Assessment of Plant extracts and their In vitro efficacy against Potato early Blight incited by Alternaria solani

Botanicals obtained from the plants are well known for the suppression of inimical plant pathogens. the present study explores the efficacy of five locally available plant extracts for their antifungal activity against the early blight of potato incited by Alternaria solani. the extracts include Datura stramonium, Allium sativum, Azadirachta indica, Eucalyptus globulus, and Lantana camara. All extracts reduced mycelial growth and conidial germination of A. solani. In vitro studies showed that extracts obtained from A. sativum and A. indica have significant inhibition of mycelial growth of A. solani (88.80 and 86.62 percent) at 20 percent concentration. higher concentrations of A. sativum extract caused a higher reduction of A. solani radial growth on potato dextrose agar medium. extracts obtained from A. sativum and A. indica at 20 percent concentration, were found most effective for inhibition of conidial germination (85.50 and 80.04 percent) respectively of A. solani. Observations by scanning electron microscope (seM) showed dramatic alteration in A. solani hyphae collapsed and spores shrinked when treated with extract of A. sativum at a 20 per cent concentration. the qualitative and quantitative analysis of various phytochemicals like flavonoids, alkaloids, saponins, tannins, steroids, terpenoids, glycosides, and phenols was showed A. sativum extract better than all the other plant extracts. Observation also revealed that 20 percent concentration of garlic extract has potential to inhibit to A. solani.

the application of various plant extracts such as Azadirachta indica, Allium sativum, Urtica spp., Ruta graveolens and Melaleuca alternifolia 12 .
Many studies have shown that effects of plant extract for controlling the plant pathogens under in vitro conditions 13, 14 . Chapagain et al. 15 reported the use of saponin rich extract of Balanites aegyptica fruit against A. solani. Another study by Muto et al. 16 tested 14 natural plant species against A. solani. Neem (Azadirachta indica) extract have antifungal 17 , antibacterial 18 , and antiviral 19 properties. Plants possess numerous secondary metabolites compounds showing plant defense mechanisms against pathogen 19 . Aromatic secondary metabolites like terpenoids, flavonoids, tannins, glycosides, saponins, sterols and alkaloids are synthesized by the natural plant species 20 . Availability of such bioactive compounds in plant species which are easily biodegradable and non-phytotoxic 22,23 . Current study explores plant extracts against A. solani under in vitro conditions.

MAteRiAls AND MethODs
Plant extracts used in the study were obtained from Datura stramonium, Allium sativum, Azadirachta indica, Eucalyptus globulus and Lantana camera. Plant materials and preparation of plant extracts D. stramonium, A. indica, E. globulus and L. camera were collected from the agricultural research farm. Allium sativum was obtained from local vegetable market. Apparently fresh leaf material was collected and washed with distilled water and then surface sterilized with sodium hypochlorite (NaClO) 1 percent solution for 2 mins then rinsed thrice with sterilized distilled water. Surface sterilized plant leaves were air dried in room temperature (35°C) and homogenized to make 1 to 2 mm diameter fine powder using a grinder. Aqueous extracts were prepared by decoction method 24 . In brief 20g dried fine powder of every plant sample was gently mixed with 80 ml of sterile distilled water and heated for 10 to 15 mins to remove contamination. Subsequently solution was filtrated through double layered muslin cloth, and finally centrifuged at 8,000 rpm for 25 mins. The obtained supernatant from previous step was filtered through Whatman filter paper No. 1 and sterilized in autoclaved. Sterilized solution considered as 100 percent concentration and preserved aseptically in separate sterile beakers at 4°C until experiment completion.

Collection of pure culture of pathogen
The authentic culture of A. solani was borrowed from the Indian Type Culture Collection, New Delhi, with the ITCC No. 3640. The fungus was sub-cultured and maintained on potato dextrose agar medium (PDA) at 4°C. The pathogenicity was checked under in vitro condition by detached leaf assay (Fig.1).

