Antifungal Potential of Indigenous Medicinal Plants against Myrothecium Leaf Spot of Bitter Gourd ( Momordica charantia L.)

Muhammad Abid Sobia Chohan Mirza Abid Mehmood Safina Naz Syed Atif Hasan Naqvi About the authors

ABSTRACT

Bitter gourd is of great importance due to its usage against the treatment of numerous ailments in human beings. A comprehensive survey at four localities of Southern Punjab, Pakistan was carried out to determine the severity of Myrothecium leaf spot. Maximum disease severity was at C1 (Chak 11/NP) and least at C2 (Kot Mehtab). Among isolated species Myrothecium roridum was found more prevalent and pathogenic as compared to M. verrucaria. Antifungal activity using solvent extracts of five medicinal plants (Mangifera indica, Melia azedarach, Nicotiana tabacum, Moringa oleifera and Eucalyptus globosum) were evaluated against isolated species by agar well diffusion method at various concentrations (0.01, 0.10, 1.0 and 10.0 µg / mL). N. tabacum revealed maximum zone size (13.40 mm and 8.28 mm) with ethanol and chloroform solvents respectively followed by M. azedarach (9.00mm and 6.48mm). However, least inhibition was observed with ethanol and chloroform extracts of E. globosum (6.04mm and 3.88mm zone size respectively). Ethanol extracts showed highest activity when compared to chloroform extracts. Qualitative phytochemical analysis showed that all the selected plants are rich in chemical compounds such as alkaloids, terpenoids, flavonoids and phenols whereas Saponins was only present in N. tabacum while absent in rest of the extracts.

