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Biological control methods of several vegetable leaf diseases in Greenhouse

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https://www.eduzhai.net/ Advances in Life Sciences 2012, 2(4): 98-103 DOI: 10.5923/j.als.20120204.03 Greenhouse Biological Approach for Controlling Foliar Diseases of Some Vegetables M. M. Abdel-Kader, N. S. El-Mougy*, M. D. E. Aly, S. M. Lashin F. Abdel-Kareem Plant Pathology Department, National Research Centre, Dokki, 12622, Giza, Egypt Abstract Evaluation the efficacy of bio-agents, application as foliar spray against vegetables foliar diseases incidence was carried out in open greenhouse conditions. The tested bio-agents were Trichoderma harzianum, T. Viride, Bacillus subtilis, Pseudomonas fluorescens and Saccharomyces cerevisiae were evaluated. The recorded foliar diseases, i.e. Powdery, Downy mildews of Cucumber, Cantaloupe and Pepper as well as Early, Late blights of Tomato were significantly reduced at all treat ments either alone or in co mb inations comparing with untreated plants. Application with either T. harzianum and B. subtilis showed significant reduction in diseases incidence comparing with the other applied bio-agents. The other bio-agent treatments, T. viride, P. fluorescens and S. cerevisiae recorded moderate reduction in this concern. On the light of the present study it could be suggested that the usage of bio-agents might be used as easily applied, safely and cost effective control methods against such foliar plant diseases. Keywords Cucumber, Cantaloupe, Pepper, To mato, Bio-Agents, Powdery Mildew, Downy M ildew, Early Blight, Late Blight 1. Introduction Vegetable crops grown under protected cultivation, which considered as Egypt’s largest export national inco me, facing a serious problem due to diseases infection that cause about 20-30 % loss of produced yield because of favourable environ ment for disease incidence and development. Therefore, many control practices need to be integrated in order for minimizing this figure to occur. Po wdery and Downy mildews as well as Early and Late blights are the most serious foliar d iseases attacked vegetables grown in plastic houses. Powdery mildew disease is one of the most serious plant diseases, causing large yield losses in a nu mber of crops[1]. Powdery mildew, is a serious disease affecting the leaves, stems and fruits of cucu mber (Cucumis sativus L.) g rown in greenhouses and in the field [2]. Powdery mildew of cucumber is one of the most dangerous foliar d iseases, attacking cucumber plants in Egypt and other countries[3-5]. Moreover, powdery mildew, caused by Erysiphe cichoracearum DC, is one of the most serious diseases affecting cantaloupe production, with a high severity at the time of fruit maturity[6]. Also, late and early b lights of tomato caused Phytophthora infestans and Alternaria solani were also recorded in growing greenhouse tomato[7, 8]. In order to overcome such hazardous control strategies, * Corresponding author: nehal_nrc@yahoo.com (N. S. El-Mougy) Published online at https://www.eduzhai.net Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved scientists, researchers from all over the world paid mo reattention towards the development of alternative methods which are, by defin ition, safe in the environment, non -toxic to humans and animals and are rap idly biodegradable, one such strategy is use of Biocontrol agents (BCAs) to control fungal plant d iseases. Among the BCAs, species of the genus Trichoderma is most promising and effective biocontrol agent. Trichoderma as antagonist controlling wide range of microbes[9]. Inhibition of pathogenic fungi has been reported as a result of BCAs treatment included in powdery mildew (Leveillula taurica) in sweet pepper[10] and powdery mildew in cucu mber[11]. Furthermore,[12] recorded the possibilit ies for bio logical control of powdery mildew (Sphaerotheca fuliginea, Erysiphe cichoracearum) and mildew (Pseudoperonospora cubensis) on cucumber by Enterobacter cloacae (J.) using the bio-agent Enterobacter cloacae sprays. On the other hand,[13] reported that based on the whole plant tests, foliar spray with Paenibacillus macerans-GC subgroup A, Serratia plymuthica, Bacillus coagulans, Serratia marcescens-GC subgroup A, Bacillus pumilis -GC subgroup B and Pantoea agglomerans bacterial isolates reduced the disease severity of early b light significantly when co mpared with control. These results suggest that the bacterial isolates studied have a good potential to be used as biocontrol agents of A. solani in tomato. The aim of present study is to evaluate the abilities of foliar spray with different bio-control agents for controlling Powdery and Downy mildews of Cucu mber, Cantaloupe, 99 Advances in Life Sciences 2012, 2(4): 98-103 Pepper and Early, Late blights of Tomato under open greenhouse conditions. 