Vegetables Root Rot Disease Management by an Integrated Control Measures under Greenhouse and Plastic Houses Conditions in Egypt A Review

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1 Vegetables Root Rot Disease Management by an Integrated Control Measures under Greenhouse and Plastic Houses Conditions in Egypt A Review Nehal S. El-Mougy, M. M. Abdel-Kader, S. M. Lashin Abstract The most reported areas for high production of vegetables under protected cultivation system in ARE were subjected to survey of either root or shoot systems fungal diseases at two growth stages of plant growth. Surveyed vegetable crops, i.e. Cucumber, Pepper, Tomato showing damping-off, root rot and wilt symptoms were subjected to isolation trails for the purpose of isolation the causal organisms. Rhizospheric samples of different healthy and diseased plants were collected at flowering growth stage from the same surveyed plastic houses distributed for determining the frequency occurrence of different fungi and bacteria associated with the root region of healthy and infected roots of cucumber, pepper, tomato and cantaloupe plants. The root fungal pathogens isolated from rhizospheric soil were Pythium spp., Fusarium spp. Rhizoctonia solani, Macrophomina sp. Sclerotinia spp., Sclerotium rolfsii which recorded in high frequency. Different approaches of some antagonistic fungal, bacterial and yeast agents applied as seed treatment or soil drench was evaluated against various soil-borne pathogens causing vegetables root rot disease under greenhouse conditions. The tested pathogenic fungi were Alternaria solani Fusarium solani, F. oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Pythium sp., meanwhile the tested bio-agents were Trichoderma harzianum, T. Viride and Bacillus subtilis, Pseudomonas fluorescens and Sacchromyces serevisiae. Significant reduction in the disease incidence was observed in bio-agent treatments in comparison with untreated control. Root rot incidence, at pre-emergence stage, significant effect was observed in bio-agent treatments as seed soaking comparing with soil drench treatment. The treated seeds showed a protective effect for seeds germination against the invasion by soil-borne pathogenic fungi. Meanwhile, soil drenched with different bio-agents showed more efficacy for reducing root rot incidence at post-emergence growth stage of tested vegetables, Cucumber, Cantaloupe, Tomato and Pepper. Under greenhouse conditions soil drench with furfural, Humic & Folic acid and/or bio-agents treatments were evaluated against root rot incidence of Cucumber, Cantaloupe, Tomato and Pepper in pot experiments. Applied treatments of the bio-agents in combination with the tested chemicals resulted in higher significant reduction in root rot incidence than each of them alone. Treatments of T. harzianum either alone or combined with chemicals were superior for reducing root rot disease for all tested vegetable plants followed by B. subtilis treatments comparing with the other tested ones. Moreover, the efficacy of Calcium chloride, Thyme oil and /or bio-agents as seed dressing against disease incidence was evaluated in pot experiments under artificially infested with vegetables root rot causal organisms under greenhouse 40 conditions. All applied treatments reduced significantly root rot incidence at both pre-, and post-emergence growth stages of Cucumber, Cantaloupe, Tomato and Pepper plants comparing with untreated check control. The obtained results showed that combination treatments of Calcium chloride, Thyme oil with bioagents reduced significantly root rot incidence of all grown vegetables comparing with the application of each of them alone. Also, in pot experiment the introduced bio-agents proved to continue keeping their antagonistic effect against pathogenic fungi for over one cultivation season resulted in minimize the incidence of root rot disease at both pre-, and post-emergence plant growth stages. Meanwhile, under plastic houses conditions, the efficacy of different plant resistance inducers and/or bioagents treatments against root diseases incidence of some vegetables were evaluated under plastic houses conditions. The evaluated treatments were applied as soil drench before transplanting at commercial plastic houses. The addition of a biological control agent in combination with plant resistance inducers resulted in increased symptom less plant stand over the biological agent. These methods characterized as environmentally safe, bioactive natural products which able successfully to control phytopathogenic fungi in crop production systems. The present review summarizes studies starting from survey of detected infected vegetable crops grown under protected cultivation system up to evaluate some control measures of fungicides alternatives approaches, e.g. some plant resistance inducers, essential oils and bio-control agents on the root rot incidence of some vegetables under greenhouse and plastic house conditions. This work was carried out during a project supported by the Science and Technology Development Fund (STDF), Egypt. Index Terms Bio-agents survival, biological control, cantaloupe, cucumber, essential oils, greenhouse, pepper, plant resistance inducers, plastic houses, root diseases, seed treatment, soil drench, tomato. I. INTRODUCTION Protected agriculture is considered an important means of increasing the productivity and quality of most vegetable crops. Recently, there has been an increase in interest in protected agriculture (PA) in Egypt. The demand for plastic houses has increased and their use has spread in throughout different regions, where there were protected houses belonging to Governmental agro-research stations and others belonging to public-sector cooperatives. Vegetable crops are grown worldwide as a source of nutrients and fiber

