Studies on Biological Control of Alternaria Solani

Abstract

The early blight disease caused by Alternaria solani is most common infection of tomato which drastically decrease the yield globally. Firstly, cultural conditions for Alternaria solani in vitro, were evaluated and subsequently, phylloplane fungi of tomato were examined, Five Trichoderma spp. were evaluated as biocontrol agents, eight antagonistic bacterial strain were tested for their antagonistic activity, then aqueous, ethanolic and methanolic extracts of 55 plant species were tested against A. solani and seven phenolic compounds were tested for their activity against A. solani, Finally the activity of five fungicides were tested. Cultural studies were performed to evaluate the carbon and nitrogen sources for effective growth of A. solani in PDA media. Eight different carbon sources were tested and glucose was found as the best carbon source for growth of A. solani while least growth was found in the control (without any carbon source). Among the seven nitrogen sources tested threonine followed by urea gave optimal growth of A. solani while minimum growth occurred in the control. Nine different pH of the media were investigated for growth of A. solani. Best growth was observed at a pH range of 5.0 to 7.0 whilst minimum at pH 9. Evidently, A. solani requires acidic pH for its growth. Maximum growth of A. solani required a temperature range of 25-30°C while minimum growth was observed at 5 °C temperature. Further, Alternate light and dark (12 hr each) appeared to be effective for good growth of A. solani. Morphological characters and growth were observed in nine different media. Colony characters included were: margin of colony, colour of colony and substrate colour while colony diameter measured the growth of A. solani. Maximum growth was observed on PDA, followed by corn meal agar which supported good growth of A. solani, whereas minimum growth was recorded in lactic acid. Fungi are vital components of nearly all ecosystems and affect human health and our economy in many ways. Monitoring fungal biodiversity from various systems including phylloplane is essential. The occurrence of phylloplane fungi on leaf surface of tomato (Solanum lycopersicum L.) was investigated. Nineteen fungal species were recorded from the tomato leaf surfaces sterilized leaf segments using dilution plating technique. Among these Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, xx Aspergillus terreus, Curvularia sp., Fusarium oxysporum, F. moniliformis, Macrophomina phaseolina, Dreschlera sp., Penicillium citrinum, Penicillium chrysogenium, Rhizopus stolonifer, Trichoderma harzianum, Trichoderma viride , white sterile mycelium and yellow sterile mycelium were recorded. Diversity of phylloplane assemblages was also measured. The colony interaction between the Alternaria solani and the phylloplane fungi of tomato were assessed following the model proposed by Dickinson. Alternaria solani overgrew the colony of Aspergillus flavus, A. niger, A. fumigatus and showed type A interaction while Trichoderma harzianum, T. viride inhibited the growth of A. solani and produced coiling around A. solani (type B interaction). Fusarium oxysporam, Fusarium moniliforme, Aspergillus terrus, Penicillium citrinum, and Peneicillium chrysogenum inhibited the growth of A. solani but did not produce coiling (type C). Rhizopus stolonifer and Macrophomina phaseolina met with A. solani colony at the edges. Biological control is a non chemical measure which is often as effective as chemical control. Multiple mechanisms are involved in biological controls that include, among others, competition for space and nutrients and modification of environmental conditions, induced resistance and secretion of inhibitory substances, and mycoparasitism. Trichoderma is a soil borne mycoparasitic fungus effective against many soil borne and foliar diseases. Trichoderma spp. are often used in agriculture as biocontrol agents against a number of pathogenic fungi. Culture filtrates of five Trichoderma species (viz. T. viride, T. virens, T. harzianum, T. koningii and T. pseudokoningii) were used against Alternaria solani. Culture filtrate of all the species of Trichoderma retarded the growth of A. solani but T. viride and T. harzianum most strongly suppressed the growth of Alternaria solani in vitro. Results of greenhouse and field experiments provided essentially similar results with those of laboratory method. All the Trichoderma species significantly increased the number of leaves, stem length, root length, stem fresh and dry weight and root fresh and dry weight over positive controls (plants sprayed with A. solani) but substantially less compared to negative control. Phenolic content was also measured under greenhouse condition following infection with Alternaria solani and the treatments with Trichoderma spp. Total phenolic content was significantly higher in all treated plants as compared to -ve control while maximum phenolic content was observed in A. solani together with Trichoderma viride and T. harzianum treatments. PAL and TAL activities were measured in greenhouse condition following infection with Alternaria solani and the xxi treatments with Trichoderma spp. Maximum PAL and TAL activities were recorded in A. solani infected plants where T. viride and T. harzianum were applied, while minimum in –ve control. Bacteria have potential to produce antifungal compounds because they can produce a wide variety of metabolites. The present research was undertaken to assess the antagonistic potential of eight different bacterial strains, Bacillus subtilis, B. aureus, B. thuringiensis, B. lentimorbus, B. cereus, B. coagulans, Pseudomonas fluorescens, and Rhizobium sp. against Alternaria solani. All isolates were subjected to preliminary tests, screening test, agar disc method and slide culture method against A. solani. Most of the selected bacteria showed antagonistic activity