International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 2(6), 91-96, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 91 Review PaperScreening of Antifungal Proteins from Plants of Cucurbitaceae family against Fusarium oxysporum: Potential as BiofungicidesYadav S., Tomar A.K., Yadav R.N. and Yadav S.* Department of Biophysics, All India Institute of Medical Sciences, New Delhi, INDIA Department of Chemistry, RRC, Alwar (Rajasthan University), INDIA Available online at: www.isca.in Received 6th May 2013, revised 20th May 2013, accepted 15th June 2013 AbstractNatural enemies like microbes and insects cause severe damage to the crops globally, resulting in a large economic loss. Plants of Cucurbitaceae family are known as the richest sources of carbohydrates, proteins, water and oil. Fusarium oxysporum causes the ‘fusarium wilt’ in the plants of this family and regarded as the major threat in growing cucurbits and thus, ensuing reduction in the total productivity of the crop. To overcome this problem, chemical fungicides are commonly spread over the crops which control the spreading of fungal diseases and subsequently increase the overall productivity. However, these synthetic chemicals are associated with various diseases and genetic disorders and are considered very lethal and risky for human beings, plants, animals and their environments. There are several proteins reported in the various plants with potential anti-fungal properties. We believe that these proteins can serve as useful resources for the formulation of future bio-fungicides which are natural, non-toxic and non-hazardous for us and our environment and surely merit over chemical fungicides. In this review, we have discussed about anti-fungal proteins reported in plants of Cucurbitaceae family which in near future, can be tested to get rid of ‘fusarium wilt’. Keywords: Bio-fungicides, Fusarium oxysporum, pathogen related proteins, ribosomal inactivating proteins, vicilin like proteins. Introduction One of the major problems in growing crops successfully is coming from natural enemies like fungi, bacteria, protists and insects. Plants are the wealthy source of nutrients for these types of organisms. There are approximately 250,000- 500,000 known plant species and only few of them show anti-microbial and anti-fungal activities1,2. It is well known that plants do not have their own immune system and when they are exposed to various pathogens, they defend themselves by varying defence mechanisms. In plants, the first line of defence includes the physical barriers such as cuticles and cell wall. Chemical barriers make the second line of defence which include the constitutive as well as induced compounds in response to the foreign particles. The well-known chemical barriers are phytoanticipins, saponins, phenols, quinines, defensins, peptides and proteins. The Phytoanticipins are low molecular weight compounds, produced after the microbial infections. The term ‘phytoantacipin’ was coined by Van Etten et al.. They may present on the plant surface or in vacuole and are released from the vacuole after infection with the help of the hydrolytic enzymes. Saponins are secondary metabolites and are produced in many plant species. They are reported to play important roles in plant defence. They show strong anti-fungal activity against various fungal species. Phenols and quinines make another class of anti-microbial compounds produced by few plant species that affect the vectors of plant pathogen either by excreting into the external environment or by accumulating into the dead cells4,5. Defensins, another group of defensive compounds, hamper with the pathogen food and impede their growth and development. There are a wide range of novel medicinally important proteins known which play important roles towards plant defense by inhibiting the fungal growth. Various plant parts including seeds, stems, leaves and roots of many plants contain antifungal proteins that potentially inhibit the growth of pathogenic fungi. Infact, plants are the richest source of chitinases, glucanases, ribosome inactivating proteins and pathogen related proteins that particularly needed for protecting seeds and seedlings from fungal infection6-8. It is well known that fungi are the organisms which are unable to produce their own food; thus, they are dependent on other organisms, specifically on plants and animals for fulfilling their nutritional needs. They can distress any part of the plant, kill plant in a short period of time and even sometimes destroy the whole crop. In Cucurbitaceae family, fungus Fusarium oxysporum causes the fusarium wilt which destroys the crops of family including watermelon, muskmelon, cucumber and many more. Cucurbitaceae is one of the largest families of plant kingdom and considered as the richest source of carbohydrates, proteins, water and oil. Fusarium wilt causes severe damages to the crop, resulting in huge losses in terms of yield, quality and revenues. This reduction in total productivity of family due to fungal invasion accounts for high economic loss worldwide and is a subject of major concern among International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(6), 91-96, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 92 agriculture and food biotechnologists. To overcome this problem, fungicide or the synthetic chemicals are more commonly used which further highlight a major scientific concern as they are generally considered very toxic and hazardous for human beings, plants and animals along with their surroundings.Continuous increase in global population along with urbanization, price rise, changing food habits and climatic disturbances has transformed food consumption, production and markets, causing global food crisis. This, in turn, is disturbing our society severely by pushing millions into poverty and malnutrition. Continuous increase in global population has increased the demand of food and nutritional resources which has simultaneously built a pressure on agricultural production resulting in the excessive use of chemical pesticides and fungicides in last few decades. It has caused a serious threat to environment