In vitro evaluation of fungicides against different diseases of kokum caused by Pestalotiopsis Maculans and Colletotrichum Gloeosporioides

Introduction

Kokum (Garcinia indica Choisy), a tropical fruit-bearing tree belongs to the family Clusiaceae and native to the Western Ghats of India, holds significant economic and medicinal value. It is one of the most significant fruits cultivated in the Konkan region, typically grown in arable wastelands characterized by high rainfall and a hot, humid climate. The environmental conditions in the Konkan region of Maharashtra are particularly wellsuited for kokum cultivation, making it a prominent cash crop in this area and its surrounding regions. The proximate composition of Kokum rind is moisture (80.0 g/100 g), protein (1.0 per cent), fat (1.4 per cent), tannin (1.7 per cent), pectin (0.9 per cent), total sugars (4.1 per cent), organic acids such as hydroxy citric acid, lactone and citric acid; anthocyanins, cyanidin-3glucoside and cyanidin-3-sambubioside; poly isoprenylated phenolics, garcinol and isogarcinol (traces) (Jagtap et al., 2015) [9] . When fruits are unripe, they are green in colour and when completely ripe their colour changes from scarlet to dark purple. The kokum fruit is oval, varies in size and weighs between 50 to 180 grams. The fruits are 2.5 to 3.75 cm in diameter, globule or spherical, dark purple when ripe and contain 5-8 seeds (Nayak et al., 2010) [14] . In India, kokum is widely used as a spice and serves as a natural acidulant in the preparation of juices, pickles and curries. The fruit is rich in antioxidants, which help neutralize free radicals and protect body cells from oxidative damage, while also promoting cell regeneration and repair. Kokum juice is especially favoured during the hot summer months due to its natural cooling properties. A popular beverage known as 'Amrut Kokum', made with sugar syrup, is traditionally used to prevent sunstroke. In Ayurvedic medicine, various parts of the kokum plant including the rind, leaves and other tissues are utilized to treat inflammatory conditions, rheumatic pain and digestive disorders. Kokum oil also serves as an emollient and antiseptic. It is used to reduce fever and alleviate allergic reactions. kokum is one of the important fruits of Western Ghats of India and has tremendous medicinal and nutraceutical values. It is rich in anti-oxidants. A number of recent studies have examined the potential of garcinol, a non-nutritive dietary component, against different cancer types such as breast cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer and tongue cancer (Saadat and Gupta, 2012) [19] . Despite its economic, medicinal and nutraceutical importance, kokum cultivation is affected by various fungal diseases that can adversely influence plant health and productivity. Among the pathogens associated with kokum, Pestalotiopsis maculans and Colletotrichum gloeosporioides are important fungal pathogens. Effective management of fungal diseases largely depends on the use of suitable fungicides. However, information on the efficacy of fungicides against these pathogens associated with kokum is limited. Therefore, the present investigation was undertaken to evaluate the in vitro efficacy of selected fungicides against Pestalotiopsis maculans and Colletotrichum gloeosporioides under laboratory conditions.

Materials and Methods

Isolation and proving pathogenicity of causal organisms

Naturally infected kokum leaves showing typical disease symptoms were collected from the Horticulture Nursery, College of Horticulture, Dapoli and brought to the laboratory for isolation. The diseased portions were cut into approximately 5 mm pieces containing both healthy and diseased tissues. The pieces were surface disinfected with 0.1% sodium hypochlorite, followed by three successive washes with sterile distilled water. The tissue pieces were aseptically placed on potato dextrose agar (PDA) medium and incubated at 27 ± 2°C for seven days. The emerging fungal growth was sub-cultured to obtain pure cultures. Pathogenicity was tested on one-month-old healthy potted kokum plants using the pin-prick method. Spore suspension prepared from ten-day old cultures was sprayed on leaves until runoff. Inoculated plants were covered with polythene bags to maintain humidity, while plants sprayed with sterile water served as control.

In vitro evaluation of fungicides

The present study was conducted at the Department of Plant Pathology, College of Agriculture, Dapoli, Dist. Ratnagiri. In vitro study of fungicides against Pestalotiopsis maculans and Colletotrichum gloeosporioides was carried out. Seven fungicides representing different fungicidal groups were evaluated against the two pathogens. The fungicides and concentrations used were Carbendazim 50% WP at 0.1%, Propiconazole 25% EC at 0.1%, Copper oxychloride 50% WP at 0.25%, Myclobutanil 10% WP at 0.1%, Captan 50% WP at 0.2%, Chlorothalonil 75% WP at 0.2% and Cymoxanil 8% + Mancozeb 64% WP at 0.2%. An untreated medium served as the control. The fungicides were evaluated using the Poisoned Food Technique described by Nene and Thapliyal (1993) [15] . The experiment was conducted in Completely Randomized Design (CRD) with three replications. Mycelial growth was recorded as mean colony diameter, and the percentage inhibition of mycelial growth over the untreated control was calculated. Observations were recorded seven days after incubation.

