Bio-fertilizer application: molecular and biochemical changes in infected cucumber with Phytophthora melonis

نوع مقاله: مقاله کامل


1 هیات علمی دانشگاه یزد

2 Department of Plant Protection, University of Zabol, Zabol, Iran


In this work, the effects of two commercial bio-fungicides Biosubtil (Bacillus subtilis) and the bio-fertilizer Biophosphorus (Pseudomonas species), was assayed on the disease incidence, changes in the defensive enzyme activity, the total phenolic content and the expression level of some defense-related genes using real-time PCR method. Biochemical and molecular changes were measured at four time point: 24, 48, 72 and 96 hours post inoculation (hpi) of cucumber plantlets with Phytophthora melonis. The disease incidence decreased through applications of both antagonists. The highest disease reduction (60%) was noted for Biosubtil application compared to control plants. Biochemical analysis showed that both bio-fertilizers are able to increase total protein and phenolic compounds in infected cucumber plants. A maximum increase was observed with biophosphorus application at 72hpi. High activity of Peroxidae )PO) and β-1,3-glucanase enzymes were noted for biophosphorus application at 72 hpi while maximum increase of Polyphenol oxidase (PPO) was observed at 96 hpi for this application. Altered transcript levels of various resistance genes including cucumber pathogen-induced 4 (Cupi4), Lipoxygenase (Lox), Phenylalanine ammonia lyase (PAL) and Galactinol synthase (Gal) were recorded in both treatments. A high expression level (9.5 fold) among the treated genes was observed for Lox gene at 72 hpi and an high expression level for other genes was noted at 48 hpi when cucumber plants treated with Biophosporus. The results suggest that both bio-fertilizers are able to enhance disease resistance in cucumber through induce of resistance mechanisms and could be used as a resistance inducer in the greenhouse condition.



Abkhoo J, Sabbagh S (2015) Control of Phytophthora melonis damping-off, induction of defense responses, and gene expression of cucumber treated with commercial extract from Ascophyllum nodosum. Journal of Applied Phycology 28(2): 13331-1342.

Ahmadzadeh M, Tehrani AS (2009) Evaluation of fluorescent pseudomonads for plant growth promotion, antifungal activity against Rhizoctonia solani on common bean, and biocontrol potential. Biological Control 48: 101-107.

Arfaoui A (2007) Treatment of chickpea with Rhizobium isolates enhances the expression of phenylpropanoid defense-related genes in response to infection by Fusarium oxysporum f. sp. ciceris. Plant Physiology and Biochemistry 45: 470-479.

Balasundram N, Sundram K, Samman S (2006) Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry 99: 191-203.

Blée E (2002) Impact of phyto-oxylipins in plant defense. Trends in Plant Science 7: 315-322.

Bradford M (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annalytical Biochemistry 72: 248-254.

Chen C, Belanger RR, Benhamou N, Paulitz TC (2000) Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiological and Molecular Plant Pathology 56: 13-23.

Chérif M, Asselin A, Bélanger R (1994) Defense responses induced by soluble silicon in cucumber roots infected by Pythium spp. Phytopathology 84: 236-242.

Choi D, Bostock RM, Avdiushko S, Hildebrand DF (1994) Lipid-derived signals that discriminate wound-and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. Proceedings of the National Academy of Sciences 91: 2329-2333.

Collinge D, Lyngs Jrgensen H (2009) Effects of b-1, 3-glucan from Septoria tritici on structural defence responses in wheat. Journl of Exprimental Botny 60: 4287-4300.

Collinge DB, Slusarenko AJ (1987) Plant gene expression in response to pathogens. Plant Molecular Biology 9: 389-410.

Conrath U, Pieterse CM, Mauch-Mani B (2002) Priming in plant–pathogen interactions. Trends in Plant Science 7: 210-216.

Cowan MM (1999) Plant products as antimicrobial agents. Clinical Microbiology Reviews 12: 564-582.