and has been considered as one of the root causes of several diseases, due to the presence of these chemicals in food10To overcome this co-problem, the need of hour is to find safer methods and ways to replace chemicals with naturally available means and resources which may not harm us and our environment. It is now well known that many plants produce proteins which have shown anti-microbial activity and are part of their defense system. These proteins are highly sensitive and effective against various harmful pathogenic fungi and have great potential to be used as eco-friendly alternatives against toxic synthetic chemicals. These natural compounds are known to have minimal toxic effects towards living organisms and their environment 11In this review article, we have discussed various proteins and peptides which are present in different parts of Cucucrbitaceaefamily plants and show anti-fungal activity against Fusarium oxysporum, with a concluding remark that anti-fungal proteins present in plants can be used as a replacement of harmful chemical anti-fungal agents and will surely merit over them.Anti-fungal proteins in plants of CucurbitaceaeFamily Cucurbitaceae is one of the largest and diverse plant families consisting of about 125 genera and 960 species, with worldwide distribution. The plants of this family are found in tropical and subtropical areas. The plants of family are well known for their traditional medicinal uses12-15. The major family members are melons, squashes and gourds. Different parts of the water melon are used as remedies in various types of ailments. Water melon contains almost 95% water and its high amount of water makes it a powerful diuretic diet. It is also traditionally used to treat cardiovascular disease and kidney problems. rind, root, Seeds and leaves extracts of water melon show painkilling and anti-inflammatory effects16-22. Valuable scientific studies have shown pharmacological importance of Muskmelon. In China, it is used as traditional medicine for the treatment of abdominal distension and constipation23. Bottle gourd and sponge gourd are the popular plants of this family. Since ancient times, bottle gourd is used as effective medicine in India, China, European countries and Brazil. It is a well-known diuretic, having anti-diabetic, anti-hyperlipidemic, analgesic, Hypertensive, cardio protective, anti-cancer, CNS depressant and anti-inflammatory activities24-27. Sponge gourd also possesses medicinal as well as nutritional values. It is used to treat asthma, sinusitis and fever28. Its seeds contain an abortifacient protein which has shown ribosomal inhibiting property as well as therapeutic potential against AIDS29-31In general, members of this family have always been considered a subject of research due to the fact that they are rich source of proteins. For long, proteins with many biological activities like anti-fungal, anti-bacterial, anti-viral, anti-diabetic, anti-tumor and anti-AIDS has been known from this family32-36. In last few years, several proteins have been purified from different plant parts which are traditionally linked with the plants defence system. On the basis of their nature and biological activity, we have classified anti-fungal proteins known from Cucurbitaceaefamily as pathogen related (PR) proteins, ribosomal inactivating proteins (RIPs), vicilin like proteins and others.Pathogen related proteins (PR) Certainplants, in presence of various stress conditions such as infections (by insects or fungi), exposure to certain chemicals, wounding and atmospheric conditions (e.g. UV irradiation), defend themselves by expressing constitutive and induced proteins collectively called PR proteins37,38. To inhibit the spreading of the infection, plants show a hypersensitive response induced by PR proteins and characterized by localized and rapid cell death (necrosis) surrounding the site of pathogen invasion39,40. This pathogen inhibiting property makes PR proteins and associated phenolic compounds and other factors a great tool of commercial and economical importance in agriculture and medicine.In plants, there are several types of inducible defence related proteins, classified into 17 families of PR proteins. PR proteins are widely distributed in plant kingdom and many of them have been isolated from different parts of the plants such as leaves, stem, root and seeds41-43. These low-molecular proteins (6-43kDa) are stable at very high temperature, remain soluble at low pH (3) and are highly resistant to proteases44. PR proteins exhibit strong anti-fungal and anti-microbial activities which make them essential for the proper development and function of plant as a whole or in parts. In case of certain fungi, PR proteins diffuse towards the attacking pathogen and break their chitin supported cell wall. PR proteins are further classified into different families (PR-1 to PR-5) depending on their amino acid sequences and enzymatic or biological activity.PR-1 are the most abundant proteins which show anti-fungal properties at micromolar levels against various pathogenic fungi. The acidic or basic nature, resistance to proteases and International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(6), 91-96, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 93 extracellular secretion are the characteristic features of PR-1 proteins45. PR-2 proteins weaken the fungal cell wall at the hyphal apex of filamentous molds by hydrolyzing structural (1,3) -glucan present in it. PR -3 proteins weaken the fungal cell wall by destroying the chitin polymer present in cell wall that makes fungal cells osmotically sensitive and thus, inhibit the fungal growth. PR-4 proteins inhibit the fungal growth by binding with -chitin present in attacking fungal cell wall. PR-5 proteins inhibit the hyphal growth and facilitate hyphal and spore lysis resulting into plasma membrane permeabilization and dissipation of the plasma membrane potential46, 47. These proteins are also known as Thaumatin like proteins due to homologous amino acid sequences. Several PR proteins have been purified from plants ofCucurbitaceae family. A thermo stable PR-1 protein of 40 kDa was purified from pumpkin