evaluation of different plants extracts under in vitro condition
To check the efficacy of five plant extracts against test pathogen under the in vitro condition was done with the help of poisoned food technique 25 . Different concentrations of test extracts, including 5, 10, 15 and 20 percent was added in PDA medium, mixed gently and 20 ml media poured in Petri dishes. PDA medium without plant extract was taken as control. Petri dishes containing different test extracts with PDA were placed at the centre of each plate with 5 mm diameter mycelial disk from 10 days old A. solani culture and plates were incubated at 27°C for one week and radial growth of mycelium was measured at 7 days after inoculation. This experiment was done with three replications for each treatment. To measure diameter of test pathogen colony by using two perpendicular lines in each Petri plates. The percent inhibition of test pathogen was assessed using the formula 26 .
Where, I= Per cent inhibition of mycelia growth C = Growth of mycelium in control (mm) T = Growth of mycelium in treatment (mm)

Preparation of conidial suspension
Conidial suspension was prepared by following Stammler et al. 27 with slight modification. 10 ml sterilized distilled water was added to every Petri plate and 7-day old spore colonies were scraped with the help of rubber spatula. 10 ml spore suspensions were made from each plate and conidia density was adjusted to 1.8 × 10 4 spores ml−1 with the help of haemocytometer.

inhibition of conidial germination of test pathogens
Five plant extracts were used to check the antifungal activity of aqueous extract on conidial germination of A. solani through microscopic slide technique with slight modification 28 . The plant extracts at four different concentrations (5, 10, 15 and 20%) was added as films on dried clean microscopic slide. 50 μl conidial suspension was spread over these films and sterile distilled water slide served as a control. Every slide was transfer on a glass rod in a Petri dish under moist conditions and incubated for 3 days at 27°C 29 . Five slides were replicated three times for each concentration. Lactophenol cotton blue (LCB) was added to each slide to arrest conidia germination after incubation. Each slide was observed under a light microscope at ×400 magnification to determine the germination rates of conidia. The experiment was repeated four times, with three replications every time. The percent inhibition of conidial germination for each treatment was assessed by the given formula: Percent inhibition = C − T/C × 100 Where, C = Germinated conidia in control T = Germinated conidia in treatment,

Mycelial deformation study by scanning electron microscopy (seM)
Mycelial disks treated with the plant extracts (Datura stramonium, Allium sativum, Azadirachta indica, Eucalyptus globulus and Lantana camera), among them A. sativum (20 %) and mycelial growth of A. solani was prepared by following procedure described by 30 with slight modifications. Mycelial disks were fixed with Karnovsky solution containing 2.5 percent glutaraldehyde, 2 percent paraformaldehyde in a 0.05 M sodium cacodylate buffer (pH 7.2) for two days for SEM study. The mycelial disk gently washed 3 times by same buffer solution for 35 mins, followed by fixation done with 1 per cent

week after inoculation
Journal of Pure and Applied Microbiology osmium tetroxide solution for 2 h further washed thrice using sterililized distilled water and then dehydrated in a crescent acetone solution series that is, 30, 50, 70, 85, 90 and 100 percent. The samples were then dried in a critical point dryer (Bal-tec CPD 030) with carbon dioxide. Finally, the specimens were spotted on aluminium stubs and fixed with double sided black tape and coated with 20nm gold layer with the help of vacuum evaporator (Bal-tec SCD 050). All specimens were visualized under Joel SEM-6400JSM-6360LV microscope (JEOL Ltd., Japan).

Qualitative analysis of phytochemical screening of different aqueous plant extract
The aqueous extract of five selected plants (D. stramonium, A. sativum, A. indica, E. globulus, and L. camera) was tested for antimicrobial secondary metabolites properties against test pathogen. Alkaloids 1.5 ml extract was taken for alkaloid testing. 2 ml of 1 per cent HCL solution thoroughly mixed with test extract. After mixing, solution was heated for to 2 mins and Mayer's reagent (6 drops) was added. Orange colour formation indicates the presence of alkaloids 31 . Flavonoids 1 ml of aqueous solution of test extract was gently mixed with 1 ml of 10 percent lead acetone. Develop yellow colour that showed the presence of flavonoids 32 .

