Brazilian Journal of Biological Sciences, 2019, Vol. 6, No. 12, p. 123-132. ISSN 2358-2731 https://doi.org/10.21472/bjbs.061211
Antifungal activity of selected plant extracts against Trichothecium roseum (Pers.) Link (1809) (Sordariomycetes: Hypocreales), causal organism of fungal rot of Solanum melongena L. (Solanales: Solanaceae) in Kashmir, India
Jahangir Abdullah Koka¹,*, Abdul Hamid Wani¹, Mohd Yaqub Bhat¹, Shazia Parveen¹, Mohammad Afaan Fazili² and Nusrat Ahmad¹
¹Section of Mycology and Plant Pathology. Department of Botany. University of Kashmir. Hazratba. Srinagar, 190006. Jammu & Kashmir. India. *Email: [email protected]. ²Cytogenetics and Plant Breeding Laboratory. Department of Botany. Aligarh Muslim University. Aligarh, 202002. Uttar Pradesh. India.
Abstract. Egg plant Solanum melongena L. (Solanales: Solanaceae) is an important vegetable grown in Kashmir. It is Received attacked by number of fungal pathogens in storage and in the July 11, 2018 field. These fungi caused several fungal rot diseases resulting heavy losses to the growers. Therefore, present study was Accepted February 13, 2019 carried out to study the incidence and management of fungal rot of egg plant using some selected plant extracts. It was revealed Released from the study that Trichothecium roseum (Pers.) Link (1809) April 30, 2019 (Sordariomycetes: Hypocreales) causing decaying of egg plant under storage. Study was also undertaken to evaluate the Full Text Article efficacy of some plant extract against Trichothecium roseum on inhibition of spore germination and mycelial growth under in vitro conditions. It was observed from the results that amongst the plant extracts, plant extract of Ajuga bracteosa at highest concentration was found most effective against Trichothecium roseum and cause highest inhibition in the mycelial growth and spore germination followed by plant extract of Taraxicum officinale, Mentha arvensis and Iris kashmiriana at the same concentrations. Other concentrations of plant extracts also
bought about significant reduction in mycelial growth and spore
germination of the test fungus but to a lesser extent as compared to control. 0000-0003-0040-1624 Keywords: Pink rot; Botanical fungicides; Inhibition; Mycelia Jahangir Abdullah growth; Spore germination; Brinjal. Koka 0000-0002-6157-9656 Abdul Hamid Wani 0000-0002-0582-4813 Mohd Yaqub Bhat
ISSN 2358-2731/BJBS-2018-0051/2019/6/12/11/123 Braz. J. Biol. Sci. http://revista.rebibio.net 124 Koka et al.
0000-0002-0086-2685 Shazia Parveen 0000-0003-4427-0806 Mohammad Afaan Fazili 0000-0003-3503-4641 Nusrat Ahmad
Introduction post harvest losses are often more severe (more than 30%) due to inadequate post Vegetables are important harvest handling, packaging, constituents of human diet. They are transportation and storage which may important sources of carbohydrates, result in decay and production by micro- minerals and vitamins. They are attacked organisms which become activated by many pathogens like fungi and because of the changing physiological bacteria that result in loss of fresh state of the fruit (Tripathi and Dubey, produce (Mitcham and Mitchell, 2002; 2004; Korsten, 2006; Singh and Sharma, Wani, 2011). Losses from the post- 2007). harvest diseases in fresh produce fall It has been reported that on an into two categories i.e., loss in quantity average, the oblong-fruited eggplant and quality. Loss in quantity occurs cultivars are rich in total soluble sugars, where deep penetration of decay occurs whereas the long-fruited cultivars in the infected vegetables. This is often as contain a higher content of free reducing a result of infection of the vegetables in sugars, anthocyanin, phenols, the field before harvest. Loss in quality glycoalkaloids (such as solasodine), dry occurs when the diseases affects only the matter, and amide proteins (Bajaj et al., surface of the vegetables. It may cause 1979). The glycoalkaloid contents in the skin blemishes that can lower the value Indian commercial cultivars vary from of the commercial crop. Rot diseases 0.37 mg/100 g fresh weight to 4.83 mg) cause greatest losses to the vegetables in (Bajaj et al., 1981). Brinjal is known to storage as well as in fields (Snowdon, have ayurvedic medicinal properties and 2003; Bashar et al., 2012). is good for diabetic patients. It has also Egg plant Solanum melongena L. been recommended as an excellent belongs to Family Solanaceae and is remedy for those suffering from liver indigenous vegetable crop of India complaints (Shukla and Naik, 1993). To including Kashmir. It contributes 9% of the best of our knowledge, this is the first the total vegetable production of the report of T. roseum causing pink rot on country. China is the largest producer of Brinjal in Kashmir region of Jammu and brinjal followed by India. It is grown in Kashmir, India. Various chemical India over an area of 0.4 million hectors fungicides have been used to control with an annual production of 7.8 million these fungal rot diseases, but these tonnes (Datar, 1999). Post harvest fungicides cause hazardous effect on decays of fruits and vegetables account humans and environment. Hence strong for significant levels of post harvest regulatory actions have been imposed on losses. It is estimated that about their use. So there is a strong need to 20%-25% of the harvested fruits and control these diseases in an ecofriendly vegetables are decayed by pathogens way. during post-harvest handling even in Various biocontrol fungi and developed countries (El-Ghaouth et al., extracts obtained from many medicinal 2004; Droby et al., 2009; Abano and San- plants have gained much popularity and Amaoh, 2012). In developing countries, scientific interest for their antifungal and