rind. It is relatively a high molecular weight anti-fungal protein in comparison to others. This protein is known to inhibit the growth of several pathogenic fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani and Candida albicans48,49. It has membrane permeability activity by which it disrupts the integrity of the cell plasma membrane and results in the leakage of the cytoplasmic component and thus, inhibits the fungal growth. A heat stable 14.8 kDa novel anti-fungal protein, PR-2, was identified from the Pumpkin rinds. This protein strongly inhibits the growth of Botrytis cinerea, Colletotrichum coccodes, Fusarium solani, Fusarium oxysporum,and Trichoderma harzianum at 10-20 50. This protein mainly inhibits the fungal growth by damaging the fungal cell membrane. Another potential antifungal PR protein was purified from the leaves of pumpkin and was designated as PR-5 protein. It is a 28 kDa protein which shows synergistic effects with the Nikkomycine, a chitin synthase inhibitor against Candida albicum51Ribosomal inactivating proteins (RIP)Plant RIPs arrest the synthesis of foreign proteins by inactivating fungal ribosome by N-glycosidase activity52. RIPs are further classified into three groups (Type1, Type2 and Type3) on the basis of their structure. Type -1 RIPs are single chain -glycosidases having molecular masses of 11- 30 kDa. Type-2 RIPs have two chains, an N-glycosidase (chain A) and cell binding lectin (chain B). Type-3 RIPs consist of four chains arranged in the form of dimers. RIPs have been isolated from number of plants e.g. seeds of the Caryophyllaceae saponaria ocymoides (30.2kDa) and Vaccaria pyramidata (28.0kDa)53. Hispin, a 21 kDa RIP was purified from the hairy melon seeds54. It shows potent anti-fungal activity against various pathogenic fungal species, such as Coprinus comatus, Fusarium oxysporum, Physalospora piricola, and Mycosphaerella arachidicola. -momorcharin protein is also an anti-fungal RIP, extracted and purified from Momordica charantia. It does not only inhibit the growth of fungi but also known to possess many important biological activities. It displays anti-fungal activity against Fusarium solani and Fusarium oxysporum55 Luffacylin, a 7.8kDa RIP was purified from sponge gourd seedswhich inhibits the growth of Mycosphaerella arachidicola and Fusarium oxysporum56 Luffangulin and Luffaculin are two another anti-fungal RIPs from Luffa acutangula seeds having molecular masses of 5.6kDa and 28 kDa respectively57Vicilin like proteins: There are many plant proteins known which perform dual functions like storage and defence. Vicilin like proteins form such a family, for example, a noval 8kDa peptide purified from Cucumis melo has shown anti-fungal activity against Fusarium oxysporum and has high sequence homology with vicilins58. Anti-fungal proteins are the unique components in plants which play an important role in plant defence against pathogenic fungi and protect them during different stages of growth. Recently, a low molecular weight (11 kDa) vicilin like protein was purified from the Citrullus lanatusseeds, however functional characterization of this protein is not yet established59Others: Cucurmoschin (8kDa), a novel anti-fungal peptide abundant in arginine, glutamate and glycine residues was purified from black pumpkin seeds. It is reported to inhibit mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum60. List of these above mentioned anti-fungal proeins from cucurbitaceae family against Fusarium oxysporum is given in able-1. ConclusionAs we all know, fungi are disease causing agents for plants which are major threat in growing them successfully. The fungal strain Fusarium oxysporum causes a number of disastrous diseases in plants of different families. Fusarium wilt is one of the oldest diseases of water melons, muskmelons and many other cucurbits that affect the production of fruits and seeds, causing huge economic loss worldwide. To overcome these problems, chemical pesticides are in great practice. No doubt, they are important to kill the disease causing pathogens, but definitely not sustainable for human health and friendly environment as long exposure to them is associated with various diseases, specifically carcinomas. Among number of anti-fungal plant proteins known and researched, proteins from plants of Cucurbitaceae family are the most promising because of their specific pathogen related activities. This is one of the largest plant families whose fruits and seeds are largely consumed by human beings in various forms because of their nutritional and medicinal values as well as good taste and availability. Many species of this family produce very effective anti-fungal proteins which inhibit the growth of Fusarium oxysporum. During their course of evolution, these proteins are developed by the plants to defend themselves against various pathogens. The potentials make them one of the very useful resources for the formulation of future bio-fungicides which are natural and non-hazardous for us and our environment. International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(6), 91-96, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 94 Table-1List of anti-fungal proteins isolated from plants of Cucurbitaceae family Proteins Source Anti-fungal activity against Molecular Weight (kDa) PR-1 Pumpkin rinds Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Candida albicans 40 PR-2 Pumpkin rinds Botrytis cinerea, Colletotrichum coccodes, Fusarium solani, Fusarium oxysporum,and Trichoderma harzianum 14.8 PR-5 Pumpkin leaves Candida albicum 28 Cucurmoschin Black pumpkin seeds Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum 8 Hispin Hairy melon seeds Coprinus comatus, Fusarium oxysporum, Physalo sporapiricola, and Mycosphaerella arachidicola 21 -Momorcharin protein Bitter gourd seeds Fusarium solani and Fusarium oxysporum 31.5 Luffacylin Sponge gourd seed Mycosphaerella arachidicola and Fusarium oxysporum 7.8 Vicillin like protein Muskmelon Fusarium oxysporum 8 AcknowledgmentThis work is supported by grants from Ministry of Environment & Forest (MoEF), Government of India, New Delhi. 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