Evaluation of different plant extracts for effective management of fungal rot of tomato and brinjal in Kashmir Valley

. The aim of present research was focused on the antifungal activities of Prunella vulgaris L and Paeonia suffruticosa Andrews via in vitro approach through agar well diffusion assay at three concentrations (25 µL, 50 µL and 75 µL) against fungi causing diseases in tomato and brinjal. All the concentration of plant extracts showed antimycotic activity against tested pathogenic fungi. Antimycotic activity increased with the increased concentrations of plant extracts. However, higher concentrations proved more effective than lower concentrations. It was revealed from the present study that the ethanolic and aqueous extracts of Prunella vulgaris L showed maximum antimycotic activity against Rhizoctonia solani and least inhibitory effect against Penicillium chrysogenum . It was further revealed from the present study that the ethanolic extract of Paeonia suffruticosa Andrews showed maximum antimycotic activity against Penicillium expansum and least activity against Mucor plumbeus . Whereas the aqueous extract of Paeonia suffruticosa Andrews showed maximum antimycotic activity against Rhizoctonia solani and Penicllium expansum and least inhibitory effect against Mucor plumbeus .


Introduction
Almost, all plants are attacked by a number of plant pathogenic fungi resulting in many plant diseases which reduce their yield and quality of the products. Fungal rots are world-wide in occurrence and have been reported from all parts of the world (Janisiewicz and Korsten, 2002).The destructive pathogen causing rots on tomato is present in parts of

Antifungal activity
The antifungal activity of the plant extracts was determined by agar well diffusion method as adopted by Perez et al. (1990), Alzoreky et al. (2003) and Ahmad et al. (2012). Seven day old fungal cultures grown on PDA medium were used to assess the antifungal activity of selected plant extracts. An aliquot of 100 µL inoculum from each fungal species was inoculated in 20 mL of molten SDA medium in culture tubes. The culture tubes were then homogenised manually and poured into 90 mm Petri plate. The culture plates were allowed to solidify inside the laminar airflow chamber and three wells at periphery of each Petri plate were made using sterile cork borers of 5 mm in diameter. A 2 mg/mL stock solution was made from the plant extract and then different volumes (25 µL, 50 µL and 75 µL) from that stock solution were loaded to respective wells. Hexaconazole solution (20 µL/well) was used as control in the separate well in the same petri plate. The effect of plant extracts on different rot causing fungi were evaluated and the plates were then sealed and incubated at 25 °C ± 2 °C for 4-5 days. Three replicates were made for each treatment. Antifungal potential was calculated by measuring inhibition zone diameters in millimeters (mm) with the help of standard measuring scale (Norrel and Messley 1997).

Statistical analysis
The data collected during these investigations were subjected to appropriate statistical analysis using SPSS statistical software (version 16.0). The data was statistically analyzed by one way analysis of variance (ANOVA) and comparison of the means was done by Duncan multiple comparison tests at P ≤ 0.05.Standard deviation was calculated

Effect of leaf extracts of Prunellla vulgaris L on the zone of mycelial inhibition of some rot causing fungi
It was found from the results (Table 1, Figure 1) that the ethanolic leaf extract of Prunella vulgaris L. caused maximum inhibitory activity of mycelial growth at 25 µL, 50 µL and 75 µL concentrations with zone of inhibition of 22.66 mm, 24.33 mm and 26.00 mm against Rhizoctonia solani, respectively. The inhibition in zone of mycelial growth of Aspergillus niger and Alternaria alternata was 20.00 mm, 22.00 mm, 23.66 mm and 16.33 mm, 19.00 mm, 23.33 mm due to leaf extracts of P. vulgaris at 25 µL, 50 µL and 75 µL concentrations, respectively. The moderate inhibitory activity of ethanolic extract was found against Penicillium expansum with zone of mycelial inhibition of 16.00 mm, 19.00 mm, 22.00 mm, respectively, and against Trichothecium roseum with zone of mycelial inhibition of 15.66 mm, 17.00 mm, 21.00 mm at 25 µL, 50 µL and 75 µL, respectively. However, the inhibition in mycelial growth of Mucor plumbeus was 14.33 mm, 16.00 mm, 19.00 mm at 25 µL, 50 µL and 75 µL concentrations of plant extracts of P. vulgaris. Whereas least inhibitory activity was shown against Penicillium chrysogenum with zone of inhibition as 13.00 mm, 16.33 mm and 18.33 mm at 25 µL, 50 µL and 75 µL of ethanolic leaf extracts.
Further, it was observed from results (Table 2, Figure 2) that the aqueous extract of Prunella vulgaris L. showed maximum inhibitory activity in mycelial growth against Rhizoctonia solani at 25 µL, 50 µL and 75 µL concentrations with zone of inhibition of 20.33 mm, 22.66 mm and 24.66 mm, respectively. Moderate antifungal activity of leaf extract was recorded against Aspergillus niger with zone of mycelial inhibition of 18.00 mm, 20.00 mm, 21.66 mm, against Alternaria alternata with zone of mycelial inhibition of 14.33 mm, 17.33 mm, 20.66 mm and against Penicillium expansum with zone of mycelial inhibition of 13.00 mm, 16.00 mm, 19.33 mm at 25 µL, 50 µL and 75 µL concentrations of leaf extracts, respectively. The inhibition in mycelial growth of Mucor plumbeus and Trichothecium roseum was observed as 12.33 mm, 15.33 mm, 17.00 mm and 11.66 mm, 15.00 mm, 19.00 mm at 25 µL, 50 µL and 75 µL concentrations of leaf extracts of P. vulgaris, respectively. The least mycelial inhibition found in Penicillium chrysogenum was as 11.00 mm, 13.33 mm and 16.00 mm at 25 µL, 50 µL and 75 µL concentrations, respectively.