Key words:
Momordica charantia L.; secondary compounds; solvent plant extracts

INTRODUCTION

Bitter gourd (Momordica charantia L.), also known as “bitter melon” is a tropical vine of the family Cucurbitaceae, widely cultivated in natives of tropical regions of Asia with worldwide distribution in China, Japan and Africa11 Grubben GJH. Tropical vegetable and their genetic resources. IBPGR, Rome. 1977; p. 51-52.. Bitter gourd is economically important plant having medicinal value and use against cancer, diabetes and many infectious diseases and also considered as a powerful weapon against immunodeficiency virus (HIV) 22 Sikder B. Improvement of bitter gourd (Momordica charantia L.) through breeding and biotechnology. Ph.D thesis. Department of Genetics and Breeding, University of Rajshahi, Bangladesh. 2004,33 Shetty AK, Kumar GS, Sambaiah K, Salimath PV. Effect of bitter gourd (Momordica charantia L.) on glycaemic status in streptozotocin induced diabetic rats. Pl Foods Hum Nutr. 2005; 60: 109-112.,44 Paul A, Raychaudhuri SS. Medicinal uses and molecular identification of two Momordica charantia varieties-a review. Electronic J Bio. 2010; 6(2):43-51.. The crop is having problems such as low seed germination, small sized and deformed fruit, low yield, Asynchronous flowering and attack of pests and diseases 22 Sikder B. Improvement of bitter gourd (Momordica charantia L.) through breeding and biotechnology. Ph.D thesis. Department of Genetics and Breeding, University of Rajshahi, Bangladesh. 2004. A number of diseases attack bitter gourd but Myrothecium leaf spot caused by M. roridum Tode ex Fr. and M. verrucaria (Alb. Schew) Ditm ex Fr., is a serious pathosystem in cultivated areas. The genus Myrothecium includes eight reported species55 Tulloch M. The genus Myrothecium. Mycological Papers. (IMI) 130. 1972; p.44., out of which many are saprophyte soil inhabitants 66 Costa I, Wanderley PM, Cavalcante MA, Fernandes MJ, Lima DMM. Hyphomycetes from soil of an area affected by copper mining activities in the State of Bahia, Brazil. Braz J Microbiol. 2006; 37 (3): 290-295.. M. roridum considered as an important plant pathogen, affecting more than 200 plant species of different botanical families 77 Ahrazem O, Gómez-Miranda AP, Bernabé M, Leal JA. Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides. Arch Microbiol. 2000; 173: 296-302.,88 Murakami R, Kobayashi T, Takahashi K. Myrothecium leaf spot of mulberry caused by Myrothecium verrucaria. J Gen Pl Pathol. 2005; 71: 153-155.and has been reported as a key pathogen of cucurbits causing leaf spot / blight 99 Ali S, Wahid A, Murtaza M, Nadeem A. Myrothecium leaf of bitter gourd in Pakistan. Pak J Agric Res. 1988; 9: 598-600.,1010 Shakoor S, Chohan S, Riaz A, Perveen R, Naz S, Mehmood MA, Haider MS, Ahmad S. Screening of systemic fungicides and biochemicals against seed borne mycoflora associated with Momordica charantia. Afr J Biotech. 2011; 10(36):6933-40.. The isolation of M. roridum has been reported from seeds of bottle-gourd 1111 Shakir AS, Mirza JH. Seed-bornefungi of Bottle gourd from Faisalabad and their control. Pak J Phytopathol. 1992; 4: 54-57., Indian gourd, red gourd, sponge gourd 1212 Shakir AS, Mirza JH, Sahi ST, Ahmad F. Detection of seed-borne fungi associated with sponge gourd {Luffa cylindrical (L.) Roem.}, their location in different seed components and their control. Pak J Phytopathol . 1995; 7: 140-144., pumpkin 1313 Cabral CS, Henz GP, Moreira AJ, Reis A. New cucurbitaceous hosts of Myrothecium roridum in Amazonas State, Brazil. Trop Pl Pathol. 2009;34(6):402-5. and melon 1415 Bharath BG, Lokesh S, Raghavendra VB, Prakash HS, Shetty BG. First Report of the occurrence of Myrothecium verrucaria in watermelon seeds from India. Aust Pl Dis Notes.2006; 1: 3-4.. As compared to other species M. verrucaria has showed a high incidence on seeds of watermelon 1515 Bharath BG, Lokesh S, Raghavendra VB, Prakash HS, Shetty BG. First Report of the occurrence of Myrothecium verrucaria in watermelon seeds from India. Aust Pl Dis Notes.2006; 1: 3-4. and also responsible for leaf spot disease in bitter gourd 1616 Sultana N, Ghaffar A. Pathogenesis and control of myrothecium spp., the cause of leaf spot on bitter gourd (Momordica charantia Linn.). Pak J Bot. 2009; 41: 429-433.. Both M. roridum and M. verrucaria are soil inhabiting as well as seed transmitted pathogens of wide host range. Murakami et al. (1999) 1717 Murakami R, Yasui H, Shirata A, Kato A. Production of myrotoxin-B by Myrothecium roridum isolated from Myrothecium leaf spot of mulberry in Japan. J Sericult Sci Japan. 1999; 68: 469-477. reported that M. roridum develops large necrotic lesions which verified the ability of M. roridum to produce toxins 1818 Kuti JO, Ng TJ, Bean GA. Possible involvement of a pathogen-produced trichothecene metabolite in Myrothecium leaf spot of muskmelon. Physio mol pl pathol.1989; 34(1):41-54..