Saponins
The procedure described by Saidulu et al 33 was followed for the assay 2 ml of aqueous test extract was taken and mixed with 6 ml sterilized distilled water. Formation of forth was observed after 3 mins. Steroids 0.5 ml test extract mixed with 2 ml of acetic anhydride solution and further 2 ml H 2 SO 4 was added to the reaction. The colour change violet to green or blue expressed the presence of steroids 34 . tannins Few drops of ferric chloride solution was mixed with test extract. Development of blackish colour expressed the presence of tannins in the extract 33 . terpenoids 2 ml test extract was mixed with 5 ml of chloroform and heated for 5 mins and then allowed to dry. Further 2 ml concentrated H 2 SO 4 was added and again heated on low flame for 2 mins. Greyish colour develops to show presence of terpenoids 33 .

Glycosides
Each test extract was hydrolysed with hydrochloric acid and then neutralized with NaOH solution. Red colour developed after Fehling's solution A and B were mixed and this revealed that presence of glycosides 35 . Phenols 1 ml of test extract was extracted in ethanol and then evaporated till dryness. After drying residue was dissolved in sterile distilled water and add 0.5 ml Folin-ciocalteu reagent followed by 5 ml of 20% Na 2 CO 3 solution. Bluish colour developed reveals that presence of phenols 36  formed and discarded, and purification process was repeated. After purification, 60 ml n-butanol was added and further extract were washed thoroughly twice with 15 ml of 5 percent NaCl. The solution was heated for evaporation, and then dried in hot air oven. Weighed and percentage was calculated of saponin 39 . Steroids 1 gm sample was crushed in 20 ml of methanol and filtered. 2 ml filtrate was taken and add 5 ml of chromagen solution. The solution was incubated at 35°C for 30 mins. Absorbance was recorded at 550 nm wavelength 40 . tannins 500 mg samples were added in 50 ml distilled water. The solution allows to shaken for 60 mins and filtered. 10 ml filtrate was pipetted out into the new test tube and 2 ml 0.1 M ferric chloride in 0.1 N HCl was added along with 0.008 M potassium ferrocyanide. The final absorbance was recorded at 120 nm wavelength 41 . terpenoids 1 g of extracts were taken, crushed in 50 ml of methanol and filtered. 2.5 ml filtrate was taken and 2.5 ml of 5 % phosphomolybdic acid was added afterward 2.5 ml of concentrated H 2 SO 4 were added. The mixture was incubated at 35°C for 30 mins. The final observance was recorded at 700 nm wavelength 40 . Glycosides 1 g test samples were taken and macerated with 50 ml of methanol and filtered. 1 ml supernatant was taken and 4 ml of alkaline pirate solution was added. The reaction mixture was heated for 5 mins and allowed to cool. The observance was recorded at 490 nm wavelength 34 . Phenols: 2 g test samples were taken in 100 ml diethyl ether solution and allowed to defat with help of Soxhlet apparatus for 2 to 3 h. Defatted solution was heated with 50 ml of ether for 15 mins. 10 ml of reaction was pipetted out into 50 ml capacity flask and 15 ml sterile distilled water was added. Further, 2 ml of NH 4 OH solution and 2 ml of concentrated amyl alcohol were added. The reaction was incubated at 35°C for 30 mins. Absorbance was recorded at 505 nm wavelength 41 .

statistical analysis
The analysis of data with help of oneway analysis of variance (ANOVA) and compared means for significance at p level of ≤ 0.05 through

ResUlts effects of plant extracts on mycelial growth of A. solani
All tested plant extracts were found effective against mycelial growth of A. solani as compared to control (Table 1). In general, proportional reduction of mycelia growth was showed with increasing the concentration of plant extracts from 5 to 20%. The minimum colony diameter (10.10 mm) was recorded on A. sativum amended media followed by A. indica (12.06 mm) and D. stramonium (12.76 mm) extracts at 20% concentration (Fig. 2). The full growth was recorded in control after seven days of inoculation. The maximum colony diameter (20.50 mm) was recorded on E. globulus extracts at 20% concentration compared to other plant extracts.

effects of plant extracts on conidial germination of A. solani
The results from the conidial germination assay by various plant extracts are represented in Table 2. Conidial germination was decreased gradually with increase concentration of tested plant extracts from 5 to 20 %. All plant extracts were significantly decreased the conidial germination of A. solani compared with the control. A. sativum and A. indica extracts were found best among all extracts for the inhibition for conidial germination. Percent inhibition of conidial germination was observed highest at 20% concentration of A. sativum (85.50 %) and A. indica (82.04 %) extracts. Least reduction of conidial germination was observed in E. globulus (68.24%) at 20% extract concentration. E. globulus and D. stramonium extracts were found less effective on inhibition conidial germination than the other three extracts. Inhibition started at low concentration (5 %) with low values (33.14 and 39.23 for E. globulus and D. stramonium, respectively).

