Abstract
In an approach toward the development of ecofriendly antifungal compounds for controlling major foliar fungal diseases of tea, ethanol and aqueous extracts of 30 plants belonging to 20 different families collected from sub-Himalayan West Bengal (India) were tested against the pathogens Pestalotiopsis theae. (Saw.) Stey., Colletotrichum camelliae. Mess., Curvularia eragrostidis. (P. Hennings) Meyer, and Botryodiplodia theobromae. Patouiilard. Spore germination technique was followed for evaluation of antifungal properties. Results showed that ethanol and aqueous extracts of Allium sativum. L., Datura metel. L., Dryopteris filix-mas. (L.) Schott, Zingiber officinale. Rosc., Smilax zeylanica. L., Azadirachta indica., A. Joss. and Curcuma longa. L. recorded 100% inhibition of spore germination. The antifungal component from these plants may be used in developing novel fungicides for tea gardens.
Introduction
Tea is a major plantation crop of India and is cultivated extensively throughout north east India. Fungal pathogens present a significant threat to tea leaves that requires use of chemical fungicides. However, the use of chemical fungicides for tea plants has increasingly become unpopular due to an all-round awareness of its polluting effects, leading to tighter health and environmental regulations. This in turn has created a need for alternative sourcing of fungicidal agents that can be developed for treatment of fungal tea-leaf diseases.
The traditional practice of using plant preparations to combat fungal infections has gained attention, and currently the focus is on detection of new antifungal components from plants that have no negative effect on the environment or on animal and human systems. Several authors have reported antifungal activity of plant extracts against pathogens of rice, tomato, wheat, pea, and other important crops (Rana et al., Citation1999; Sindhan et al., Citation1999; Hu et al., Citation2001). However, such reports involving tea pathogens are very few (Chakraborty et al., Citation1991).
This paper presents a study on the in vitro. effect of aqueous and ethanol extracts of 30 plant species against four major foliar fungal pathogens of tea. This is an approach to identify antifungal elements in natural sources that may be used for controlling tea-leaf diseases.
Materials and Methods
Plant material
The extracts of 30 species of plants (29 angiosperms and one pteridophyte) belonging to 20 different families have been evaluated for their antifungal activity. shows a list of the plants along with their local names and traditional uses (Rastogi & Mehrotra, Citation1995; Chatterjee & Pakrashi, Citation1997; Chopra et al., Citation1996). Some of the plant materials were collected from forest areas of the Mahananda Wild Life Sanctuary located in the terai region of the eastern Himalayas. Others were collected from local areas within and outside the campus of the University of North Bengal. Voucher specimens have been deposited in the departmental herbarium of the Department of Botany, University of North Bengal.
Table 1.. List of plants tested in this study.
Preparation of extracts
Fresh plant materials were washed thoroughly with sterile distilled water and allowed to dry at room temperature. The dried materials were ground and extracted separately with sterile distilled water and ethanol (0.5 g/ml). The extracts were filtered through double-layered cheesecloth and centrifuged at 10,000 × g. for 30 min. The supernatants of aqueous extracts were sterilized by passing through a Millipore filter (0.2 µm). All extracts were stored at 4°C and used for bioassay.
Fungal culture
The plant extracts were tested against four important fungal pathogens. The four fungal pathogens, Pestalotiopsis theae. (Saw.) Stey., Colletotrichum camelliae. Mass. [imperfect state of Glomerella cingulata. (Stonem) S. & v. S.], Curvularia eragrostidis. (P. Hennings) Meyer, and Botryodiplodia theobromae. Patouiilard were isolated from infected leaves of tea plants. They were identified and, after verification of Koch's postulates, are maintained in the laboratory in potato destrose agar (PDA) slants at 4°C.
Effect of plant extracts on spore germination
Plant extracts (30 µl) were placed at two spots 3-cm apart on each microscopic slide taken in duplicate. Spore suspension was prepared from 12-day-old cultures of the fungus in sterile distilled water and added (30 µl) to the plant extracts. Control slides contained appropriate solvents in place of plant extracts. The slides were incubated at 30°C in a humid chamber. After 24 h, they were stained with cotton blue/lactophenol mixture and observed (500 spores) under the microscope. Germination, if any, was noted, and the percentage inhibition was calculated.
Statistical analysis
The mean values of the data for inhibition of spore germination of each extract were compared with control using Student's t.-test.