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Antifungal activity of selected plant extracts against Trichothecium roseum 125
antibacterial activities (Parveen et al., made to study the efficacy of some plant 2016; Koka et al., 2017). They are extracts against Trichothecium roseum believed to be less hazardous than causal organism of fungal rot of brinjal in chemical fungicides and can therefore be Kashmir. used as an alternative to control fungal The present study was carried rot diseases (Jobling, 2000). The use of out to evaluate the effect of different these plant extracts for inhibition of concentrations of plant extracts on fungal diseases is an important step mycelia growth and spore germination of towards the assessment of the degree of Trichothecium roseum causing pink rot of variability among the diverse natural brinjal. flora (Khandare and Vasait, 2017). The antifungal activities of these plant Materials and methods extracts are attributed to different chemical compounds like phenols, To investigate the fungi which flavonoids, isoflavonoids, coumarins, cause the rotting of brinjal fruits in pyrones, alkaloids, etc. present in these Kashmir Valley, diseased brinjal fruits plants which effect the growth of were collected in separate polythene pathogenic fungi (Jantasorn et al., 2016). bags from different fields, markets, Hence these plant extracts may have godowns and storage houses of Kashmir potential as a new natural fungicide for valley. These samples were either used management of fungal rot pathogens. So immediately or stored at 10 °C in the in an approach towards ecofriendly laboratory for different pathological management strategy, an attempt was studies.
Figure 1. Infected brinjal fruit. Figure 2. Culture of T. roseum Figure 3. Conidiophore with on PDA (Trichothecium possess conidia of T. roseum (100x) pinkish colored colonies). (Conidiophore was simple hyphae, septate in their lower half and bear clusters of conidia at the tip. Conidia of T. roseum were smooth and clavate).
Small portions of rotted tissues The casual pathogen was identified on were cut out aseptically from the the basis of symptoms caused by the diseased brinjal fruits and transferred to fungus on brinjal fruits, cultural and Potato Dextrose Agar medium (PDA). microscopic characteristics. The fungus
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on Potato Dextrose Agar medium (PDA) To prepare various after 48 h of inoculation at 24 °C ± 2 °C, concentrations of plant extracts leaves produced white colonies and then due to were collected, cleaned and cut into conidial production the colonies turn small pieces before being dried under light pink in colour (Figure 1, 2, and 3). shade at room temperature. The dried Pure colony cultures were material was ground to fine powder obtained by sub-culturing the fungal using a mechanical blender. Dry leaf growth in separate Petri plates powder (200 g) was packed in Soxhlet containing the same medium. A pink rot apparatus and extracted with distilled fungus was identified by their water at 80 °C-85 C. The extracts were morphological, reproductive and cultural filtered through Whatmann filter paper characteristics (Ellis, 1971; Barnett and No. 1 and the solventᵒ was removed under Hunter, 1972; Watanabe, 2002; Gilman, reduced pressure at 35 °C-45 °C using 2008). rotavapor. For pathogenicity, pathogens The dried extract (5 mg/5 mL) were re-inoculated after isolation onto was considered as standard solutions (S) the healthy brinjal fruits (Tomkin and and stored at 4 C in storage vials for Trout, 1931). Then all the five brinjal experimental use (Kaul, 1997). Then fruits were kept in clean polythene bags other concentrationsᵒ such as S/2, S/5 and incubated at 25 °C + 2 °C for ten and S/10 were obtained by adding days. These pathogenicity tests were appropriate amount of sterilized distilled used for the identification of plant water to the standard concentration. pathogens and to confirm the detection These concentrations were evaluated for of a particular disease. Identification of their effect on the mycelial growth by the disease and the pathogen was done food poisoning technique (Adams and following Koch’s postulates. Parameters Wong, 1991). 1 mL from each such as symptoms caused by these fungi concentration of the plant extract was on the healthy brinjal fruits, cultural mixed with nine ml of autoclaved and characteristics of the pathogens and cooled PDA just before pouring into Petri microscopic features of the pathogens plates. The medium was then dispensed were studied. uniformly into 90 mm sterile Petri plates and then inoculated with 5 mm mycelial Preparation and evaluation of disc of the pathogen from 10 day old different concentrations of plant fungal culture. Three replicates were extracts maintained for each concentration Different concentrations (i.e., S, including the control without any S/2, S/5 and S/10) of aqueous extracts of treatment. The Petri plates were leaves of Ajuga bracteosa Wall ex Benth, incubated at 25 °C ± 2 °C and Taraxacum officinale Weber ex Wiggers, observations of the mycelial growth of Mentha arvensis L. and Iris kashmiriana test fungus were recorded after seven Baker were evaluated for their effect on days of incubation. the inhibition in mycelial growth and in The percent inhibition in fungal spore germination of Trichothecium growth due to various treatments at roseum isolated from decayed brinjal different concentrations was computed fruits. as follows:
Mycelial growth inhibition (%) = {(dc – dt) / dc} × 100
Where: dc = average diameter of fungal colony in control, and dt = average diameter of fungal colony in treatment group.