Qualitative screening of phytochemicals in aqueous extract
Phytochemical analysis for tested extract revealed that the presence of secondary metabolites showed anti-microbial properties against Alternaria solani (Table 3). A. sativum extract was containing all major secondary metabolites, except steroids. The L. camara

Quantitative assessment of phytochemicals of different aqueous plant extract
The results represented in Table 4 for quantitative analysis of phytochemicals in tested plant extracts. The results revealed that maximum flavonoids content (1.12 %) was recorded in A.

Mycelium and conidia of A. solani under scanning electron microscopy
Based on mycelial growth assay results, A. sativum extract was further investigated by scanning electron microscopy (SEM) against A. solani. Results obtained from SEM revealed that the hyphae of the pathogen exposed to A. sativum plant extract were collapsed, short branching, and caused curling in the hyphae, whereas no such changes were observed in the hyphae developed in the control treatment. Moreover, A. sativum extract results revealed that no conidia formation, shorter germ tubes, and caused shrinking in conidia. The untreated control did not cause any spore shrinking (Fig.3).

DisCUssiON
In the current study we demonstrated that antimicrobial compounds were present in five plant extracts. The plant extracts inhibited mycelial growth of A. solani and conidial germination under in vitro condition. Various plants have been reported to comprise antifungal activity and they have shown encouraging results under in vitro conditions [42][43][44][45] . The current in vitro study revealed that among the five tested plant extracts, A. sativum and A. indica resulted significant reduction in conidial germination and growth of A. solani. Gradual increase in concentrations of plant extracts represents gradual decrease in conidial germination of the pathogen. Plant extracts evaluated against A. solani in the current study also showed significant results 46 . Reports suggested antifungal activity of A. sativum against harmful phytopathogens 47,48 . Significant inhibition was observed with A. sativum extract at higher concentration (20%) against the A. solani. Investigation of the efficacy of A. sativum by Scanning Electron Microscope also showed higher inhibition of test fungus and this finding is in agreement with that of Baka and Rashad 49 . Plant extracts used at higher concentration have capability to reduce growth of phytopathogenic fungi 50 . Plant extracts from A. sativum also comprised botanical compounds such as Allicin, which checked the growth of pathogen mycelium and also inhibited spore germination 51 .
Plant extracts were analyzed for potential antifungal phytochemicals. Various secondary metabolites were detected including alkaloids, flavonoids, saponins, tannins, phenols, terpenoids, glycosides and steroids. The extract from A. sativum was most potential in inhibiting the growth of A. solani compared to other plant extracts, because presence of phytochemicals in huge amounts. Plant derived secondary metabolites like flavonoids have antifungal, antibacterial and antiviral activity used in higher concentrations 52  in plant defense mechanism against invasion by harmful microorganism and insects 56 .

CONClUsiON
The present investigation showed that among five tested plant extracts, A. sativum and A. indica resulted significant reduction in mycelial growth of A. solani, reduction in conidial germination or no conidia formation, formation of shorter germ tubes and shrinking in conidia. Various secondary metabolites were also detected in the plant extracts. The research concludes that plant extracts such as Allium sativum, Azadirachta indica, Datura stramonium, Eucalyptus globulus, and Lantana camara at higher concentrations have capability to reduce pathogen growth and spore germination of inimical plant pathogenic fungi because of presence of several antifungal secondary metabolites such as alkaloids, saponins, terpenoids, flavonoids and phenols. This biocontrol method is environmentally safe, eco-friendly, long lasting, and minimizing the use of hazardous chemicals for management of Alternaria solani in potato.

ACKNOWleDGMeNts
The authors are grateful to Professor and Head, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi for providing all necessary facilities to conduct experiments.

CONFliCt OF iNteRest
The authors declare that there is no conflict of interest.

AUthORs' CONtRiBUtiON
SK was responsible for methodology, investigation and wrote the manuscript. LB helped in performing data analysis. RC supervised the experiment and reviewed the manuscript and made suitable changes. The author(s) read and approved the final manuscript.