Chemical fungicides are being used by farmers world over for the management of fungal disease resulted in increasing production cost and pose threat to human health and environment 1919 Bowers JH, Locke JC. Effect of botanical extracts on the population density of Fusarium oxysporum in soil and control of Fusarium wilt in the greenhouse. Pl Dis. 2000; 84:300-305.. However, botanicals or biopesticides are considered to be one of the best substitutes of expensive and hazardous fungicides 2020 Joseph B, Muzafar A, Vinod K. Bioefficacy of Plant Extracts to Control Fusarium solani f. sp. melongenae Incitant of Brinjal Wilt. Global J Biotech & Biochem. 2008; 3 (2):56-59.. As an alternative to chemical fungicides, botanicals have been reported against fungal diseases of plants. Since plant products being rich in active chemical components can be obtained from any plant part viz., seeds, roots, leaves, flowers, bark etc. 2121 Cragg GM, David JN. Natural product drug discovery in the next millennium. J Pharm Biol 2001; 39: 8-17.,2222 Ivanova D, Gerova D, Chervenkov T, Yankova T. Polyphenols and antioxidant capacity of Bulgarian medicinal plant. J Ethnopharmacol. 2005; 96:145-150.. Phytochemical screening to obtain the knowledge of chemical constituents present in plants and their antimicrobial activities have been reported by earlier scientists 2323 Aqil F, Ahmad I, Mehmood Z. Antioxidant and free radical scavenging properties of twelve traditionally used Indian medicinal plants. Turkish journal of Biology. 2006; 30(3):177-83.,2424 Parekh J, Chanda S. Phytochemicals screening of some plants from western region of India. Pl Arch. 2008; 8: 657- 662.,2525 Chanda S, Dave R. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: An overview. Afr J Microbiol Res. 2009; 3(13): 981-996.. These secondary metabolites defend host plants against attack of microorganisms, insects and herbivores 2626 Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12: 564-582.. In Pakistan, to our best of knowledge no work is reported towards the ecofriendly management of leaf spot disease of bitter gourd. The aims of present investigations were to evaluate the inhibitory effects of organic solvent extracts of indigenous medicinal plants against leaf spot disease of bitter gourd for disparity in their phytochemical properties.

MATERIALS AND METHODS

Sample collection

A comprehensive survey of rural areas of District Rahim Yar Khan, Pakistan (27°40'-29°16' north latitudes and 60°45'-70°01' east longitudes) from four different localities (C1= Chak 11/NP, C2= Kot Mehtab, C3= Muhammad Pur and C4= Chak 165/P) was conducted during 2014-15. From each locality 20 plants were randomly selected and tagged, naturally infected bitter gourd leaves showing typical symptoms of leaf spot disease were preserved in clean brown paper bags and transported to the Mycology laboratory of Plant Pathology Department, Multan.

Disease Severity

Forty infected leaves from already tagged plants were randomly selected from each locality for the assessment of disease severity with the help of 0-4 disease rating scale suggested by Vir and Grewel (1974) 2727 Vir S, Grewel JS. Physiologic specialization in Ascochyta rabiei, the casual organism of gram blight. Ind Phytopath. 1974; 27: 524-526. where 0= no symptoms on leaves, 1= <5% infection on leaves, 2 = 5-25% infection on leaves, 3=25-75% infection on leaves, 4= >75% infection on leaves. Percent Disease Index was calculated by the modified formulae suggested by Sultana & Ghaffar (2009) 1616 Sultana N, Ghaffar A. Pathogenesis and control of myrothecium spp., the cause of leaf spot on bitter gourd (Momordica charantia Linn.). Pak J Bot. 2009; 41: 429-433.:

Isolation and morphological characterization of fungal strains

The infected bitter gourd leaf segments (1cm x1cm) were washed with tap water, surface sterilized with 70% ethanol for 1 min and rinsed in five changes of sterile distilled water. Isolation procedure was carried out on Potato Dextrose Agar medium (PDA- Difco) as described by Dhingra & Sinclair (1985)2828 Dhingra OD, Sinclair JB. Basic Plant Pathology Methods. CRC Press Inc, Boca Raton, Florida, 1985; Pp.132.. Five leaf segments were aseptically plated on solidified PDA and incubated at 25oC for one week to facilitate the mycellial growth. Purification of culture was done by single hyphal tip technique, stored at 4oC on PDA slants and identified according to the cultural and morphological characters. Morphological characterization of sporodochia, phialides and conidia of resultant fungi were also studied. Size of conidia were measured (length x width) and compared with characters illustrated by Barnett & Hunter (1987) 2929 Barnett HL, Hunter BB. Illustrated genera of imperfect fungi. Minnesota: APS press; 1998; p. 241.. Pathogenicity test was also carried out on healthy seedlings to confirm Koch’s postulates.

Plant materials

Fresh and healthy leaves of selected medicinal plants (Mangifera indica, Melia azedarach, Nicotiana tabacum, Moringa oleifera and Eucalyptus globosum) were collected from local area, identified and authenticated from Department of Botany, Bahauddin Zakariya University, Multan.