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The plant extracts of tested created in 100 mm Petri plates by plants were also evaluated for their covering both sides of the Petri plates effect on the spore germination of with moist filter paper to maintain Trichothecium roseum. Spore suspension enough humidity. Three replicates were was prepared from 10 days old fungal maintained for each treatment including culture. A drop about 0.1 mL of spore the control. The slides were examined suspension was then placed in a cavity after 24 h by hand tally methods at glass slide containing a drop (about different microscopic fields. Percent 0.1 mL) of different concentration of spore germination for each was recorded plant extract and then incubated at using formula given by Kiraly et al. 25 °C ± 2 °C for 24 h in a moist chamber (1974).
Percent spore germination = (No. of spores germinated/Total no. of spores examined) × 100
Statistical analysis of the plant extracts. Among the plant Statistical analysis was carried extracts used Ajuga bracteosa at highest out using SPSS statistical software concentration ‘S’ was found most (version 16.0). Data was analyzed by one effective against Trichothecium roseum way analysis of variance (ANOVA) and and cause highest inhibition in the comparison of the means was done by mycelial growth (42.52%) followed by Duncan multiple comparison tests at Taraxacum officinale (34.03%), Mentha P 0.05. arvensis (29.75%) and Iris kashmiriana (25.54%) at the same concentration. Results≤ Other concentrations of plant extracts also bought about significant Effect of different reduction in the mycelial growth but to a concentrations of plant extracts on lesser extent. In different concentrations the mycelial growth of T. roseum of Ajuga bractosea, the inhibition in It was revealed from the results mycelial growth ranges from 42.52% to (Table 1) that different concentration of 23.37% and in T. officinale, the inhibition plant extracts caused significant in mycelial growth ranges from 34.03% inhibition in the mycelial growth of to 4.21%, respectively. Likewise, the Trichothecium roseum as compared to inhibition in mycelial growth in different control. However, the maximum concentrations of Mentha arvensis ranges inhibition in mycelial growth was found from 29.75% to 14.87% and in Iris at highest concentration ‘S’ followed by kashmiriana the inhibition ranges from lower concentrations S/2, S/5 and S/10 25.54% to 19.15%, respectively.
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Table 1. Effect of different concentrations of plant extracts on the mycelial growth of Trichothecium roseum. Mycelial growth (mm)
S S/2 S/5 S/10 Control 9.00 ±1.00c 10.00±1.0c 11.66±0.57b 12.00±1.00b Ajuga bracteosa 15.66±0.57a (45.52%) (36.14%) (25.54%) (23.37%) 10.33±0.57b 11.33±1.15b 14.33±0.57a 15.00±1.00a Taraxicum officinale 15.66± 0.57a (34.03%) (27.65%) (8.49 %) (4.21 %) 11.00±1.00 c 12.00±1.00b bc 12.66±1.15bc 13.33±0.57b Mentha arvensis 15.66± 0.57a (29.75%) (23.37%) (19.15%) (14.87 %) 11.66±0.57b 12.00±1.00b 12.33±2.08b 12.66±1.52b Iris kashmiriana. 15.66± 0.57a (25.54%) (23.37%) (21.26%) (19.15%) *Each value is mean of 3 replicates ± SD. Figures in parenthesis is the mycelial growth inhibition (%)
Effect of different concentrations of plant extracts also concentrations of plant extracts on bought about significant reduction in the spore germination of T. roseum spore germination but to a lesser extent. It was observed from the results In A. bracteosa, the inhibition in spore (Table 2) that different concentrations germination varies from 23.40% to (S, S/2, S/5 and S/10) of plant extracts 18.28% in different concentrations. In caused significant reduction in spore different concentrations of Taraxicum germination of T. roseum compared to officinale the inhibition varies from control. Among the plant extracts used, A. 27.42% to 23.23% and in Mentha bractosea at highest concentration (S) arvensis the inhibition varies from was found most effective and caused 33.48% to 26.49%, respectively. highest reduction in spore germination Likewise, the inhibition in the spore followed by T. officinale, M. arvensis L. germination varies from 37.09% to and Iris kashmiriana, respectively, at the 29.99% in different concentrations of Iris same concentration. Other kashmiriana, respectively.