2. Materials and Methods 2.1. Plant Materials Trasplants of Cucumber (cv. Alpha), Cantaloupe (cv. Yatherb 7), To mato (cv. Castel Rock) and Pepper (cv. Ca lifornia ) we re used in the present study. 2.2. Bio-agents The tested antagonistic fungi were Trichoderma harzianum, T. Viride and the bacteria Bacillus subtilis, Pseudomonas fluorescens and the yeast Saccharomyces cerevisiae. These antagonists were isolated fro m the rhizosphere of cucumber, cantaloupe, tomato and pepper grown in plastic houses under protected cultivation systems and showing root rot disease symptoms[14]. The present bio-agents proved their antagonistic ability against the above mentioned pathogens under in vitro conditions. 2.3. Bio-agents Inocul a Preparati on The antagonistic bacteria (B. subtilis, P. fluorescense) were grown on nutrient broth mediu m while yeast (S. cerevisiae) was grown on NYDB med iu m[15]. All tested bacteria and yeast incubated in a rotary shaker at 200 rp m for 24 h at 28 ±2℃. The bacterial and yeast cells were harvested by centrifugation at 6,000 rp m for 10 min, washed twice with 0.05 M phosphate buffer at pH 7.0, and re-suspended in sterilized distilled water. The concentrations of both bacteria and yeast cells in the suspensions were adjusted to 1x105-106 cells per millilitre (Cfu / mL) with the aid of a haemacytometer slide. Meanwhile, antagonistic fungi were grown on PDA mediu m[15] incubated for 72 h at 25 ±2℃. Fungal conidia were harvested by scraping the surface of the colonies with a spatula and transferred to sterilized distilled water and filtered through nylon mesh, then spore suspension was adjusted 1x104-105 spore per millilit re (Cfu / mL) with the aid of a haemacytometer slide. 2.4. Greenhouse Experi ments Evaluation the efficacy of bio -agents application as foliar spray against vegetables foliar diseases incidence, the fo llowing procedures was carried out in open greenhouse, National Research Centre, Egypt. Transplants of Cucumber, Cantaloupe, To mato and Pepper were p lanted in natural loamy soil as three transplants per pot and five pots per replicates in each particular fo liar treat ment. Foliar spraying with tested bio-agents, i.e. T. harzianum, T. viride, B. subtilis, P. fluorescens and S. cerevisiae were applied twice with two weeks intervals starting one week fro m transplanting. One week after the second antagonistic foliar application, foliar artificial infestations with Sphaerotheca fuliginea and Peronoplasmopara cubensis the causal fungi of Cucu mber, Cantaloupe and Pepper Powdery and Downy mildews, re- spectively as well as Alternaria solani the causal fungi of Tomato Late blight was carried out as spraying of pathogen suspension (2x104cfu/mL). Phytophthora infestans the causal of To mato Early blight was applied as soil drench through soil irrigation with fungal suspension (2x104cfu/ mL) after[16] at the rate of 50ml/Pot. These mentioned pathogenic fungi were obtained fro m collected diseased vegetables grown in different locations of commercial protected cultivated plastic houses at the same growing season. Fungal suspensions was carried out in distilled water and also adjusted to 1x104-105 spore per millilit re (Cfu / mL) with the aid o f a haemacytometer slide. Another set of soil planted with tested vegetables transplants sprayed only with folia r diseases incidents was kept as control check treat ment. Appearance of different foliar d iseases incidence were recorded periodically and the average accumulated percentages were calcu lated after 60 days of transplanting, the experimental period. Statistical anal ysis All experiments were set up in a co mplete randomized design. One-way ANOVA was used to analyze d ifferences between applied treatments and disease incidence. A general linear model option of the analysis systemSAS [17] was used to perform the ANOVA. Duncan’s mult iple range test at P ≤ 0.05 level was used for means separation[18]. 