Extraction

The leaves samples were thoroughly washed with tap water, followed by distilled water and finally with ethanol (Merck) to eliminate any traces of contaminants. Blot dried leaves of each sample were then dried in the oven at 50oC for 24 hours, homogenized to fine powder and stored in airtight bottles. Ten gm of powdered material was used in Soxhlet apparatus (J.P.Selecta-Spain) with 100 mL solvents (ethanol and chloroform) for 48 hours. The extract solutions were centrifuged at 6000 rpm for 10 min, filtered with filter paper (Whatman No.1) and concentrated over a water bath at 40oC. After complete solvent evaporation extract residues were sealed in dark bottles at 4oC for further use by Modified method of Nycee et al. (2012)3030 Neycee MA, Nematzadeh GHA, Dehestani A, Alavi M. Assessment of antifungal effects of shoot extracts in chinaberry (Melia azedarach) against 5 phytopathogenic fungi. Intr J Agri Crop Sci. 2012; 4 (8): 474-477..

Agar well diffusion assay

Antifungal tests were carried out by Agar well diffusion method (Perez et al., 1990)3131 Perez C, Paul M, Bazerque P. An antibiotic assay by the agar well diffusion method. Acta Biologiae et Medicine Experimentalis. 1990; 15: 113-115.. An aliquot of 100µL spore suspension (106spores/mL) was streaked on the surface of petri plates having PDA medium. Wells of 8 mm in diameter were punched in the seeded agar plates with the help of sterile cork borer. About 100µL of plant extracts alone and in combination (1:1v/v) with certain concentrations (0.01, 0.10, 1.0 and 10.0 µg / mL) were added into each well and allowed to diffuse for 1hr at room temperature. Dimethyl Sulfoxide (DMSO) was used as a control and treated plates were incubated at 25oC for 72 hours. Antifungal activity was evaluated by measuring the size of zone with a ruler (mm). All the tests were performed in a Completely Randomized Design (CRD) with five replicates.

Phytochemical Screening

Plant extracts were subjected to phytochemical screening using the methods of Sofowora, (1993)3232 Sofowora A. Medicinal plants and Traditional Medicine in Africa. Spectrum Books, Ibadan; 1993; p. 150 and Harborne, (1973)3333 Harborne JB. Phytochemical Methods; A guide to modern techniques of plant analysis. Second Edition, London, Chapman and Hall, 1973; p. 221- 232..

Alkaloids (Wagner’s test): 20mg of ethanolic extract was warmed with 2% sulphuric acid (H2SO4) for 1-2 min, filtered and treated with few drops of wagner’s reagent. Presence of reddish brown precipitation or turbidity indicated the presence of alkaloids.

Tannins (Ferric chloride test): About 20mg plant extract was dissolved in ethanol, few drops of 0.1% ferric chloride added and observed for formation of blue black coloration.

Steroids: 2mL of acetic anhydride and conc. H2SO4 added to 50mg ethanolic plant extract. Blue green ring indicates the presence of steroids.

Terpenoids (Salkowski Test): About 5mL extract of each sample was mixed in (2mL) chloroform and (3mL) conc. H2SO4was added carefully to form a layer. Formation of reddish brown coloration at interface indicates the presence of terpenoids.

Saponins: 20mg of powdered sample was boiled in 5ml distilled water and shaken vigorously for a stable persistent froth. Three drops of olive oil was mixed vigorously with frothing and observed for formation of emulsion.

Flavonoids: Powdered sample (20mg) was heated with 10ml of ethyl acetate for 3min and filtered. 4mL of filterate was mixed with 1mL of dilute ammonia solution. A yellow coloration that disappears on addition of conc. Hydrochloric acid indicated the presence of flavonoids.