Table 2. Effect of different concentrations of plant extracts on the spore germination of Trichothecium roseum. Spore germination (%)
S S/2 S/5 S/10 Control Ajuga bracteosa 18.28±1.67ⅽ 18.78±1.05ⅽ 22.40±2.50b 23.40±1.43b 36.05±2.57a Taraxicum officinale 23.23±2.88b 23.66±1.52b 26.02±3.57b 27.42±2.50b 36.05±2.57a Mentha arvensis 26.49±1.29 28.33±2.88b 33.01±2.86a 33.48±2.80a 36.05±2.57a ⅽ Iris kashmiriana 29.99±3.33ƅ 32.91±1.84c 35.32±0.81b 37.09±0.88ab 39.41±0.83a ԁ ԁ *Each value represents the mean spore germination %age of 3 replicates ± SD.
Discussion brinjal. Such studies on fungal rot of brinjal have been carried out for the first It was clear from the results that time in Kashmir Valley. However, some brinjal fruits in storage are attacked by earlier studies have been carried out on Pink rot fungi and caused Pink rot of the fungal rot of brinjal in India and all
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over the world. In the present study Allium sativum extracts against some plant extracts were evaluated for F. oxysporum have also been reported by their antifungal activity against the Thakur et al. (1995). The variation in fungus causing Pink rot of brinjal. T. inhibition among test extracts could be roseum is Isolated from apples (Zabka et due to variation in the components of al., 2006) and eggplants (Pandey, 2010). antifungal chemicals in different plant Mycotoxins (roseotoxin B and species (Gautam et al., 2003). trichothecin) production was carried Sengul et al. (2009) studied that from T. roseum by Engstrom et al. (1975) the methanol extract of Inula aucherana, and Ghosal et al. (1982). Shamim Shamsi Fumaria officinalis, Crocus sativus, Vicum (2008) reported the association of this album, Tribulus terestris, Polygonatum fungus with chickpea Cicer arietinum L. multiflorum, Alkanna tinctoria and Antifungal activities of these Taraxacum officinale were proved to plant extracts are attributed to different possess considerable antimicrobial chemical compounds like phenols, potentiality against a number of flavonoids, isoflavonoids, coumarins, microorganisms. The methanolic extract pyrones, alkaloids, etc. present in these had shown better antimicrobial activity plants which effect the growth of compared to aqueous extract. Balkan et pathogenic fungi (Jantasorn et al., 2016). al. (2017) screened the fifty plant species Hence these plant extracts may have for their antifungal effects against potential as a new natural fungicide for T. roseum. Anthemis arvensis, Origanum management of fungal rot pathogens. vulgare, Sambucus ebulus and Thymus Presently, Mancozeb proved longicaulis powders totally inhibited the effective in reducing the losses caused by mycelia growth of T. roseum at 10% Trichothecium roseum. Parveen et al. (w/v). Chelidonium majus and (2013) observed the effect of fungicides Clinopodium vulgare powders were and plant extracts on fruit rot pathogens, effective to T. roseum, with a percentage viz. Alternaria alternata and Mucor of inhibition of mycelia growth higher piriformis. Likewise, some other studies than 70%. also revealed the antimycotic activity of some plant extracts against fungi causing Conclusion fungal rot of vegetables (Oyelana et al., 2011; Raji and Raveendran, 2013; Pawar, It is concluded from the study 2013). that these plant extracts showed Dal Bello (2008) reported that antifungal activity and can possibly be Trichothecium roseum causing exploited in the management of postharvest fruit rot on tomato fruit for pathogenic fungi to prevent the first time in Argentina. The fungus biodeterioration in an eco-friendly way has been cited causing fruit rot on many but after further investigation. crops including tomato (Welch et al. 1975; Farr et al., 2007). It is also clear Acknowledgement from the above study that the plant extracts of all the tested plants proved The authors are highly thankful effective against Trichothecium rot to the Head Department of Botany, pathogen, Trichothecium roseum. Gupta University of Kashmir, for providing the et al. (1996) reported that extracts of necessary facilities for the smooth Calotropis gigantea and Azadirachta research and also to Curator, Centre of indica were most effective against Biodiversity and Plant Taxonomy, Fusarium oxysporum Schlecht inhibiting Department of Botany, University of the mycelial growth by 78.5% and Kashmir, Srinagar, J & K in proper 73.2%, respectively. Antifungal identification of the plant. properties of Azadirachta indica and
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