2 in the human diet. In Egypt, the most common cultivated vegetables under PA system are Cucumber, sweet and hot Pepper, Tomato, Cantaloupe and Eggplant [1]. Under plastic houses conditions such vegetables are reported to be infected with different root and foliar diseases, i.e. dampingoff, root rot, white rot, wilt, downy and powdery mildew [2,3,4]. The impact of plant pests on the aspiring producer of greenhouse vegetables is direct and significant. The prospective producer must understand that the greenhouse condition is a paradise for both the crop and the accompanying pests that afflict it. Plastic houses have led to changes in the microclimate of protected crops. Restricted air exchange results in the atmospheric humidity being much higher inside insulated greenhouses than conventional ones which encourage several plant diseases and cause physiological disorders. Climate change could alter stages and rates of development of the pathogen, modify host resistance, and result in changes in the physiology of hostpathogen interactions [5]. Fungal plant pathogens can cause devastation in these crops under appropriate environmental conditions. The challenges for producers in managing these diseases are ever-increasing, as consumer demand for yearround production of fresh vegetables with reduced or no pesticide residues continues to grow. Concerns over the potential impact of disease management practices including the use of fungicides on the environment or on consumer health have prompted producers to examine alternative methods to combat fungal diseases. There is a growing need to develop alternative approaches for controlling plant diseases. Root and stem rot diseases caused by pathogens which survive in soil are responsible for serious losses in vegetables crop yield, e.g. Tomato [6], Cantaloupe [7] and Pepper [8]. It was also, recorded that [9] Fusarium stem and root rot of cucumber was observed at four commercial greenhouses in Leamington, Ontario, Canada causing losses of 25-35%. Vegetable producers confronted with the challenges of managing fungal pathogens have the opportunity to use fungi, bacteria and yeasts as biological control agents. Several commercially available products have shown significant disease reduction through various mechanisms to reduce pathogen development and disease. Plant diseases need to be controlled to maintain the quality and abundance of food, feed, and fiber produced by growers around the world. Different approaches may be used to prevent, mitigate or control plant diseases. Beyond good agronomic and horticultural practices, growers often rely heavily on chemical fertilizers and pesticides. Such inputs to agriculture have contributed significantly to the spectacular improvements in crop productivity and quality over the past 100 years. However, the environmental pollution caused by excessive use and misuse of agrochemicals, as well as fear mongering by some opponents of pesticides, has led to considerable changes in people s attitudes towards the use of pesticides in agriculture. Today, there are strict regulations on chemical pesticide use, and there is political pressure to remove the most hazardous chemicals from the 41 market. Additionally, the spread of plant diseases in natural ecosystems may preclude successful application of chemicals, because of the scale to which such applications might have to be applied. Consequently, some pest management researchers have focused their efforts on developing alternative inputs to synthetic chemicals for controlling pests and diseases. Among these alternatives are those referred to as biological control. The application of biological controls using antagonistic microorganisms has proved to be successful for controlling various plant diseases in many countries [10]. However, this is not an easy method, and it is costly to apply; however it can serve as the best control measure under greenhouse conditions. Trichoderma harzianum introduced to the soil, was able to reduce root rot incidence of faba bean plants significantly more than the fungicide Rizolex-T [11]. In recent years, several attempts have been made to overcome this obstacle by applying antagonistic microorganisms. Trichoderma spp. are well documented as effective biological control agents of plant diseases caused by soil-borne fungi [12, 13, 14]. Many investigators [15,16] observed that the application of wheat bran colonized by T. harzianum to soil infested with R. solani and S. rolfsii, reduced the incidence of root diseases caused by these pathogens. As for antagonistic bacteria [17] found that seed treatment with Bacillus spp. was actively controlled three fungal root diseases of wheat. Also, Pseudomonas cepacia or Pseudomonas fluorescens applied to pea seeds act as biological control agent against Pythium damping-off and Aphanomyces root rot and was able to reduce diseases incidence [18, 19]. Considerable researches has been done to investigate antagonistic microbes for use in seed treatments as reported by several workers [20,21,22,23]. In this regard, many crops are susceptible to seed and seedling root rot caused by soil-borne fungi or by pathogens carried on the seed. Biological seed treatments may provide an alternative to chemical control of many soil and seed-borne pathogens. Bio-priming, a seed treatment system that integrates the biological and physiological aspects of disease control, involves coating the seed with fungal or bacterial bio-control agents. Furthermore, soil drench with T. harzianum was significantly able to reduce the incidence of bean root rot of bean and pepper wilt diseases [11,24]. Furthermore, with the knowledge of the adverse effects of synthetic fungicides worldwide, attention is rapidly, being shifted to non-synthetic, safer alternatives. The present review focuses on finding compounds that are safe to humans and the environment, e.g. chemical resistance inducers and/ or bio-agents. In this regards, plant products are characterized as having a wide range of volatile compounds could be used as alternative antibacterial and antifungal treatments [25]. It is evident from reviews by several investigators that Humic and Fulvic acids have been early recorded to have appositive effect against plant pathogens and their cells biological activities [26,27,28]. On the other hand, furfural is a naturally occurring compound, and recently used as a new pesticide active ingredient