Results
shows the effect of ethanol and aqueous extracts of 30 plants on spore germination of four major foliar fungal pathogens of tea. Among these, extracts from nine plants showed significant inhibition against all the four tested fungi. Spore germination of Pestalotiopsis theae. was totally inhibited (100% inhibition) by ethanol and aqueous extracts from Datura metel., Allium sativum., and Zingiber officinale.. In addition, aqueous leaf extracts from Azadirnachta indica. and ethanol extracts from Smilax zeylanica. and Dryopteris filix-mas. were equally effective. The germination of spores of Colletotrichum camelliae. was totally inhibited by ethanol and aqueous extracts from Allium sativum. bulbs and also by aqueous extracts from Dryopteris filix-mas. and ethanol extracts from Curcuma longa. and Datura metel. plants. Curcuma longa. extracts were similarly effective against Curvularia eragrostidis.. Spore germination of Botryodiplodia theobromae. was completely inhibited only by ethanol extracts from Allium sativum. bulbs. However, extracts from several plants, which were effective against other fungal pathogens, recorded more than 90% inhibition of spore germination. Altogether, barring a few exceptions, the ethanol extracts seemed to be more effective than the aqueous extracts.
Table 2.. Effect of different plant extracts on spore germination of Pestalotiopsis theae. (P.t..), Colletotrichum camelliae. (C.c..), Curvularia eragrostridis. (C. e..), and Botryodiplodia theobrome. (B. t..).
Discussion
Considering the need for alternative biorational fungicides in tea gardens, it was believed to be worthwhile to evaluate the antifungal effects of locally available plant extracts. The results were encouraging, as several plants showed total inhibition of spore germination. Because, for the pathogenic fungi, spore germination is a determining factor at the onset of host colonization, many of these extracts have a definite potential for new effective fungicides.
Among the different plants whose extracts were found to be effective, Allium sativum. bulbs showed maximum potential because both its aqueous and ethanol extracts showed either 100% or more than 90% inhibition of spore germination of all the fungal pathogens. Antifungal properties of Allium sativum. are well-known (Muhsin et al., Citation2000; Srinivasan et al., Citation2001), especially against human pathogens (Samuel et al., Citation2000) and also against plant pathogens (Sindhan et al., Citation1999). It contains different antimicrobial components like allicin, E.-and Z.-ajoene, iso-E.-10-devinylajoene, and so forth, which are effective against bacteria, yeasts, and phytopathogenic fungi (Prithiviraj et al., Citation1998; Yoshida et al., Citation1999).
Leaf extracts from Datura metel. also showed high antifungal efficiency against all the tested pathogens. In a field study, Asha and Kannabiran (Citation2001) observed that aqueous leaf extracts sprayed at 8 days after sowing protect chilli seedlings against Colletotrichum capsici. (Sydow) Butler and Bisby up to 35 days after sowing. During the current study, though both aqueous and ethanol extract of Datura metel. showed significant activity (P < 0.01), the ethanol extract was found to be more effective in inhibiting spore germination in vitro..
Rhizome extracts of Zingiber officinale. showed high antifungal activity against Pestalotiopsis theae. and Colletotrichum camelliae.. This plant is reported to possess insect growth inhibition, antifeedant, antibacterial, and antifungal properties (Singh et al., Citation1991; Agarwal et al., Citation2001; Martins et al., Citation2001). Curcuma longa. also showed significant activity that is in agreement with findings reported in literature (Jayaprakasha et al., Citation2001). In this case, the aqueous extract was more effective than the ethanol extracts. The antimicrobial properties of Azadirachta indica. leaves, which showed high antifungal activity against Pestalotiopsis theae. and Botryodiplodia theobromae., are well established (Parveen & Alam, Citation1996). Literature studies on other plants with more than 90% inhibition of spore germination revealed that antimicrobial property of Cassia tora. (Kitanaka & Takido, Citation1986), Dryopteris filix-mas. (Asolkar et al., Citation1992), and Psidium guajava. (Ali et al., Citation1996) have been reported earlier. However, there are no previous reports on antifungal activity of Leea indica., Piper peepuloides., and Smilax zeylanica. against phytopathogens.
In spite of the fact that all the plants were tested for their antifungal efficiency by using crude and concentrated extract, the possibility of development of effective fungicides from plant sources to control tea-leaf diseases is evident from our initial screening. Among the four tested pathogens, all are not equally controlled by the same plant extract. Hence, to control all the pathogens, a bioformulation involving extracts from more than one plant may be necessary for effective application in the field. Thus, many of the tested plants may be used for developing new, safer, and effective fungicides.
Acknowledgments
The authors wish to thank Prof. A.P. Das, Department of Botany, University of North Bengal, for identification of some plants. Financial assistance received from CSIR, New Delhi, in the form of a project [38(0987)// EMR–II] is gratefully acknowledged.
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