Antifungal Potential of Plant Extracts against Seed-borne Fungi Isolated from Barley Seeds (Hordeum vulgare L.)

Barley is an important rabi season crop of India and plays a major role in barley production all over the world. Ranking of barley is next to the maize, wheat and rice both in acreage and in production of grain. It is very nutritious and a rich source of Vitamin ‘B’ complex and Protein. The cultivated crops are infected by one or more fungal pathogens causing economic losses. The majority of the diseases are caused by seed-borne fungi. These seed-borne pathogens are resulting into losses or death of crop plants. The damage caused by plant parasitic pathogens and seed-borne fungi considered as worldwide and has extensive host range. Due to this they cause potentially serious constrains to crop productivity.


Introduction
Barley is an important rabi season crop of India and plays a major role in barley production all over the world. Ranking of barley is next to the maize, wheat and rice both in acreage and in production of grain. It is very nutritious and a rich source of Vitamin 'B' complex and Protein. The cultivated crops are infected by one or more fungal pathogens causing economic losses. The majority of the diseases are caused by seed-borne fungi. These seed-borne pathogens are resulting into losses or death of crop plants. The damage caused by plant parasitic pathogens and seed-borne fungi considered as worldwide and has extensive host range. Due to this they cause potentially serious constrains to crop productivity.
The most important unit of the crop is the seed; which should be of high quality and pathogen free. Healthy seeds free from pathogens are used for sowing to achieve desired germination, emergence, healthy seedlings and plant population [1]. Seed-borne fungi result in heavy losses in crop yield and seed quality. The most abundant seed-borne fungi, Alternaria spp. is ubiquitous and includes both plant pathogenic and saprophytic species that may damage crops in the field and cause post-harvest decay. Certain grains are frequently reported to be infected by several species of Alternaria, in particular A. alternata Keisler, cause a disease called black point consisting of a discoloration of the germ and of the seed which is due to its mycelium and spore mass [2]. The disease can be a major problem in barley in those areas where heavy rainfall occurs during the early stages of kernel development [3]. A. alternata the most frequently isolated fungi were determined by plating the seeds on agar medium or on moistened filter paper [4]. The presence of fungal mycelium in different parts of black spotted kernels has been determined by microscopical observations [5]. Biological control of plant pathogens is preferred in comparison with the hazardous chemical based products. More use of fungicides like organo-mercurials, carbamates etc. have posed serious health problems to human and environment so there is need to search of natural biodegradable source of bio-fungicides have always been quest for the researchers for control of fungal diseases of plants. Phytometabolites and plant based pesticides appear to be one of the better alternatives for the control fungal diseases, as they are known to have minimal environmental problems and less danger to consumers in contrast to synthetic pesticides [6]. Exploitation of phyto-metabolites in crop protection and prevention of biodeterioration grains caused by fungi appear to be promising. Research on a more sustainable agriculture system and eco-friendly is the need of the hour, as there is a growing concern on the deteriorating quality of the environment as a result of intensive agriculture. So with this view, the present study was undertaken to select plants that could be effective in the development of new mechanism for the control of diseases caused by fungi to the plants of economic importance.

Collection of seed sample
Barley (Hordeum vulgare L.) variety Narendra barley-2 was collected from the quarsi agricultural farm of Aligarh district of (UP) India. The sample was collected in gunny bag and stored at room temperature 28 o C till the processing. The sample was examined for the seed-borne mycoflora according to the international rules for seed testing (ISTA).
Standard blotter and agar plate methods are usually followed where seeds are incubated for a definite period under specific conditions. The associated fungi are identified based on their morphological and habit characters on seed surface and colony characters on the medium. in the test. Three blotters of the size of the petri-plate are dipped in sterilized water and placed in the petri-plate after dropping off extra water. Untreated 10 seeds are plated at equal distance in each petriplate; three hundred seeds of barley sample were examined. The plates are incubated for alternate periods of 12 hours light and 12 hours darkness at 28 ± 2 o C. The plates are removed on the eighth day. After incubation, the fungi developed on seeds are examined under different magnification of a stereomicroscope and identified.

Agar-plate method
The agar plate method is another popular method for the detection of seed-borne mycoflora, in which seeds are plated on an agar medium and sterilized petri-plates of 9 cm diameter containing potato dextrose agar media were used. In each petri-plate 10 seeds were placed. Three hundred seeds of barley were examined. The plated seeds are usually incubated for 8 days at 28 ± 2 o C under 12 th alternating cycles of light and darkness. At the end of the incubation period, fungi growing out from the seeds on the medium are examined and identified. Identification is based on colony characters and morphology of sporulating structures under a compound microscope.

Preparation of aqueous plant extracts
For the study, fresh leaves were collected, detached and surface sterilized with 1% mercuric chloride and washed first in tap water then in distilled water and blotter dried. 100 g of fresh sample was chopped and then crushed in a surface sterilized mortar and pestle by adding 100 ml distilled water (1:1 w/v). The extract was filtered through two layers of muslin cloth and was used as stock solution in the above experiment.