3 intended for the use as a fumigant to control root infesting plant parasitic nematodes and fungal plant diseases. Moreover, [29] reported that most of drip irrigation treatments reduced populations of Pythium ultimum and F. oxysporum and increased stem height compared with the nontreated controls. Metham sodium, furfural + metham sodium, sodium azide, and chloropicrin significantly reduced the incidence of Liatris stem rot caused by Sclerotinia sclerotiorum. Moreover, the concern of pesticides use with respect to human health and environment has brought increasing interest in alternatives use by avoiding negative effect on the environment. Essential oils are known for their natural components, such as mono terpenes, diterpenes, and hydrocarbons with various functional groups. Many other researchers have reported antifungal activities [30,31,32] of essential oils in food applications, pharmaceutical research and other areas as plant disease control. The present article reviewed surveying the root diseases infection and associated rhizospheric microorganisms of some vegetables grown under protected cultivation system at different Governmental agro-research stations plastic houses located at different locations throughout Egypt. Moreover, the review focuses on recorded compounds that are safe to humans and the environment, e.g. some plant resistance inducers, essential oils as well as bio-control agents which may provide an alternative control of many soil and seed-borne pathogens. The objective of the present work was to evaluate fungicide alternatives and /or bio-agents against root rot incidence when used as seed and soil treatment under greenhouse and plastic house conditions. Survival and activity of introduced bio-agents to the soil was also considered. II. SURVEYING OF VEGETABLE FUNGAL DISEASES THROUGHOUT DIFFERENT PROTECTED CULTIVATION LOCATIONS The most reported areas for high production of vegetables under protected cultivation system in ARE were subjected to survey of either root or shoot systems diseases at two growth stages of plant growth, i.e seedling (30-60 days after transplanting) and maturity ( days after transplanting). The percentage of different diseases incidence was recorded at different commercial greenhouses distributed in five governorates, i.e. Giza, Cairo, Kalubia, Ismaelia and Behiera. The average percentages of root and foliar diseases infections were calculated as the number of infected plants in relative to the total number of examined plants. Crop monitoring is the continually on-going surveillance to detect the presence of a pest or disease at the very early stages of development of the disease or pest population, before economic damage has occurred. Therefore, the research team involved in working the crop monitoring are enough qualified of the common disease problems and what to look for to detect the presence of disease symptoms in certain crop. 42 The recorded diseases of surveyed different vegetable crops, i.e. Cucumber, Pepper, Tomato and Cantaloupe grown under PA in different governorates in Egypt [33] were damping-off, root-rot, white rot and wilt. The obtained results revealed that the surveyed plants at early stages (30-60 days after transplanting) showed root infections expressed at highest records with wilt infection followed by root-rot and damping-off. Cantaloupe infection with damping-off, root rot and wilt diseases was only recorded at plastic houses located at Ismaelia governorate. It is observed that the recorded data indicate that all cultivars of various surveyed vegetable crops grown under protected cultivation system are susceptible to disease infection with both soilborne and airborne plant pathogens at all surveyed locations. In this regard, it was recorded that the most important root diseases in greenhouses systems are caused by fungi of various species of Pythium and Phytophthora. These fungi are known collectively as water moulds and are important pathogens in soils in field [3]. Moreover, disease organisms, insects, and nematodes can cause serious problems in plastic houses. Without a real winter period, populations of pests continue to build, and many are sustained throughout the year. With this mild climate comes the adaptability of both temperate and tropical pests, thus presenting a large number of potential problems for greenhouse crops [2]. These reports are in a harmony with the present results of our study. Surveyed production areas were examined for the initial symptoms of disease during routine crop maintenance activities, whereas, the earlier a disease is found and identified. The most recorded common diseases affecting greenhouse vegetables were also previously reported in Florida [3,4,34,35,36]. III. ISOLATION DIFFERENT SOILBORNE MICROORGANISMS ASSOCIATED WITH HEALTHY AND INFECTED VEGETABLES Soil microorganisms, fungi and bacteria, associated with the root region of healthy and infected roots of Cucumber, Pepper and Tomato plants grown in plastic houses were isolated [33]. They added that the obtained results showed the frequency occurrence of different fungi in assayed rhizospheric soil samples. The root fungal pathogens e.g. Pythium spp., Fusarium spp. Rhizoctonia solani, Macrophomina sp. Sclerotinia spp., Sclerothium rolfsii were recorded in high frequency comparing with other fungal genera Alternaria spp., Aspergillus spp., Penicillium spp. and Trichoderma spp. The genus Fusarium represented in highest records followed by the genus Rhizoctonia and Sclerotium respectively at all assayed samples. Meanwhile, the pathogens referred to genus Pythium, Macrophomina and Sclerotinia were represented in a lesser frequency. Vegetable crops, i.e. Cucumber, Pepper, Tomato showing damping-off, root rot and wilt symptoms were subjected to isolation trails for the purpose of isolation the causal organisms. The isolated fungi were previously recorded as the main causal of root diseases of surveyed vegetables [37,38,39,40,41,42]. (Concerning the total bacterial counts,