Daayf F, Bel-Rhlid R, Bélanger RR (1997) Methyl Ester ofp-Coumaric Acid: A Phytoalexin-Like Compound from Long English Cucumber Leaves. Journal of Chemical Ecology 23: 1517-1526.

de Ascensao AR, Dubery IA (2003) Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f. sp. cubense. Phytochemistry 63: 679-686.

Dordas C (2009) Role of nutrients in controlling plant diseases in sustainable agriculture: a review. Agronomy for Sustainable Development 28(1): 33-46.

Elad Y (2000) Biological control of foliar pathogens by means of Trichoderma harzianum and potential modes of action. Crop Protection 19:709-714.

Esmaeili Shirazifard A, Banihashemi Z (2009) The role of Phytophthora melonis and P. drechsleri in damping-off of Cucumber in Iran. Iranian Journal of Plant Disease 44: 54-72. (In Persian).

Expert J, Digat B (1995) Biocontrol of Sclerotinia wilt of sunflower by Pseudomonas fluorescens and Pseudomonas putida strains. Canadian Journal of Microbiology 41: 685-691.

Feussner I, Wasternack C (2002) The lipoxygenase pathway. Annual Review of Plant Biology 53: 275-297.

Gupta P, Ravi I, Sharma V (2013)Induction of β-1, 3-glucanase and chitinase activity in the defense response of Eruca sativa plants against the fungal pathogen Alternaria brassicicola. Journal of Plant Interactions 8: 155-161.

Harborne JB (1984) Phenolic compounds. In: In: Harborne JB (ed.), Phytochemical methods. 3th edition. Springer Science & Business Media, UK. pp. 37-99.

Hausbeck MK, Lamour KH (2004) Phytophthora capsici on vegetable crops: research progress and management challenges. Plant Disease 88:1292-1303.

Ho H (1986) Phytophthora melonis and P. sinensis synonymous with P. drechsleri. Mycologia7: 907-912.

Hofmann J, El Ashry AEN, Anwar S, Erban A, Kopka J, Grundler F (2010) Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. The Plant Journal 62: 1058-1071.

Khosrowfar F, Banihashemi Z (2004) Role of weeds on the survival of Phytophthora drechsleri the causal agent of cucurbit damping-off in Fars province. Iranian Journal of Plant Pathology 40: 105-126. (In Persian).

Kim MS (2008) Galactinol is a signaling component of the induced systemic resistance caused by Pseudomonas chlororaphis O6 root colonization. Molecular Plant-Microbe Interactions 21: 1643-1653.

Kloepper JW, Ryu C-M, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94: 1259-1266.

Kloepper JW, Schroth MN (1978) Plant growth promoting rhizobacteria on radishes. In: IVth International Conference on Plant Pathogenic Bacteria, August 27 - September 2, Angers, France. 882.

Li Y, Minerdi D, Garibaldi A, Gullino ML (2009) Molecular detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii artificially inoculated with zoospores. Journal of Phytopathology 157:438-445.

Lifshitz R, Kloepper JW, Kozlowski M, Simonson C, Carlson J, Tipping EM, Zaleska I (1987) Growth promotion of canola (rapeseed) seedlings by a strain of Pseudomonas putida under gnotobiotic conditions. Canadian Journal of Microbiology 33: 390-395.

Matern U, Grimmig B, Kneusel RE (1995) Plant cell wall reinforcement in the disease-resistance response: molecular composition and regulation. Canadian journal of Botany 73:511-517.

Meena B, Radhajeyalakshmi R, Vidhyasekaran P, Velazhahan R (2000) Effect of foliar application of Pseudomonas fluoresencens on activities of phenylalanine ammonia-lyase, chitinase and β-1,3–glucanase and accumulation of phenolics in rice. Acta Phytopathologica et Entomologica Hungarica 34:307-315.

Mittler R (2002) Oxidative stress, antioxidants and stress tolerance Trends in Plant Science 7: 405-410.

Moghaddam MRB, Van den Ende W (2012) Sugars and plant innate immunity. Journal of Experimental Botany 63(11): 3989-3398.

Nawar H, Kuti J (2003) Phytoalexin synthesis and peroxidase activity as markers for resistance of broad beans to chocolate spot disease. Journal of Phytopathology 151: 564-570.

