectively (Fig 2). Mean values in rows and column sharing similar small and capital letters do not differ significantly P > 0.05

DISCUSSION
Fusarium wilt produced by Fusarium oxysporum f.sp melongenae is major issue-in cropping regions, in Pakistan; under favorable environment it can cause almost 70% yield losses (Ashfaq et al., 2014). Fusarium wilt indicates signs of a yellowing of the leaves and the fall of apical bud, which kills the whole crop. Soil inhabiting fungi which colonized the senescing tissues of the infected plant and could persist in the soil for many years (Joseph et al., 2008). A subsequent wilt and death of older leaves may occur. Xylem vessel of infected tissue show brown discoloration (Rani et al., 2008). The most pronounced symptom is epinasty of leaves and partial stunting of eggplant by Fusarium wilt (Altinok, 2005). For soilinhabiting diseases-namely Fusarium wilt, usingresistant varieties was good and appropriate method (Naik et al., 2007). Use or resistant host minimizes the disease incidences and avoid-application of fungicides on-soil due to its toxic effects (Russo and Howard, 2002).
Current study indicated that the disease-uptake caused soil and air temperature rise of 19% to 70%, soil-humidity and wind-speed while the comparative moisture rise causes diseases to decrease, with other soil and environmental conditions such as minimum land and aerial-temperature. These-results werematched with Karimi et al.,(2012) and- Mehmood et al.,(2013) results, respectively,-who evaluated that temperature of soil and air alongwith-soil moisture leads towards significant disease development-because of thermopolic-nature-of pathogen (Land et al., 2006;Chand and Khirbat, 2009).
Plant extracts are being used because they are alternative to the fungicidal chemicals, plants extracts are names botanical pesticides as they are not detrimental to plants, human beings and animals (Asthana et al., 2001). These bio-pesticides are-utilized in various formulations namely sprays, dip solutions, fumigants, cakes and water solutions (Siripornivisal and Ngamchawee, 2011). Current study shows five different plants-were used-against Fusarium wilt disease as botanical pesticides (Azadirachta indica, Datura stramonium, Zingiber officinales, Eucalyptus globulus, and Allium sativum). From all there extracts the Z. officinales exhibited outstanding resultscompared to other extracts-of plants. These extracts have lectins, trypsin and proteinase inhibitors and antifungal properties (Goyal et al., 2007;Bijina et al., 2011). They inhibit germination of Fusarium oxysporum spores. Results of this research also matched with observations of- Jamil et al., 2007, Neela et al., (2014 who demonstrated-anti-fungal-properties of-many extracts-of plants against pathogen Fusarium oxysporum casual agent of Fusarium wilt disease. If resistant varieties are unavailable and-application of fungicides is an essential method to control soil inhabiting pathogens to prevent yield losses during epidemically widespread of diseases. Evaluation-offungicides-has been a-good way-to manage infectious diseases for protection of large amount-of money-andenvironment (Song et al., 2004). During contemporary studies, three-fungicides (Flumax, Shincar and Topsin-M) with recommended dose have been checked against Fusarium wilt. Among these,-the Shincar showed significant results. Shincar (Carbendazim) disturbs metabolism and delays-growth-and-development of Fusarium wilt-pathogen (Iqbal et al., 2010). It-formsstrong bonds-with chemical force to pathogens microtublules and change its ionic concentrations (Magnucka et al., 2007). Results of current studies were also favored by results of Naik et al., 2007 whotested carbendazim, benomyl, Thiophanate methyl (Topsin-M) and triadimefon against Fusarium oxysporum and observed carbendazim revealed significant results when compared to other fungicides.

Conclusion
Five plant extracts and three fungicides are used in current research. It is concluded that Zingiber officinale (Ginger) and Shincar (carbendazim) are statistically significant antifungal agents to minimize losses and disease incidence caused by Fusarium wilt of eggplant.