STATISTICAL ANALYSIS

The datasets of disease severity, isolation frequency and size of inhibition zone were subjected to analysis of variance (ANOVA) and treatment means were evaluated with Fisher’s Least Significance Difference (LSD) test at 5% level of significance (Steel et al., 1997)3434 Steel RGD, Torrie JH, Dicky DA. Principles and Procedures of Statistics: A Biometrical Approach, 3rd edition, 1997; pp: 352-358 McGraw Hill Book Co. Inc., New York. using Analytical Software (2005).

RESULTS

Four different locations of District Rahim Yar Khan were evaluated for the disease severity of Myrothecium leaf spot and disease symptoms were observed as: yellowing, chlorosis and appearance of brown concentric rings on necrotic areas of leaves, which later coalesce to give the blighted appearance. In advance stages necrotic and chlorotic areas increased in size resulting into curling and death of leaves. Isolates of each species were identified on the basis of morphological characteristics of conidia, conidiophores, sporodochia and phyialides.

Leaflets with characteristics leaf spot symptoms yielded two isolates; M. roridum and M. verrucaria with varying percentages of PDI. Laboratory experiments confirmed that isolates of M. roridum and M. verrucaria were pathogenic. Maximum disease severity 63.80% was observed at locality C1 (Chak 11/ NP) followed by 37.50, 23.80 and 20.60 % at C3, C4 (Muhammad Pur, Chak 165 P) and C2 (Kot Mehtab) respectively (Fig 1).

Fig 1:
Percent disease severity of Myrothecium leaf spots on different locations

Considering the isolation frequency of leaf samples collected from four different localities yielded two isolates of Myrothecium species. Leaflets from C1 and C3 have both species of Myrothecium (Myr1, Myr2, Myv1, and Myv2) whereas locality C2 samples possess only Myr1 and Myr2. Overall maximum frequency percentage of Myrv1 was isolated from samples of locality C2 followed by C3, C4 and C1 with 80.00, 65.00, 60.00 and 55.00 % respectively while least was of Myv2 only from two localities C2 and C1 with 10 and 5% respectively (Table 1).

Table 1:
Frequency isolation (%) of leaf spot disease of bitter gourd form infected plants

The organic solvent extracts had antifungal effects on the growth of M. roridum. Ethanol extraction was superior as compared to chloroform. Ethanol extract of Nicotiana tabacum; in particular, showed maximum inhibition zone size (22.20 mm) of M. roridum using 10 µL concentration followed by 13.80, 13.60, 12.80 and 12.20 mm zones of inhibition formed by Melia azedarach, Moringa oleifera, Mangifera indica and Eucalyptus globosum respectively, compared to control (Dimethyl sulphoxide) with zero inhibition zones. It was observed that the zone size increased with an increase in concentration of the extracts. Overall N. tabacum showed maximum inhibitory effects towards mycelia growth of M. roridum at all concentrations with 13.40 mm inhibition zone followed by 9.00, 7.64, 6.32 and 6.04 mm zone of inhibition in M. azedarach, M. oleifera, M. indica and E. globosum respectively (table 2).

Table 2:
Inhibitory effect of various ethanolic plant extracts at different concentrations on mycelial growth of Myrothecium roridum using agar well diffusion method.

Correspondingly chloroform extracts of N. tabacum also showed better inhibitory effects on mycelia growth of M. roridum with maximum (17.2 mm) inhibition zone at 10 µL concentration followed by 12.4, 12.2, 11.2 and 10.2 mm inhibition zone size produced by M. azedarach, M. oleifera, E. globosum, and M. indica respectively compared to untreated control. In general N. tabacum performed best forming 8.28 mm inhibition zone followed by M. azedarach (6.48 mm), M. oleifera (5.88 mm), M. indica (5.16 mm) and E. globosum (3.88 mm) inhibition zones (table 3).

Table 3:
Inhibitory effect of various plants extracts at different concentrations extracted with chloroform on mycelial growth of Myrothecium roridum using agar well diffusion method.

Preliminary qualitative phytochemical analysis showed that plant extracts have fair amount of secondary metabolites. Considering the presence or absence of these compounds in medicinal plants utilized, it was observed that alkaloids, terpenoids and flavonoids were present in all plants extracts except E. globosum while tannins were found in N. tabacum, M. indica and E. globosum. Steroids were present in M. azedarach and M. oleifera, whereas, saponins were only present in N. tabacum but absent in rest of the plant extracts (table 4).