4 results [33] showed that the rhizosphere samples of cucumber, pepper, tomato and cantaloupe were differed in their total bacterial counts. Results also revealed that the four main bacterial groups were common in most plants rhizosphere. In rhizospheric samples collected from Giza plastic houses showed a high records of bacterial count in cucumber rhizosphere, compared to the bacterial count in pepper and tomato samples, respectively. The isolated bacteria are identified according to specific characteristics as Bacillus sp., Agrobacerium sp. and non fluorescent Pseudomonas. In this regards, plant pathogenic bacteria are dominant soil-borne microorganisms which cause many serious diseases of plants throughout the world [43] causing relatively damage and economic cost [44]. Survival of plant pathogenic bacteria in nature occurs most commonly in plant debris left on the soil surface, in and on seeds, in soil, and in association with perennial hosts [45]. Knowledge of survival is usually essential to intervene in dissemination and for disease management. They added that Dissemination commonly occurs by windblown soil and sand particles that cause plant wounding, particularly during or after rains or storms. IV. SURVIVAL AND ANTAGONISTIC ACTIVITY OF INTRODUCED BIO-AGENTS TO THE SOIL Biological control of soil-borne plant diseases is growing in importance as the demand for more environmental friendly management strategies for plant pests increases. The application of biological control using antagonistic microorganisms proved to be successful for controlling various plant diseases in many countries [10]. Fungi in the genus Trichoderma are among the most widely commercialized bio-control agents for soil-borne diseases of crops [46,47,48]. Species of Trichoderma are present in nearly all soils and other diverse habitats [49]. The ability of several Trichoderma spp. isolates to control various plant pathogens, such as Pythium spp., Fusarium spp., Rhizoctonia solani, Verticillium spp., Sclerotium rolfsii, Botrytis cinerea, Armillaria spp., Botryoshaeria spp. and others, has been demonstrated [12,13,14,50,51]. As for antagonistic bacteria, [17] found that seed treatment with Bacillus spp. was actively controlled three fungal root diseases of wheat. Also, Pseudomonas cepacia or Pseudomonas fluorescens applied to pea seeds act as biological control agent against Pythium damping-off and Aphanomyces root rot and was able to reduce diseases incidence [18,19]. Also, Bacillus sp. gave a highly antagonistic effect against some pathogenic fungi including Fusarium solani [53]. It is critical that artificially introduced inoculum is able to colonize, establish, compete and survive in the complex soil environment. Satisfactory bio-control by Trichoderma spp. is apparently dependent on the attainment of some minimum threshold populations in the soil [54] and the continued presence of living cells of the antagonist, as well as a food base [16]. Artificial antagonist inoculum tends to decline in natural soils, and could degenerate to undetectable levels within a relatively short time [54]. 43 Degeneration of active biomass may be accelerated by edaphic factors, such as soil drying [55] or nutrient depletion. Failure of antagonist inoculum to survive and accord long term disease control is a major hindrance to the adoption of biocontrol strategies in the management of soilborne plant diseases [56]. Conversely, indigenous populations of phytopathogens are often well established and adapted, and may persist in the soil for long periods, from where they could multiply and cause disease. In practice, booster applications of antagonists are often necessary to prolong the biocontrol effect. Repeated applications could significantly raise production costs. Pot experiment was carried out in the open greenhouse of Plant Pathology Dept., National Research Centre, Egypt for evaluating the survival and activity of bio-control agents which previously introduced to the soil, as soil treatments, one season before comparing with fresh ones against root rot disease incidence [57]. Cucumber, Cantaloupe, Tomato and pepper plants were used in this experiment as a model of widespread vegetable crops mainly grown under protected cultivation system in Egypt and showed susceptibility to attack with root rot pathogens [33]. They found that incidence of pre-, and post-emergence root rot show that the previously bio-agent applied treatments caused a significant effect on root rot incidence at both plant growth stages of grown vegetables comparing with control. At the first cultivation season, pre-, and post-emergence stages treatment with the bio-agent, T. harzianum caused the highest protection for seeds germination against the invasion by soil-borne pathogenic fungi followed by the others, T. viride, B. subtilis and P. fluorescence, respectively in descending order. The yeast, S. serevisiae showed the lowest protection in this concern. Similar trend was observed at the second cultivation growing season. Regarding the obtained results in the second cultivation season and comparing them with results obtained in the previous cultivation season, it could be observed that survival of both pathogenic and antagonistic microorganisms was proved. Although, the percentage of root rot incidence in control check treatment was lower in second cultivation season than the first one, the bio-agent treatments also showed lesser disease incidence. This phenomenon could be explained as the count of introduced microorganisms into soil, either pathogenic or antagonistic, facing the environmental conditions which reflecting on their counts but not activity to reach their establishment stage which called soil equilibrium community. They stated that survival of both pathogenic and antagonistic microorganisms was proved. Although, the percentage of root rot incidence in control check treatment was lower in second cultivation season than the first one, the bio-agent treatments also showed lesser disease incidence. This phenomenon could be explained as the count of introduced microorganisms into soil, either pathogenic or antagonistic, facing the environmental conditions which reflecting on their counts but not activity to reach their establishment stage which called soil equilibrium