The percentage inhibition of mycelial growth was calculated by using the formula given by Vincent (1947) [23] . -------------------× 100 C

Per cent Growth Inhibition (I) = C -T -

Where, C = Growth (mm) of the test fungus in untreated control plate T = Growth (mm) of the test fungus in treated plates.

Results and Discussion

Isolation and proving pathogenicity of causal organisms Diseased kokum leaves yielded two distinct fungal isolates on PDA medium after seven days of incubation at 27 ± 2 °C. Based on cultural and morphological characteristics, the isolates were identified as Pestalotiopsis maculans and Colletotrichum gloeosporioides. Pathogenicity testing confirmed that both isolates were capable of producing characteristic symptoms on artificially inoculated kokum leaves within 10-12 days, whereas the uninoculated control plants remained healthy. The respective pathogens were successfully re-isolated from the artificially infected leaves, and their cultural and microscopic characteristics corresponded with those of the original isolates, thereby confirming Koch's postulates. The pathogenicity of C. gloeosporioides was comparable with the findings of Abera et al. (2016) [1] . and Golakiya et al. (2020) [6] . Similarly, the pathogenicity of Pestalotiopsis spp. has been established by Ismail et al. (2013) [8] . and Bhagariya (2019) [2] .

In vitro evaluation of fungicides against Pestalotiopsis maculans

The results presented in Table No [7] . reported complete inhibition of Pestalotiopsis palmarum with Propiconazole 25% EC, while Devi et al. (2017) [5] . recorded complete inhibition of Pestalotiopsis mangiferae and Kyada (2006) [11] , recorded 99.77% inhibition of Pestalotiopsis guepinii with propiconazole. The present findings are further supported by Nakum (2017) [13] . and Das et al. (2020) [3] , who reported cent per cent inhibition of Pestalotiopsis anacardii and P. mangiferae, respectively, by Cymoxanil 8% + Mancozeb 64% WP under in vitro conditions. Likewise, Bhagariya (2019) [2] . reported that propiconazole was among the most effective systemic fungicides, while Cymoxanil 8% + Mancozeb 64% effectively suppressed the mycelial growth of Pestalotiopsis clavispora. [21] , Ranjitha et al. (2019) [18] , Sharma et al. (2019) [20] . and Vandana et al. (2021) [22] , who reported cent per cent (100%) inhibition of C. gloeosporioides by propiconazole under in vitro conditions. Patil et al. (2010) [17] . also reported complete inhibition by propiconazole, while carbendazim recorded 91.67% inhibition. Similar observations were made by Dev et al. (2016) [4] , who reported cent per cent inhibition of C. gloeosporioides with both propiconazole and carbendazim under in vitro conditions. Comparable results were also reported by Kothikar and Koche (2017) [10] . in Colletotrichum dematium and by Padghan et al. (2023) [16] . in Colletotrichum capsici, where propiconazole also exhibited cent per cent inhibition. Mishra and Pan (2008) [12] , reported cent per cent (100%) growth inhibition of both Pestalotia psidii and Colletotrichum gloeosporioides by Propiconazole 25% EC under in vitro conditions, supporting its high efficacy against both pathogens.

Plate 1: In vitro evaluation of fungicides against Pestalotiopsis maculans

Conclusion

The present investigation demonstrated significant variation in the in vitro efficacy of the tested fungicides against Pestalotiopsis maculans and Colletotrichum gloeosporioides associated with kokum. Propiconazole 25% EC at 0.1% concentration was the most effective fungicide against both pathogens and completely inhibited their mycelial growth. Cymoxanil 8% + Mancozeb 64% WP at 0.2% also produced complete inhibition of P. maculans. Carbendazim 50% WP showed comparatively high activity against both pathogens.

Acknowledgement

AcknowledgmentsThe authors gratefully acknowledge the Department of Plant Pathology, College of Agriculture, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, for providing the laboratory facilities and necessary support for conducting the present investigation.

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