Effect of leaf extracts of Paeonia suffruticosa Andrews at different concentrations on the zone of mycelial inhibition of some rot causing fungi.
It was revealed from results (Table 3, Figure 3) that the ethanolic leaf extract of Paeonia suffruticosa Andrews showed maximum inhibitory activity at 25 µL, 50 µL and 75 µL concentrations with zone of mycelial inhibition of 23.00 mm, 25.00 mm and 26.66 mm against Penicillium expansum, respectively. The inhibition in mycelial growth of Rhizoctonia solani and Alternaria alternata was 22.33 mm, 25.00 mm, 28.33 mm and 21.00 mm, 24.00 mm, 26.00 mm at 25 µL, 50 µL and 75 µL concentrations of ethanolic leaf extracts of P. suffruticosa, respectively. The inhibition in mycelial growth of Aspergillus niger was 21.33 mm, 23.00 mm, 25.00 mm at 25 µL, 50 µL and 75 µL concentrations of ethanolic leaf extracts of P. suffruticosa, respectively. The moderate antifungal activity of ethanolic extract was shown against Penicillium chrysogenum and Trichothecium roseum with zone of mycelial inhibition as20.00 mm, 21.66 mm, 24.00 mm and 19.33 mm, 22.00 mm, 25.00 mm at 25 µL, 50 µL and 75 µL concentrations of leaf extracts, respectively. The least inhibition in mycelial growth was found in case of Mucor plumbeus with zone of inhibition as19.66 mm, 22.00 mm and 24.33 mm at 25 µL, 50 µL and 75 µL concentrations respectively of ethanolic leaf extracts of P. suffruticosa.
Further it was found from the results (Table 4, Figure 4) that the aqueous extract of Paeonia suffruticosa Andrews showed maximum inhibitory activity at 25 µL, 50 µL and 75 µL concentrations with zone of mycelial inhibition of 20.66 mm, 23.33 mm, 25.33 mm and 19.33 mm, 21.33 mm, 25.00 mm against Rhizoctonia solani and Penicillium expansum respectively. The aqueous leaf extract of Paeonia suffruticosa showed moderate antifungal activity against Alternaria alternata and Aspergillus niger with the zone of mycelial inhibition as 19.00 mm, 22.00 mm, 24.33 and 19.00 mm, 21.00 mm, 23.33 mm at 25 µL, 50 µL and 75 µL concentrations respectively. However, the zone of inhibition in mycelial growth of Trichothecium roseum and Penicillium chrysogenum was 19.00 mm, 20.66 mm, 23.33 mm and 18.00 mm, 19.66 mm, 22.33 mm at 25 µL, 50 µL and 75 µL concentrations of leaf extracts respectively. Whereas the least mycelial inhibition was shown in Mucor plumbeus with the zone of inhibition as 17.66 mm, 20.00 mm and 23.33 mm at 25 µL, 50 µL and 75 µL, respectively of leaf extracts. Each value is mean of 3 replicates ± SD. Mean values followed by different superscript in a column are significantly different (p ≤ 0.05).

Discussion
It was clear from the results that extracts of two medicinal plants Prunella vulgaris and Paeonia suffruticosa brought about significant inhibition in the mycelial growth at their different concentration. Higher concentration proved effective than lower concentration. In the present study some plant extracts were evaluated for their antimycotic activity against the fungus causing rot of tomato and brinjal. These two test plant species proved highly effective in reducing the mycelial growth of fungi causing rot diseases of tomato and brinjal fruits. Such study has been carried for the first time on the extracts of Prunella vulgaris and Paeonia suffruticosa. However, extracts of other plants have been evaluated for their antimycotic activity in a similar way. In a similar study, efficacy of plant extract of Maesa lanceolata, var. goulungensis against many fungal plant pathogens such as Phytophthora cryptogea, Trichoderma virens, Aspergillus niger, Phoma sp., Fusarium oxysporium, Pythium ultimum, Cochliobolus heterostrophus, Rhizoctonia solani, Sclerotium rolfsii and Pyrenophora teres under in vitro conditions was reported by Okemo et al. (2003). Abass (2007) studied the effect of leaf extracts of Henna Lawsonia inermis at different concenterations on some plant pathogenic fungi namely Rhizoctonia solani, Thielaviopsis paradoxa, Fusarium oxysporum f. sp. melonis and Mauginiella scaettae. Several workers tested the antifungal activity of aqueous, ethyl alcohol and acetone extracts of Garlic Allium sativum L., onion Allium cepa L., leek Allium porrum L., Ocimum basilicum L. and Allium sativum L. against Aspergillus niger, Colletotrichum gloeosporioides and other different fungi (Misra and Dixit, 1976;Irkin and Korukluoglu, 2007;Ogbebor et al., 2007). Webster et al. (2008) screened 14 plants for their antifungal activity against various pathogenic fungi and concluded that Fragaria virginiana, Epilobium angustifolium and Potentilla simplex show a promising antifungal potential. Baka (2010)

Conclusion
The present study indicates that the inhibitory effect of plant extracts which have been evaluated for the first time on these rot causing fungi in Kashmir may be attributed to the presence of some partially effective antifungal ingredients, in the plant extracts of all the test plants and thus may have potential to be used as the new natural fungicide for the management of fungal rot diseases.