5 community. Therefore, the introduced bio-agents continue keeping their antagonistic effect against pathogenic fungi for over one cultivation season resulted in minimize the incidence of root rot disease at both pre-, and postemergence plant growth stages. They conclude that the introduced bio-agents continue keeping their antagonistic effect against pathogenic fungi for over one cultivation season resulted in minimize the incidence of root rot disease at both pre-, and post-emergence plant growth stages. Results reported by [57] are also confirmed by several researchers [11,58,59]. Biological control of seedling diseases using antagonistic fungi and bacteria has received increasing attention. There are several methods for introducing and delivering bio-agent to the cultivation soil. In this regards, antagonists applied to seeds prior to planting colonize the rhizosphere of seedlings and thus are present at or near the pathogen infection court, where they act by producing antifungal or antibiotic compounds, through hyper parasitism, or by competitively colonizing sperm sphere and rhizosphere substrates [60]. Seed treatment is an attractive delivery system either fungal or bacterial bioprotectants [61]. Bio-protectants applied to seeds may not only protect seeds [12] but also may colonize and protect roots [62]. On the other hand, [20] added a suspension of the bacterium Pseudomonas fluorescens to 1.5% methyl cellulose coated, surface sterilized sweet corn seed prior to hydrating the seeds between moistened paper towels. The two methods were used to protect tomato and sweet corn against Pythium damping-off. In the study carried by [57] the obtained results revealed long term antagonistic ability against soil borne pathogens for over one cultivation season through mixing bio-agents with cultivation soil. In this concern it was reported that temporal population dynamics and survival of antagonists are relevant for the management of disease epidemic cycles arising from amplification of pathogen inoculum. Facultative parasites are capable of saprophytic multiplication, even in the absence of a crop, hence increasing the amount of primary inoculum, which may aggravate disease epidemics in future crops [63]. Also, [64] reported that infusion of pea seed with the fungicide metalaxyl before coating it with conidia of T. harzianum improved survival of conidia in the rhizosphere compared with the survival in the rhizosphere from seed that received conidia only. Furthermore, [65] studied the survival of two bacterial strains in two soils of different texture cropped with wheat. They found that B. subtilis populations declined rapidly in both soils and then stabilized at the levels of added spores. P. fluorescens showed a slow, steady decline in both soils; survival was better in the finer-textured soil, a silt loam, than in the coarser loamy sand. For both bacteria, some translocation to deeper soil layers was observed. No significant rhizosphere effects were detected in either of the two soils. Also, [66] reported that the survival of B. subtilis NB22-1, was investigated in four different soils. After a gradual decline, the bacterial viable cell number stabilized at a level of colony forming units/g-dry soil 44 irrespective of soil differences. The best survival of B. subtilis MBI 600 occurred as spores in sterilized soil, and spore applications to pasteurized soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off [67]. V. LONG ACTIVITY OF STORED FORMULATED BIO-AGENTS AGAINST SOILBORNE PLANT PATHOGENIC FUNGI Fungal disease control is achieved through the use of fungicides which is hazardous and toxic to both people and domestic animals. This leads to environmental pollution. Therefore, a more balanced, cost effective and eco-friendly approach must be implemented and adopted by farmers. Biological control is an innovative, cost effective and ecofriendly approach. Use of natural enemies to control disease is termed biological control. Biological control is an alternative to the use of chemical pesticides. Biological fungicides may act to suppress the population of the pathogenic organisms through competition with pathogenic organisms. Stimulated plant growth, which may allow plants to quickly outgrow any pathogen effects, or damage the pathogen by means of toxins produced [68]. Evaluation the long term viability and activity of some stored antagonistic fungal and bacterial agents against some root rot pathogens was carried out in vitro[69]. They added that viability and antagonistic ability of stored formulated bio-agents were tested periodically throughout ten months of storage. They found that carriers of (Sawdust) and (Sawdust + CMC) were the most suitable tested carries for keeping the viability of B. subtilis, P. fluorescens and T. harzianum with no significant reduction all over the storage period up to ten months. Meanwhile, these antagonists started to lose their viability significantly when formulated on (Sawdust + Talc powder + Chitosan) carrier after the third month of storage followed by the carrier (Sawdust + Chitosan) and (Sawdust + Talc powder) after the fifth month and finally the carrier (Sawdust + CMC +Talc powder) after six months of storage. The inhibitory effect of stored antagonistic fungi and bacteria against the linear growth of root rot pathogenic fungi was evaluated in vitro. The tested pathogenic fungi were Sclerotium rolfsii, Rhizoctonia solani, Fusarium solani, F. oxysporum, Sclerotinia sclerotiorum, S. minor, Macrophomina phaseolina Pythium sp. and Alternaria solani. No significant differences were observed in the antagonistic ability of B. subtilis, P. fluorescens and T. harzianum either stored on (Sawdust) and (Sawdust + CMC) or fresh cultures against tested pathogens. Also, the antagonistic ability of B. subtilis, the highest tested antagonist, against tested pathogenic fungi showed lesser reduction for losing antagonistic ability when formulated on (Sawdust), (sawdust + CMC) carriers and stored for ten months comparing with formulation on (Sawdust + Chitosan), (Sawdust + Talc powder), (Sawdust + CMC +Talc powder) and (Sawdust + Talc powder + Chitosan) carriers and stored for the same period. Similar trend was

