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Stevia rebaudiana Bertoni, a natural sweetener contains two main sweetest compounds, stevioside (ST) and rebaudioside A (R-A), tasting about 300 and 450 times sweeter than sucrose, respectively. This commercially important plant also suffers a leaf spot disease caused by the fungus Alternaria alternata in various districts of Tamil Nadu, India. In the present study, ten isolates of fluorescent pseudomonads were evaluated for their ability to control leaf spot in stevia (Stevia rebaudiana Bertoni). These isolates were characterized as Pseudomonas fluorescens based on biochemical tests. Among these isolates, P. fluorescens isolate AUPF6 and AUPF5 showed the maximum inhibition of mycelial growth of Alternaria alternata. They also increased plant growth in stevia apart from reducing the leaf spot incidence under greenhouse condition. The isolates AUPF6 and AUPF5 were further tested for their ability to induce production of defense related enzymes and chemicals in plants. Earlier and increased activities of phenylalanine ammonia lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) were observed in P. fluorescens AUPF6 and AUPF5 pretreated stevia plants challenged with Alternaria alternata. Moreover, higher accumulation of phenolics was noticed in plants pretreated with P. fluorescens isolates AUPF6 and AUPF5 challenged with Alternaria alternata . Thus, the present study shows that in addition to direct antagonism and plant growth-promotion, induction of defense-related enzymes involved in the phenyl propanoid pathway collectively contributed to enhance resistance against the invasion of Alternaria alternata in stevia.
Stevia rebaudiana Bertoni, Alternaria alternata, leaf spot, Pseudomonas fluorescens, defense related enzymes.
Stevia rebaudiana is a natural perennial herb, commonly referred to as honey leaf, candy leaf and sweet leaf. It grows upto 1m height and is widely cultivated for its sweet leaves . Stevia is the world’s only natural sweetener with zero calories, low carbohydrates and zero glycemic index. Stevia extract is known to be 300 times sweeter than sugar and has gained attention with the rise in demand for low-carbohydrate, low-sugar food alternatives and its use can be a boon to diabetics and weight losers . Stevia sweeteners are becoming increasingly popular over the last several years because of consumer demand for all natural foods and beverages especially for ones low in calories. It is used as a sweetener even in confectionery, beverages, food industry and cosmetic industry .
Among the several constraints of Stevia cultivation, diseases are a major one. The diseases include Root rot disease caused by Sclertoium rolfsii , Leaf spot disease caused by Alternaria alternata . Powdery mildew by Erysiphe cichoracearum DC, Damping-off by Rhizoctonia solani Kuehn. and Stem rot by Sclerotium dephinii Welch . Among these diseases, leaf spot disease cause severe yield loss and stevioside content. Although fungicides have shown promising results in controlling the leaf spot disease, phytotoxicity and fungicide residues are major problems leading to environmental pollution and human health hazards. Development of fungicide resistance by Alternaria spp. further discourages its use for disease control . Sanitation using sterile or clean water supplies, application of organic compost and regulation of watering and temperature during seedling growth contributed to the management of the disease to some extent. Thus, existing control measures are not effective for the control of damping-off disease. Biological control is an alternative approach to the chemical fungicides and it may be a safe, effective and ecofriendly method for plant disease management. Soil has enormous untapped potential antagonistic microbes i.e. Trichoderma spp. and fluorescent pseudomonads which show antagonistic effects against soil-borne plant pathogenic organisms. The saprophytic pseudomonads associated with plants include P. fluorescens, P. putida and P. aeruginosa. The use of fluorescent pseudomonads is gaining importance for plant growth-promotion and biological control. Fluorescent pseudomonads control fungal pathogens through different modes of action such as competition for nutrients and space , antibiosis , production of siderophores  and lytic enzymes . In addition, induction of resistance by fluorescent pseudomonads is an additional mechanism by which these bacteria protect several crop plants against pests and diseases . The objectives of the present study are (i) to characterize and evaluate the fluorescent pseudomonads isolated from different crops grown in different regions of Tamil Nadu against leaf spot disease in Stevia under greenhouse and field conditions. (ii) to study the induction of various defense-related genes encoding proteins implicated in strengthening of plant cell walls by P. fluorescens in response to infection by Alternaria alternata
Many of the fluorescent pseudomonads, predominantly P. fluorescens, were isolated from suppressive soil for the management of soil-borne diseases. The present study indicates that the all the strains isolated from rhizhosphere of different crops belonged to P. fluorescens. The majority of the fluorescent pseudomonads were found to be P. fluorescens in rhizosphere soils in Australia . The present study also indicates all the fluorescent pseudomonads isolated from different crops cultivated in India belonged to P. fluorescens. Rosales et al. (1993) reported that some of the isolates within each genus of bacteria isolated from rice rhizosphere could be differentiated phenotypically and through protein profile studies.