Table 4:
Analysis of phytochemicals extracted from medicinal plant extracts.

DISCUSSION

Myrothecium leaf spot of bitter gourd is considered to be the most serious pathosystem of cucurbis in the cultivated areas. During the current research, Myrothecium infestation on various locations of the District Rahim Yar Khan was assessed. We observed Myrothecium species to produce brown to dark brown leaf spots which later on coalesce to give blighted symptoms in the field on the host plants and it seems 3535 Belisario A, Forti E, Corazza, Van Kestsren HA, 1999. First report of Myrothecium verrucaria from muskmelon seeds. Plant Path. 1999; 83: 589. a serious threat to vegetable growers of the area. During the in vitro studies of the collected diseases samples of the different locations we found M. roridum and M. verrucaria both pathogenic and responsible for leaf spot in bitter gourd. We calculated less isolation frequency of M. verrucaria revealed that it is a weak pathogen of bitter gourd grown in this area. Our findings are in agreement with the results of earlier workers whom reported M. roridum and M. verrucaria both as plant pathogens while later is a weaker one 77 Ahrazem O, Gómez-Miranda AP, Bernabé M, Leal JA. Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides. Arch Microbiol. 2000; 173: 296-302.. Myrothecium species are soil- inhabiting fungi but due to facultative parasitic nature also able to cause disease in aerial parts of economically important host plants 66 Costa I, Wanderley PM, Cavalcante MA, Fernandes MJ, Lima DMM. Hyphomycetes from soil of an area affected by copper mining activities in the State of Bahia, Brazil. Braz J Microbiol. 2006; 37 (3): 290-295.,3636 Domsch KH, Gams W, Anderson T. Compendium of soil fungi. 2nd Edition. IHW Verlag: Eching, Germany. 2007; p. 672.,3737 Worapong J, Sun J, Newcombe G. First report of Myrothecium roridum from a gymnosperm. North American Fungi. 2009; 4:1-6.,3838 Zhao YJ, Li BJ, Shi YX, Xie XW. First Report of Myrothecium Leaf Spot of Common Bean in China Caused by Myrothecium roridum. Pl Dis . 2010; (1):127.. However, M. roridum also identified as a virulent seedborne pathogen 1515 Bharath BG, Lokesh S, Raghavendra VB, Prakash HS, Shetty BG. First Report of the occurrence of Myrothecium verrucaria in watermelon seeds from India. Aust Pl Dis Notes.2006; 1: 3-4.. Rain splashes, irrigation water and dew are dissemination agencies of both Myrothecium species. Prolonged hot and humid weather favors the foliar attack of Myrothecium3939 Zitter TA, Hopkins DL, Thomas CE. Compendium of cucurbit diseases. APS Press; 1996.. Two isolates; M. roridum and M. verrucaria were isolated and identified and compared with work of earlier scientists 3636 Domsch KH, Gams W, Anderson T. Compendium of soil fungi. 2nd Edition. IHW Verlag: Eching, Germany. 2007; p. 672.. According to some scientists large necrotic lesions are due to necrotrophic nature of fungi that produce toxins in host tissues resulting into death of host tissues and the fungal pathogen colonize over these necrotic areas 4040 Bean GA, Fernando T, Jarvis BB, Bruton B. The isolation and identification of trichothecene metabolites from a plant pathogenic strain of Myrothecium roridum. J nat prod. 1984; (4):727-9.,88 Murakami R, Kobayashi T, Takahashi K. Myrothecium leaf spot of mulberry caused by Myrothecium verrucaria. J Gen Pl Pathol. 