6 also observed with the other formulated antagonists P. fluorescens and T. harzianum. Referring to the obtained results they conclude that the antagonistic ability not depended on the counts or population of the antagonist but mainly on its active viability. These findings were confirmed with other reports. Various carriers and polymers have been used to increase the survival rate of the organism with mixed success. Some of the biological control agents are adversely affected by the combination with some traditional chemical seed protection. These products come in dry formulations as dusts, dry spores, and gum/talc powders. Many liquid formulations are also available for sprays, dips, fluid drilling gels and solid matrix priming. These may be designed for large-scale application or planter box treatments [70]. The market for biological control products is not only determined by agricultural aspects such as the number of diseases controlled by one bio-control product in different crops but also by economic aspects as costeffective mass production, easy registration and the availability of competing means of control including fungicides. Shelf life is a very important parameter to be considered in the development of a formulation, because most products will have to be stored for long periods of time before they can be marketed and later applied. More recently commercial formulations of biological controls have been developed which have consistently given good control of some plant diseases [71]. Also, [70] reported that the effect of storage time at room temperature on the viability of Trichoderma spp. in the prepared formulations showed that more than 40% viability of the colonies was recorded at room temperature storage after 4 months. The lowest viability was observed in all fungal formulates after 4 months. Similar results were obtained by [72] who declared that formulations of Trichoderma spp. after stored at ambient conditions for 6 to 8 months. However, [73] mentioned that no viability was observed in different soils at 30 C after 9 weeks, whereas there was viability in all soils at 4 C even after 24 weeks. In nature wide range of organic substrates could be used for the solid-state fermentation for mass multiplication. Solid fermentation media consisting of inert carriers with food bases was used for mass production of bio-control agents [74]. The media with relatively low microbial content would be suited for solid-state fermentation and for the amendment of bio-control agents. Solid substrates include straws, wheat bran, sawdust, moistened bagasse, sorghum grains, paddy chaff, and decomposed coir pith, farmyard manure and other substrates rich in cellulose for inoculums production. In the present study formulated bio-agents on base of sawdust and sawdust + CMC was found to be the most suitable carriers tested for keeping both viability and antagonistic ability of stored both fungi and bacteria bio-agents for up to ten months (300 day). Also, Vidhyasekaran and Muthamilan, 1995 recorded that Pseudomonas fluorescens strains showed inhibitory action against the chickpea (Cicer arietinum) wilt pathogen Fusarium oxysporum f. sp. ciceris under in vitro studies. 45 They assessed the efficacy of various carriers in sustaining the population of these strains during storage and found that in talc-based and peat-based formulations the bacteria survived even up to 240 days of storage although the population declined from 30 days, while chickpea seeds treated with talc-based formulations, P. fluorescens survived on the seeds for at least 180 days. Furthermore, growth population and viability of antagonists primed seeds during storage were reported also by several workers. Also, it was found [65] that T. harzianum strain T-22 increased 10-fold during matrix priming of tomato and cucumber seeds. Viability of the encapsulated T. harzianum remained high for at least six months when stored at 5 o C. The suppressive ness of Zeolite- and peat-based of Paenibacillus sp. and Streptomyces sp. formulation stored at room temperature or at 4 o C was retained for over six months [76]. On the other hand several carriers for formulating bio-agents had been reported. Formulations of fluorescent Pseudomonas were developed through liquid fermentation technology. The fermented biomass was mixed with different carrier materials (Talc/ Peat/ Kaolinite/ Lignite/ Vermiculite) and stickers [77]. Also, [78] developed talc based formulation of P. fluorescens for the management of rice blast caused by Pyricularia grisea, in which methyl cellulose and talc was mixed at 1: 4 ratio and blended with equal volume of bacterial suspension at a concentration of cfu/ml. Talc based strain mixture formulation of fluorescent pseudomonades was developed by mixing equal volume of individual strains and blended with talc [79]. Talc based strain mixtures were effective against rice sheath blight and increased plant yield under field conditions than the application of individual strains. Talc and peat based formulations of P. chlororaphis and B. subtilis were prepared and used for the management of turmeric rhizome rot [80]. One school of thought explains that CMC is added as a sticker at 1:4 ratios to talc. Though it is effective in disease management, it would lead to the increase in the production cost, which would prevent the growers to adopt the technology. More over another school of thought explain that CMC and talc should be used at 1:100 ratios. Hence feasibility of the technique and shelf life of the product has to be evaluated to make the technology as a viable component in disease management so as to promote organic farming. So far, few biological control agents have achieved success under field conditions. Among the hundreds of organisms identified as potential biological disease control agent, only few have resulted in proving commercially acceptable control of these diseases [81]. A fungal biocontrol preparation for control or prevention of plant fungal diseases comprises sporulated fungal biomass and a carrier preferably is vermiculite. Different formulations have been used in control soil-borne pathogens, these are, fungal spores [82], and powdery preparations of fungal mycelium [83]. A bio-control formulation with agricultural potential should possess several desirable characteristics such as: easy preparation and application, stability, adequate shelf life,