Antifungal activity of botanicals on the growth of fungi by poison food method
Leaf extracts of five plants viz., Eucalyptus globulus, Calotropis procera, Melia azedarach, Datura stramonium, Acalypha indica were evaluated against mycelial growth of Alternaria alternata. These plant extracts were used at 5, 10 and 20% concentrations for which 5, 10 and 20 ml of stock solution was mixed with 95, 90 and 80 ml of sterilized molten PDA media. The medium was thoroughly shaken for uniform mixing of leaf extract. 20 ml of agar media was poured into sterile petriplates and allowed to solidify. Five mm of agar disk of test fungi were cut from 8-10 days old culture plate by using sterile cork borer and placed in the centre of petri-plate containing different concentration of plant extract. There were three replicates of each treatment. The petriplates without plant extracts serve as control. The inoculated plates were incubated at 28 o C for seven days. The percentage inhibition of mycelial growth was calculated as per formula given by Vincent.

C-T 1 = C
Where I= Percent Inhibition; C= Growth of pathogen in control and T= Growth of pathogen in treatment.

Seed treatment with plant extracts
For the seed treatment with five plant extracts, three hundred moderately infected barley seeds were treated with each of the plant extracts involving soaking the seeds in the 20% concentration (as it was found to be most effective in poison food technique) for 12 hours. Treated seeds were dried on blotter sheets overnight under room temperature and then placed on the standard blotter and on agar-plate method and incubated for 8 days under 12 th alternating cycles of light and darkness at 28 ± 2 o C. After incubation seeds were examined for fungal development. Seeds soaked in distilled water served as control. Relative frequencies of seed-borne fungi and percent germination of seeds were calculated.
The Relative frequency of the fungus was calculated by the following formula: No. of seeds containing a particular fungus ×100 Total seeds used No. of seeds germinated % germination = ×100 Total number of seeds

Data analysis
Data was analyzed by one-way analysis of variance (ANOVA) and LSD was calculated at p=0.05 for significance. The analysis was performed with the software R (R Development Core Team 2011).

Antifungal assay
The results presented in Table 1 revealed that leaf extracts of all five plants significantly inhibited mycelial growth of A. alternata at all the tested concentrations. Leaf extract of E. globulus at 20% concentration caused highest inhibition of mycelial growth of A. alternata (52.6%) followed by C. procera (50.88%), M. azedarach (48.21%) and D. stramonium (47.42%), whereas the lowest inhibition (37.52%) of mycelial growth was recorded at 5% leaf extract of A. indica as compared to control.

Discussion
Seed is the most important unit of crop production and its health plays important role in agriculture, which determines the plant population and final yield. One of the major constraints that deteriorate the seed quality is the seed-borne fungi present inside or on the surface of seeds [7]. Leaf extracts of many higher plants have been reported to possess antifungal activity under laboratory trails [8]. In the present investigation antifungal activity of five plants extracts viz., E. globulus, C. procera, M. azedarach, D. stramonium and A. indica was assesed   at the rate of 5%, 10% and 20% concentration against the mycelial growth and sporulation of A. alternata by poison food technique. Similar investigation on antifungal activity of plant extracts against seed-borne mycoflora was reported by many workers [9][10][11]. Among these E. globulus leaf extract was found more effective in inhibition of mycelial growth against A. alternata than other plants. Antifungal activity of E. globulus leaf extract at different concentrations against seed-borne fungi was also checked and found to be effective [12,13] because of essential oil a mixture of terpenoids, aromatic phenols and many other compounds such as 1, 8-Cineole proved to have pesticidal properties [14].
Further seed treatment was done at 20% concentration as found effective in poison food technique. Seed treatment is the safest and the cheapest way to control the seed-borne fungal diseases and is used to prevent biodeterioration of grains [15]. Seed treatment with E. globulus leaf extract was found most effective in reduction of seed-borne incidence and improvement of germination in both standard blotter and agar plate methods. It is known that plants synthesize a variety of bioactive compounds in plant tissues like alkaloids, flavonoids, tannins, terpenoids, saponins, and other compounds, reported to have in vitro antifungal properties [16]. These antifungal compounds stop or inhibit the development of mycelia growth, inhibition of germination or reduce sporulation of fungal pathogens, it is considered that these compounds obtained from plants are biodegradable and safe for use as a substitute for disease control in a traditional production system [17,18]. These are also evidences from the earlier workers, that plants possess the antifungal activity that can play a pivotal role in the management of the plant disease which are cheap, locally available, and biodegradable and environment friendly.

Conclusion
From both the standard blotter method and agar plate method, A. alternata was found most frequently isolated fungi. Among the various plant extracts tested in poison food technique, E. globulus leaf extract at 20% concentration was found most effective in inhibition of mycelial growth of A. alternata.. Seed treatment with all the plant extracts at 20% concentration which were found effective in poison food technique was also found to be effective in both standard blotter methods and agar plate methods. Among all the tested plants E. globulus leaf extract at 20% concentration was found more effective in reducing seed-borne incidence and improved germination in both methods. Further studies should be taken to find the exact mechanism of action by which extracts exert their antifungal effect and to find the active compounds responsible for plant biological activity.