against A. solani. Out of eight bacterial species, Bacillus subtilis and Pseudomonas fluorescens gave effective biocontrol against A. solani. Whereas, B. thuringiensis and B. coagulans reduced 50 % growth compared to control in slide culture technique, Bacillus subtilis and Pseudomonas fluorescens, completely inhibited the spore germination of A. solani. Bacillus subtilis and Pseudomonas fluorescens were also tested against A. solani under greenhouse and field conditions. Both the bacterial strains significantly reduced the growth of A. solani and showed significant increase in number of leaves, stem and root length, fresh and dry weights of root, dry weights of stem and root compared to positive control but maximum growth was found in negative control. Total soluble phenolic content was measured under greenhouse condition following infection with Alternaria solani and bacterial treatments with the result that the phenolic content was appreciably higher in all treated plants as compared to -ve controls. Fifty five plant species were selected from local flora and gardens for evaluation of their antifungal potential using aqueous, ethanolic and methanolic extracts (generally leaves) against Alternaria solani in Potato Dextrose Agar (PDA). Poison food technique (for aqueous extract) disclosed that nineteen plants out of fifty five species inhibited the radial growth of Alternaria solani by more than 50 percent. On the other hand, thirteen plants in ethanolic extract and eight plants in methanolic extract reduced the growth of A. solani by above fifty percent. Carica papaya, Clerodendron inerme, Tephrosia purpurea, Capsicum annum, Gliricidia sepium(stem), Cleome viscosa, Euclyptus lanceolatus, Caesalpinia bonduc, Cassia fistula (fruit), Azadirachta indica and Cassia alata showed maximum acitivity against A. solani in all three extracts. Plants with greater activity in all three extract were xxii selected for greenhouse and field experiments. Though aqueous extracts of all the eleven plants selected were effective in disease control, the order of pathogen control and plant growth varied. Pigment content in tomato leaves was also measured under greenhouse condition following infection with Alternaria solani and treatment with plants extract and chlorophyll a, b and carotene were greater in the order - ve control > Tephrosia purpurea > Clerodendron inerme > Capsicum annum > Carica papaya > Cleome viscosa > Eucalyptus lanceolatus > Caesalpinea bonduck > Gliricidia sepium > Cassia alata > Cassia fistula > Azadirachta indica while pigment levels were lower in +ve control. The phenolic compounds derived from plants can be used as natural fungicide against common pathogens of plants. Plants are rich source of a wide variety of bioactive metabolites including phenolic compounds, tannins, glycosides, terpenoids, alkaloids, and flavonoids that are reported to have in vitro antifungal properties. The antifungal activities of phenolic compounds were studied against Alternaria solani. Seven phenolic compounds including Catechol, trans Cinnamic acid, Caffeic acid, p-Caumaric acid, Ferulic acid, Gallic acid and Salicylic acid were tested against Alternaria solani. Determination of antifungal activity was performed by plate diffusion method with three concentrations 0.5, 1.5 and 3 g-l for each phenolic compound and the water controls. Subsequent to addition of phenolics either the media were re-autoclaved or not. However, the results of both methods were closely similar. The results indicated that out of seven, four phenolic compounds i.e., Salicylic acid, Catechol, trans Cinnamic acid and p-Coumaric acid inhibited the growth of A. solani more effectively than did others particularly at higher concentration. More or less similar results were obtained in greenhouse and field experiments. All phenolic compounds showed significant effect on growth characteristic of tomato plant (number of fruit and leaves, stem and root length, fresh and dry weight of stem and root) over positive control but maximum growth was recorded in negative control plants. Further, Chlorophyll a, b and carotene were measured under greenhouse condition following infection with Alternaria solani and the treatments with phenolic compounds. Pigment contents were greater in the order -ve control > Salicylic acid > Catechol > trans Cinnamic acid > p-Caumaric acid while markedly lower in +ve control. Five fungicides including Score, Cabritop, Carbofuran, Carbendazim and Aliette were evaluated for their efficiency against Alternaria solani under labouratory, xxiii greenhouse and field condition. The fungicides Score, Cabritop, Carbendazim and Aliette decrease the growth of A. solani at the concentration of 0.5, 1, 3, 5 and 7μg ml-1. The fungicides Score and Aliette reduced the spore germination of A. solani at the concentrations of 0.5, 1 and 3 μgml-1. Effect of Cabritop was not significant at low concentration (0.5μgml-1) but it had significant effect at higher concentrations compared to positive controls. The fungicides Score, Cabritop, Aliette and Carbofuran at the concentration of 3μg ml-1, significantly reduced disease severity by 50%, 50%, 40% and 60% (as per scale) respectively compared to the +ve controls. The fungicides Score and Aliette were further checked in greenhouse and field experiments and found adequately effective against A. solani. Both the fungicides Allete and Score significantly increased the number of leaves, stem and root length, stem fresh weight and root fresh weights, stem dry weights and root dry weights over positive controls, while growth of negative control (water control) was, in general, substantially higher compared to fungicide treatments. Whereas, pigment contents (Chlorophyll a, b and carotene) were high in the order -ve control > Score > Alette while, pigment levels were substantially lower in +ve control. The results obtained using various biocontrol and chemical agents are discussed in detail.