3. Results and Discussion Results in Table (1) and Fig (1) represented the efficacy of foliar spraying with b io-agents against vegetables foliar diseases incidence under greenhous e conditions. Presented data revealed that Powdery and Downy mildews were d etected only on Cucumber, Cantaloupe and Pepper while Early and Late blights were recorded on Tomato plants. Data also showed that all applied bioagents significantly reduced the recorded foliar diseases comparing with untreated control. Application with either T. harzianum and B. subtilis showed significant reduction in diseases incidence comparing with the other applied bio-agents. The recorded percentage of Powdery mildew in b io-agents spray application ranged between 13.3-33.3% in Cucu mber, 13.3-30.0% in Cantaloupe and 16.6-33.3% in Pepper comparing with 50.0, 43.3 and 40.0% in control treat ment, respectively. The other bio-agent treatments, T. viride, P. fluorescens and S. cerevisiae recorded moderate reduction in Powdery mildew incidence ranged between 20.0-26.6% of the tested vegetables. Similar trend was also observed for Downy mildew in Table (1). The recorded disease incidence were 16.6-30.0% of Cucumber, 13.3-33.3% of Cantaloupe and 16.6-36.6% of Pepper co mparing with 53.3, 40.0 and 46.6% in control treatment for sprayed plants in respective order. To mato plants recorded Early and Late blights infection as 16.6-30.0% and 16.6-33.3%, co mparing with unsprayed plants which showed diseases incidence calculated as 33.3% and 36.6% for both diseases, respectively. In this concern several workers conducted with bio- con- M . M. Abdel-Kader et al.: Greenhouse Biological Approach for Controlling Foliar Diseases of Some Vegetables 100 trol applications against plant diseases control were reported. Bio logical control o f vegetable foliar d iseases by different micro-organis ms is a part of important researches over the world. Overall, reduction of pesticide use is one objective, on the other hand the decline of residues on agriculture products is another issue and here the biocontrol of foliar diseases can have a significant effect. Biological control using natural products or antagonistic micro-organisms proved to be successful for controlling various plant path ogens in many countries[19]. It is still not expensive and is easy in application, however it can serve as the best control measure under restricted conditions. In addition, its application is safe, un-hazardous for human and avoids environmental pollution[20]. In this regard, [21] reviewed that during the past ten years, over 80 biocontrol products have been marketed world wide. A large percentage of these have been developed for greenhouse crops. Products containing Trichoderma, Ampelomyces quisqualis, Bacillus, Uloclad iu m and Pseudomonas are being developed to control the prima ry foliar d iseases, Botrytis and powdery mildew in greenhouses could predominate over chemical pesticides. In the present study, spraying vegetables, Cucu mber, Cantaloupe, tomato and Pepper with the bio-agents, T. harzianum, T. viride, B. subtilis, P. fluorescens and S. cerevisiae was effect ively ab le to reduce the foliar diseases comparing with untreated control. These results were in agreement with several previous reports. A field experiment was conducted at Rahuri, Maharashtra[22], India to investigate the efficacy of the culture filtrates of T. viride, T. harzianum, T. hamatum, T. longiflorum and T. koningii and the fungicide carbendazim against the powdery mildew (Leveillula taurica) of guar. They found that all treat ments recorded beneficial effects on growth parameters and disease control. Also, [23] recorded that Trichoderma harzianum which can be regarded as a model to demonstrate biocontrol under commercial conditions and the mechanis ms involved. He added that this biocontrol agent controls the foliar pathogens, Botrytis cinerea (gray mold), Pseudoperonospora cubensis (downy mildew), Sclerotinia sclerotiorum (foliar blight) and Sphaerotheca fusca (powdery mildew) in cucu mber under comme rcia l greenhouse conditions. Similarly, [24] evaluated culture filtrates of T. viride, T. harzianum, T. hamatum, T. Iongiforum and T. lconlngll for the management of powdery mildew o f Cluster bean caused by LevelIlula taurica. They found that culture filtrates of Trichoderma spp. either alone or in co mbination were found effective against powdery mildew. A lso, Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity caused by Sphaerotheca fusca on greenhouse cucumber by up to 97%[25,26]. Fu rthermore, [27] stated that foliar application of Pseudomonas fluorescens combined with a half of the recommended dose of