7 abundant viable propagules, and low cost [84]. The formulation should be amenable for application to both phylloplane and rhizosphere, depending on the pathogens and plants to be controlled. Formulation of the bio-agents to reduce incidence of the diseases caused by soil-borne pathogens in the field is of great importance in bio-control of such diseases. Therefore the reported work by [69] was aimed also to determine the efficacy of application of formulation contained the B. subtilis, P. fluorescens and T. harzianum as seed treatment against root rot diseases of Cucumber and Pepper under greenhouse conditions. In this study, the efficacy of using stored formulated various antagonistic fungi and bacteria as seed treatment against root rot incidence of cucumber and pepper was evaluated in pots experiment using soil artificially infested with the disease incidents under greenhouse conditions. They added that all the tested fresh and ten months stored bio-agents showed interesting highly significant effect causing high reduction of root rot incidence at both pre-, and post-emergence stages of plant growth comparing with the check treatment. Their results revealed that all the tested fresh and stored bio-agents showed interesting highly significant effect causing high reduction of root rot incidence at both pre-, and postemergence stages of plant growth comparing with the check treatment. No significant differences were observed between fresh applied bio-agents cultures and stored ones formulated on (Sawdust) and (Sawdust + CMC) carriers. Meanwhile stored bio-agents on (Sawdust + Chitosan); (Sawdust +Talc powder); (Sawdust + CMC +Talc powder) and (Sawdust +Talc powder + Chitosan) carriers showed less significant protective effect against root rot disease incidence. They showed low significant pre-emergence root rot incidence ranged between % for applied fungal and bacterial, respectively comparing with untreated and check treatments which recorded as 54.6 and 51.9% for cucumber and pepper, in respective order. Similar trend concerning postemergence root rot incidence was observed. All treatments varied in their effect on disease incidence. Treated seeds showed higher significant reduction on disease incidence than untreated ones. Moreover, bacterial treatment showed superior effect on disease incidence ( %) followed by fungal treatment ( %). Also, [69] reported that it is also interesting to note that, more reduction in disease incidence was observed at post-emergence stage of plant growth than at pre-emergence. This observation could be attributed to their sensitivity to the fluctuations in environmental conditions and are inconsistent in their performance. Similar observation was also reported by several investigators. It was recorded that [85] 60-75% of the cotton crop in US is treated with B. subtilis for the management of soil borne pathogens encountered in cotton ecosystem. Among several PGPR strains Bacillus based products gains momentum for commercialization. Because, Bacillus spp., produce end spores tolerant to extremes of a biotic environments such as temperature, ph, pesticides and fertilizers [85]. Furthermore, seed treatment of pigeon pea 46 with talc based formulation of fluorescent pseudomonads at the rate of 4g/kg of seed followed by soil application at the rate of 2.5 kg/ha at 0, 30, and 60 days after sowing controlled pigeon pea wilt incidence under field conditions. The additional soil application of talc based formulation improved disease control and increased yield compared to seed treatment alone [86]. Delivering of P. fluorescens as seed treatment followed by three foliar applications suppressed rice blast under field conditions [78]. Combined application of talc based formulation of fluorescent pseudomonads comprising of Pf1 and FP7 through seed treatment, seedling dip, soil application and foliar spray suppressed rice sheath blight and increased plant growth better than application of the same strain mixture either through seed, seedling dip or soil (Nandakumar et al., 2001). Biological control of plant pathogens is becoming an important component of plant disease management practices. In case study of [69] the used fungal and bacterial antagonists proved their highly inhibitor effect against root rot pathogens under in vitro and in vivo conditions. These results are also confirmed by several researchers [11,58,59]. VI. DIFFERENT APPROACHES OF INTRODUCING BIO-AGENTS TO THE SOIL FOR CONTROLLING ROOT ROT PATHOGENS Root rot in vegetables strikes quickly and then ruin a whole crop. However the largest instruction course of actions is preventative measures, as therapy with fungicide does not normally work. Biological control of seedling diseases using antagonistic fungi and bacteria has received increasing attention. Antagonists applied to seeds prior to planting colonize the rhizosphere of seedlings and thus are present at or near the pathogen infection court, where they act by producing antifungal or antibiotic compounds, through hyper parasitism, or by competitively colonizing sperm sphere and rhizosphere substrates [60]. (Application of biological control using antagonistic microorganisms had been recorded by several investigators. In this concern, [87] evaluated different approaches of some antagonistic fungal, bacterial and yeast agents applied as seed treatment or soil drench against various soil-borne pathogens causing vegetables (cucumber, cantaloupe, tomato and Pepper) root rot disease under greenhouse conditions. In this study, the tested pathogenic fungi were Alternaria solani Fusarium solani, F. oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Pythium sp., meanwhile the tested bio-agents were Trichoderma harzianum, T. Viride and Bacillus subtilis, Pseudomonas fluorescens and Sacchromyces serevisiae. They reported that the applied biogent treatments either as seed soaking or soil drench caused a significant effect on root rot incidence at both plant growth stages of grown vegetables comparing with control. Regarding root rot incidence at pre-emergence stage, a significant effect was observed in bio-agents treatment as seed soaking comparing with soil drench treatment. The treated seeds showed a protective effect for seeds germination against the invasion by soil-borne pathogenic fungi. Their data also revealed that the antagonist T.