After isolating and identifying the fluorescent pseudomonads, selecting an effective isolate is the first and foremost important step in biological control. P. fluorescens isolate AUPF6 showed the maximum inhibitory effect on mycelial growth. Fluorescent pseudomonads having antagonistic activity and increasing the plant growth would certainly be promising in evaluating suitable isolates in biological control. In addition to direct antagonism, P. fluorescens AUPF6 increased the activities of various defense-related enzymes and chemicals in response to infection by the pathogen. It is well known that all plants are endowed with defense genes which are quiescent in nature and appropriate stimuli or signals are needed to activate them. It has been reported that application of P. fluorescens triggers/activates plants’ latent defense mechanisms in response to infection by pathogen. Inducing a plant’s own defense mechanism by prior application of a biological agent is a novel strategy in plant disease management. In the present study, it has been observed that seeds treated with P. fluorescens isolate AUPF6 increased the activities of various defense-related enzymes which lead to the synthesis of defense chemicals in the plants. PAL plays an important role in the biosynthesis of phenolic phytoalexins. When groundnut plants were sprayed with P. fluorescens, increase in activity of PAL was observed. Cucumber plants treated with Pseudomonas corrugata had initially higher levels of PAL and levels were lower after challenging the plant with Pythium aphanidermatum. Increase in mRNAs encoding PAL and chalcone synthase were recorded in the early stages of the interaction between bean roots and various rhizobacteria. De Meyer et al. (1999) reported that rhizosphere colonization by P. aeruginosa 7NSK2 activated PAL in bean. roots and increased the salicylic acid levels in leaves. Increased activity of PAL was observed in P. fluorescens treated tomato and pepper plants (during flowering stage) in response to infection by F. oxysporum f. sp. lycopersici and C. capsici . In the present study, increased activity of PAL was recorded in P. fluorescens isolate AUPF6 treated stevia plants challenged with the pathogen, reached maximum on the fifth day after challenge inoculation and was maintained at higher levels throughout the experimental period. In plants inoculated with the pathogen alone the activity declined greatly on the third day after challenge inoculation. Invasion of root tissues by the pathogen might have resulted in decreased activity of PAL whereas earlier and increased activity of PAL due to P. fluorescens isolate AUPF6 treatment might have prevented fungal invasion and thus the activity was maintained at the higher levels. PO and PPO catalyze the last step in the biosynthesis of lignin and other oxidative phenols. In the present study, foliar treatment with P. fluorescens induced the activities of PO and PPO. In bean, rhizosphere colonization of various bacteria induced PO activity. The higher PO activity was noticed in cucumber roots treated with P. corrugate challenged with Pythium aphanidermatum. The native-PAGE study indicates that PO and PPO isoforms were prominently expressed in P. fluorescens isolate AUPF6-treated leaves in response to infection by the pathogen. PPO transcript levels increased in young leaves of tomato when mature leaflets were injured.
Phenolic compounds may be fungitoxic in nature and may increase the mechanical strength of the host cell wall. In the present study, seed treatment with P. fluorescens isolate AUPF6 resulted in increased accumulation of phenolic substances in response to infection by the pathogen. M’Piga et al. (1997) reported that P. fluorescens isolate 63–28 induced the accumulation of phenolics in tomato root tissues. The hyphae of the pathogen surrounded by phenolic substances exhibited considerable morphological changes including cytoplasmic disorganization and loss of protoplasmic content. Accumulation of phenolics by prior application of P. fluorescens in pea has been reported against P. ultimum and F. oxysporum f. sp. pisi (Benhamou et al., 1996). Benhamou et al. (2000) reported that an endophytic bacterium, Serratia plymuthica induced the accumulation of phenolics in cucumber roots following infection by P. ultimum. P. fluorescens Pf1 isolate induced the accumulation of phenolic substances and PR-proteins in response to infection by C. capsici in pepper . Since several defense-related genes encoding proteins are synthesized in ISR by fluorescent pseudomonads, it has been hypothesized that induced resistance by P. fluorescens isolate AUPF6 is related to multigenic/polygenic (horizontal) resistance in plants which is effective against multiple pathogens/races of pathogens. Tuzun (2001) described that constitutive accumulation of defense-related gene products was an integral part of both multigenic resistance and ISR. In cucumber, rhizobacteria induced resistance against cucumber mosaic virus (CMV) and tomato mottle virus (ToMoV) . Induced resistance by fluorescent pseudomonads has broad spectrum activity against several fungal, bacterial and viral diseases .
In conclusion, fluorescent pseudomonads isolated from soils from Tamil Nadu belonged to P. fluorescens group. Application of talc-based formulation of P. fluorescens consistently reduced the incidence of leaf spot disease in Stevia. Prior treatment of stevia with P. fluorescens triggered the plant-mediated defense mechanism in response to infection by Alternaria alternata. Thus, it has been found that P. fluorescens isolate AUPF6 shows broad-spectrum protection against stevia leaf spot pathogen Alternaria alternata.
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