azo xystrobin is of practical significance, since an applicat ion of fungicide alone requires three to four further following sprays for an effective control of downy and powdery mildews of cucu mber. Table 1. Effect of spraying ant agonist ic bio-agent s against veget ables foliar diseases under open greenhouse condit ions P lant Cucumber Cant alo up e Tomato P epp er Bio-agent T. harzianum T. viride B. subtilis P. fluorescens S. cerivisae Control T. harzianum T. viride B. subtilis P. fluorescens S. cerivisae Control T. harzianum T. viride B. subtilis P. fluorescens S. cerivisae Control T. harzianum T. viride B. subtilis P. fluorescens S. cerivisae Control Powdery mildew 13.3 f 26.6 d 16.6 f 33.3 c 23.3 d 50.0 a 13.3 20.0 de 16.6 f 30.0 c 20.0 de 43.3 ab ND ND ND ND ND ND 16.6 f 26.6 d 20.0 de 33.3 c 26.6 d 40.0 ab Foliar diseases (%) Downy mildew Late blight 16.6 f ND* 26.6 d ND 16.6 f ND 30.0 c ND 23.3 d ND 53.3 a ND 13.3 f ND 26.6 d ND 16.6 f ND 33.3 c ND 16.6 f ND 40.0 ab ND ND 20.0 e ND 26.6 c ND 16.6 f ND 33.3 b ND 23.3 d ND 36.6 a 16.6 f ND 20.0 de ND 33.3 c ND 36.6 c ND 23.3 d ND 46.6 ab ND Early blight ND ND ND ND ND ND ND ND ND ND ND ND 16.6 e 26.6 c 20.0 d 30.0 b 20.0 d 33.3 a ND ND ND ND ND ND Mean values within columns for each diseas e followed by the same letter are not significantly different (P ≤ 0.05) * ND = Not detected 101 Advances in Life Sciences 2012, 2(4): 98-103 Cucumber foliar diseases (%) Powdery mildew Downy mildew Late blight Early blight 80 60 40 20 0 Cantaloupe foliar diseases (%) 80 60 40 20 0 Tomato foliar diseases (%) 60 50 40 30 20 10 0 Pepper foliar diseases (%) 80 60 40 20 0 T. harzianum T. viride B. subtilis P. fluorescens S. cerevisiae Figure 1. Reduction in vegetables foliar diseases in response to spraying with antagonistic bio -agents under open greenhouse conditions Also, [13] suggest that Paenibacillus macerans, Serratia plymuthica, Bacillus coagulans, Serratia marcescens-GC, Bacillus pumilis and Pantoea agglomerans the bacterial isolates as foliar spray reduced the disease severity of early blight significantly when compared with control and have a good potential to be used as biocontrol agents of A. solani in tomato. The mode of action of biocontrol mechanisms was explained by many investigators. 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[28,29]. Most of these substances belong to lipopeptides, especially fro m surfactin, iturin and fengicin classes. Not so much is known about the mechanism of antifungal activity of these substances produced by Bacillus sp. M . M. Abdel-Kader et al.: Greenhouse Biological Approach for Controlling Foliar Diseases of Some Vegetables 102 Some o f them (iturin and surfactin) are able to modify bacterial surface hydrophobicity and, consequently, microbial adhesion to surfaces (to myceliu m)[28]. A lso, antibiotics of the iturin group we re found to act upon the sterol present in the cytoplasmic membrane of the fungi[29,30]. Moreover, Saccharomyces cerevisiae was used as biocontrol agent and systemic resistance mechanis ms [31]. A local isolate of S. cerevisiae was reported to have a reduction potential against radial growth of pathogenic fungi Macrophomina phaseolina and Fusarium solani, the cause of root rot diseases in tomatoes and eggplants[32]. The obtained results in the present study showed that foliar spray with biogants can have a considerable activity against Powdery and Downy mildews of Cucu mber, Cantaloupe, Pepper and Early, Late blights of To mato under open greenhouse conditions. Their non chemical propert ies suggest potentials for commercial formu lation and application which could suggested as a broad spectrum use against foliar pathogens under plastic greenhouses conditions. ACKNOWLEDGMENTS This work was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No. 1059. REFERENCES [1] Kiss, L., 2003, A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest M anage. Sci., 59, 475-483. [2] Bettiol, W., Harllen, S.A.S., Ronielli, C.R., 2008, Effectiveness of whey against zucchini squash and cucumber p owdery mildew. Sci Hortic., 117, 82-84. [3] M osa, A.A., 1997, Effect of foliar application of phosphate on cucumber powdery mildew. Annals Agric. Sci. Ain Shams Univ. Cairo, 42,241-255. [4] Reuveni, M ., Agapov, V., Reuveni, R., 1997, A foliar spray of micronutrient solutions induced local and systemic protection against powdery mildew (Sphaerotheca fuliginea) on cucumber plants. Europen. 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