Nemati M, Navabpour S (2012) Study on quantitative expression pattern of oxalate oxidase and β-1, 3 glucanase genes under Fusarium graminearum treatment in wheat by quantitative Real time PCR. International Journal of Agriculture and Crop Sciences 4:443-447.

Nicholson RL, Hammerschmidt R (1992) Phenolic compounds and their role in disease resistance. Annual Review of Phytopathology 30:369-389.

Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology 16:115-125.

Paul D, Sarma Y (2005) Pseudomonas fluorescens mediated systemic resistance in black pepper (Piper nigrum L.) is driven through an elevated synthesis of defence enzymes. Archives of Phytopathology and Plant Protection 38:139-149.

Phuntumart V, Marro P, Métraux J-P, Sticher L (2006) A novel cucumber gene associated with systemic acquired resistance Plant Science 171: 555-564.

Porta H, Rocha-Sosa M (2002) Plant lipoxygenases. Physiological and Molecular Features Plant Physiology 130:15-21.

Rahman M, Punja ZK (2005) Biochemistry of ginseng root tissues affected by rusty root symptoms. Plant Physiology and Biochemistry 43: 1103-1114.

Ramamoorthy V, Raguchander T, Samiyappan R (2002) Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici. Plant and Soil 239: 55-68.

Ramjegathesh R, Samiyappan R, Raguchander T, Prabakar K, Saravanakumar D (2013) Plant–PGPR Interactions for Pest and Disease Resistance in Sustainable Agriculture. In: Dinesh Kumar Maheshwari(ed.), Bacteria in Agrobiology: Disease Management. Springer Nature, Switzerland. pp. 293-320

Reuveni R, Shimoni M, Crute I (1991) An association between high peroxidase activity in lettuce (Lactuca sativa) and field resistance to downy mildew (Bremia lactucae). Journal of Phytopathology 132: 312-318

Sabbagh S, Roudini M, Panjehkeh N (2017) Systemic resistance induced by Trichoderma harzianum and Glomus mossea on cucumber damping-off disease caused by Phytophthora melonis. Archives of Phytopathology and Plant Protection:1-14.

Sabbagh SK, Valizadeh S (2016). Effect of bio-fertilizers on greenhouse cucumber resistant to damping-off disease caused by Pythium aphanidermatum and increase of yield component. Biological Control of Pests and Plant Diseases 5:111-122.

Shah J (2003) The salicylic acid loop in plant defense Current Opinion in Plant Biology 6: 365-371

Shirzad A, Fallahzadeh-Mamaghani V, Pazhouhandeh M (2012) Antagonistic potential of fluorescent pseudomonads and control of crown and root rot of cucumber caused by Phythophtora drechsleri. The Plant Pathology Journal 28:1-9.

Siedow J (1991) Plant lipoxygenase: structure and function. Annual Review of Plant Physiology and Plant Molecular Biology. 42:145-188.

Silva M, Guerra-Guimarães L, Loureiro A, Nicole M (2008) Involvement of peroxidases in the coffee resistance to orange rust (Hemileia vastatrix). Physiological and Molecular Plant Pathology 72:29-38.

Simons TJ, Ross A (1970) Enhanced peroxidase activity associated with induction of resistance to Tobacco mosaic virus in hypersensitive. Tobacco Phytopathology 60: 383-384.

Šukalović V, VeljovićJovanović S, Maksimović JD, Maksimović Pajić Z (2010) Characterisation of phenol oxidase and peroxidase from maize silk. Plant Biology 12:406-413.

Van Loon L, Bakker P, Pieterse C (1998) Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36:453-483.

Wu Z, Wang XR, Blomquist G (2002) Evaluation of PCR primers and PCR conditions for specific detection of common airborne fungi Journal of Environmental Monitoring 4:377-382.

Zhang W, Dick W, Hoitink H (1996) Compost-induced systemic acquired resistance in cucumber to Pythium root rot and anthracnose. Phytopathology 86:1066-1070.