2005; 71: 153-155.. Though virulence of M. roridum was demonstrated on large number of plants but different M. roridum isolates revealed variability in virulence but highly aggressive behavior was observed in tomato and cucumber 4141 Quezado Duval AM, Henz GP, Paz-Lima ML, Medeiros AR, Miranda BE, Pfenning LH, Reis A. New hosts of Myrothecium spp. in Brazil and a preliminary in vitro assay of fungicides. Braz J Microbiol . 2010; 41(1):246-52.. N. tabacum was found rich in all compounds except steroids and thus possessed excellent fungicidal activity (Taiga et al., 2008)4242 Taiga A, Suleiman MN, Sule W, Olufolaji DB. Comparative in vitro inhibitory effects of cold extracts of some fungicidal plants on Fusarium oxysporium mycelium. Afr J Biotech . 2008; 7(18). which proved that N. tabacum was more fungitoxic compared to other plant extract due to the subsistence of tannins in addition to Saponins and flavonoids. These results corroborated with the outcome of other scientists 4343 Liu IX, Durham DG, Richards ME. Vancomycin resistance reversal in enterococci by flavonoids. J Pharm Pharmacol, 2001; 53: 129-132.,4242 Taiga A, Suleiman MN, Sule W, Olufolaji DB. Comparative in vitro inhibitory effects of cold extracts of some fungicidal plants on Fusarium oxysporium mycelium. Afr J Biotech . 2008; 7(18).. Furthermore, phytochemical studies of various plant extracts showed that fungicidal activity is due to presence of tannins, alkaloids, flavonoids and saponins 3232 Sofowora A. Medicinal plants and Traditional Medicine in Africa. Spectrum Books, Ibadan; 1993; p. 150,4444 Singh R, Singh S, Kumar S, Arora S. Evaluation of antioxidant potential of ethyl acetate extract/fractions of Acacia auriculiformis. A Cunn Food Chem Toxicol. 2007; 45:1216-1223.. Out of five plants extracts used, all of them showed antimicrobial activity but N. tabacum and M. azedarach were found more promising 4545 Maji MD, Chattopadhyay S, Kumar P, Saratchandra B. In vitro screening of some plant extracts against fungal pathogens of mulberry (Morus spp.). Arch Phytopath Pl Prot. 2005; 38(3):157-64.,4646 Vaghasiya Y, Chanda SV. Screening of methanol and acetone extracts of fourteen Indian medicinal plants for antimicrobial activity. Turk J Biol. 2007; 31: 243-248.,4747 Jana S, Shekhawat GS. Phytochemical analysis and antibacterial screening of in vivo and in vitro extracts of Indian medicinal herb: Anethum graveolens. Res J Med Plant. 2010; 4: 206-212.. Plants with more phenolic contents show efficient antioxidant activity 4848 Brighente IMC, Dias M, Verdi LG, Pizzolatti MG. Antioxidant activity and total phenolic content of some Brazilian species. Pharm Biol. 2007; 45: 156-161.. Similarly, according to Ali et al., (2008)4949 Ali SS, Kasoju N, Luthra A, Singh A, Sharanabasava H, Sahu A, Bora U. Indian medicinal herbs as sources of antioxidants. Food Res Int. 2008; 41: 1-15. plant possesses natural antioxidants in the form of phenolic compounds such as phenolic acids, tocopherol, flavonoids etc. These results are substantiated with the outcome of this study. There is a need to explore more plants possessing antimicrobial potential to use in our organic farming system for cleaner environment.