8 harzianum showed significant superior effect to reduce diseases incidence followed by B. subtilis. Also, the antagonists T. viride and P. fluorescence occupied significantly the second degree for reducing root rot incidence. The treatment with S. serevisiae had the lowest effect on disease incidence, although it significantly lesser than check control treatment. This observation was true with all vegetables tested. In this regards, the antagonistic fungi were more actively than the two antagonistic bacteria for reducing root rot incidence at pre-emergence stage of plant growth. Furthermore, data also showed that the lowest root rot incidence was recorded with Cantaloupe and cucumber followed by Tomato and Pepper plants. Data also revealed that the pathogenic fungi S. rolfsii, R. solani, F. solani, F. oxysporum, Pythium sp., M. phaseolina and S. sclerotiorum showed more response to antagonistic fungi and bacteria which reflected in recorded minimization of the root rot incidence. They added that soil drenched with different bioagents showed more efficient for reducing root rot incidence at post-emergence growth stage of tested vegetables. In this concern, the obtained data revealed that percentage of root rot incidence highly reduced in soil drenched with T. harzianum comparing with seed soaking treatment with the same fungus for tested vegetables, Cucumber, Cantaloupe, Tomato and Pepper. Similar observations were also recorded in soil drench treatment with T. viride, B. subtilis, P. fluorescens and S. serevisiae. Furthermore, T. harzianum and B. subtilis drenched soil showed interested results against root rot pathogens that complete reduction (100%) in disease incidence was recorded for Cucumber and Cantaloupe in infested soil with the most tested pathogenic fungi comparing with % infection recorded in control check treatment. The case study of [87] showed that bio-agents applied as soil drench was the most favorite method of application resulted in the best control records. They conclude that the obtained reduction in invaded vegetable plants with root rot pathogens may be attributed to the high accumulative inoculum potential of the introduced bio-agents into the root region, before sowing and throughout the growing season as well, where they are predicted to have a direct impact on already established pathogens population. Similar explanation was reported by [11], who stated that, soil treatment with biocide T. harzianum showed better reduction in root rot incidence of bean followed by seed coating with the biocide. He added that, these differences could be due to the initial inoculum of T. harzianum introduced into the soil. Moreover, the high fungal population density introduced through soil treatment technique enables the fungus to adapt itself against environmental conditions [64] resulting in dominance of high population of the introduced fungi in the plant rhizosphere. The use of microorganisms that antagonize plant pathogens (biological control) is risk-free when it results in enhancement of resident antagonists. Moreover, the combination of such biological control agents (BCAs) with reduced levels of fungicide (integrated control) promotes a degree of disease suppression similar to that achieved with full fungicide treatment [89]. Moreover, the application of biological controls using antagonistic 47 microorganisms has proved to be successful for controlling various plant diseases in many countries [10,59,61,62,90]. Furthermore, for the effective biological control of soilborne plant pathogens, a major consideration has been given to proliferation of the antagonist after introduction into the soil. Among the desirable attributes of a successful antagonist is its ability to produce inoculum in excess and to survive, grow, and proliferate in soil and the rhizosphere [90]. Various actinomycetes, bacteria, and fungi, which show antagonism to P. capsici, exist in soils where peppers are grown [91,92,93]. In particular, some antagonistic rhizobacteria such as Burkholderia cepacia [92] and Pseudomonas aeruginosa [93] were very effective against Phytophthora blight in pepper plants under laboratory and greenhouse conditions. Similarly, application of B. cepacia granules into soil provided better suppression of Phytophthora blight on red-pepper seedlings, as compared to direct drenching with Burkholderia cepacia suspensions [94]. Soil drenches and dipping of seedling roots with the antagonist suspensions were found to be more effective in disease suppression than the coating and dipping pepper seeds [92]. Also, [95] reported that Trichoderma are present in all soil and they are the most cultural fungi. Trichoderma species are strongly antagonistic to other phytopathogenic fungi. They produce hydrolytic enzymes which are believed to play an important role in the parasitism of phytopathogenic fungi. The study of [87] demonstrate that the use of bio-agent treatments, B. subtilis, P. fluorescens and S. serevisiae either as seed soaking or soil drench could reduce root rot incidence of tested vegetables grown in soil artificially infested with disease incidents. These results are in agreements with previous reports in this concern [59,96]. Several workers explained the mode of action of antagonistic bacterial and yeast isolates. In this regards, the potential of Bacillus sp. to synthesize a wide variety of metabolites with antifungal activity is known and in recent years it has been a subject of experiments [97]. Most of these substances belong to lipopeptides, especially from surfactin, iturin and fengicin classes. Not so much is known about the mechanism of antifungal activity of these substances produced by Bacillus sp. Some of them (iturin and surfactin) are able to modify bacterial surface hydrophobicity and, consequently, microbial adhesion to surfaces (to mycelium) [97]. Antibiotics of the iturin group were found to act upon the sterol present in the cytoplasmic membrane of the fungi [98]. Biological control of Aspergillus niger by Bacillus subtilis was also investigated by [99]. They demonstrated that the bacterial cells initially adhered to the fungus, multiplied and extensively colonized the surface. Rapid growth of bacterial cells on the surface resulted in damage of fungal cell walls. These aspects appear essential in association with the antifungal properties of Bacillus sp. used in the biological control of plant diseases. Moreover, although the biocontrol activity of antagonistic bacteria and yeasts has been demonstrated on a variety of commodities, the mode of action of these microbial bio-control agents has not been fully elucidated. In the case of bacterial antagonists, it has been suggested that their bio-control activity may be partly associated with

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