CONCLUSION

Use of plant extracts to control fungal pathogen responsible for blight of bitter gourd is ecofriendly approach and an effective alternative to toxic chemical fungicides. Studies are in progress, in the greenhouse and in open fields, to evaluate antifungal effect of these natural substances on bitter gourd development and on qualitative and quantitative fruit production.

ACKNOWLEDGMENTS

We are very thankful to Department of Plant protection, Pest Warning and Quality Controls of Pesticides, Sadiqabad, Pakistan for assisting us in field surveys during the study.

CONFLICT OF INTEREST DISCLOSER

There is no conflict of interest among authors regarding the current manuscript.

REFERENCES

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    Grubben GJH. Tropical vegetable and their genetic resources. IBPGR, Rome. 1977; p. 51-52.
  • 2
    Sikder B. Improvement of bitter gourd (Momordica charantia L.) through breeding and biotechnology. Ph.D thesis. Department of Genetics and Breeding, University of Rajshahi, Bangladesh. 2004
  • 3
    Shetty AK, Kumar GS, Sambaiah K, Salimath PV. Effect of bitter gourd (Momordica charantia L.) on glycaemic status in streptozotocin induced diabetic rats. Pl Foods Hum Nutr. 2005; 60: 109-112.
  • 4
    Paul A, Raychaudhuri SS. Medicinal uses and molecular identification of two Momordica charantia varieties-a review. Electronic J Bio. 2010; 6(2):43-51.
  • 5
    Tulloch M. The genus Myrothecium. Mycological Papers. (IMI) 130. 1972; p.44.
  • 6
    Costa I, Wanderley PM, Cavalcante MA, Fernandes MJ, Lima DMM. Hyphomycetes from soil of an area affected by copper mining activities in the State of Bahia, Brazil. Braz J Microbiol. 2006; 37 (3): 290-295.
  • 7
    Ahrazem O, Gómez-Miranda AP, Bernabé M, Leal JA. Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides. Arch Microbiol. 2000; 173: 296-302.
  • 8
    Murakami R, Kobayashi T, Takahashi K. Myrothecium leaf spot of mulberry caused by Myrothecium verrucaria. J Gen Pl Pathol. 2005; 71: 153-155.
  • 9
    Ali S, Wahid A, Murtaza M, Nadeem A. Myrothecium leaf of bitter gourd in Pakistan. Pak J Agric Res. 1988; 9: 598-600.
  • 10
    Shakoor S, Chohan S, Riaz A, Perveen R, Naz S, Mehmood MA, Haider MS, Ahmad S. Screening of systemic fungicides and biochemicals against seed borne mycoflora associated with Momordica charantia. Afr J Biotech. 2011; 10(36):6933-40.
  • 11
    Shakir AS, Mirza JH. Seed-bornefungi of Bottle gourd from Faisalabad and their control. Pak J Phytopathol. 1992; 4: 54-57.
  • 12
    Shakir AS, Mirza JH, Sahi ST, Ahmad F. Detection of seed-borne fungi associated with sponge gourd {Luffa cylindrical (L.) Roem.}, their location in different seed components and their control. Pak J Phytopathol . 1995; 7: 140-144.
  • 13
    Cabral CS, Henz GP, Moreira AJ, Reis A. New cucurbitaceous hosts of Myrothecium roridum in Amazonas State, Brazil. Trop Pl Pathol. 2009;34(6):402-5.
  • 14
    Fish WW, Bruton BD, Popham TW. Cucurbit host range of Myrothecium roridum isolated from watermelon. Amr J Pl Sci. 2012; 3(03):353.
  • 15
    Bharath BG, Lokesh S, Raghavendra VB, Prakash HS, Shetty BG. First Report of the occurrence of Myrothecium verrucaria in watermelon seeds from India. Aust Pl Dis Notes.2006; 1: 3-4.
  • 16
    Sultana N, Ghaffar A. Pathogenesis and control of myrothecium spp., the cause of leaf spot on bitter gourd (Momordica charantia Linn.). Pak J Bot. 2009; 41: 429-433.
  • 17
    Murakami R, Yasui H, Shirata A, Kato A. Production of myrotoxin-B by Myrothecium roridum isolated from Myrothecium leaf spot of mulberry in Japan. J Sericult Sci Japan. 1999; 68: 469-477.
  • 18
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  • Erratum

    In Article “Antifungal Potential of Indigenous Medicinal Plants against Myrothecium Leaf Spot of Bitter Gourd (Momordica charantia L.)”, with the number of DOI: http://dx.doi.org/10.1590/1678-4324-2017160395, published in journal Brazilian Archives of Biology and Technology, vol. 60, the 01 page.
    That read:
    “http://dx.doi.org/10.190/1678-4324-2017160395”
    Read:
    “http://dx.doi.org/10.1590/1678-4324-2017160395”

Publication Dates

  • Publication in this collection
    2017

History

  • Received
    03 Feb 2016
  • Accepted
    14 July 2016
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