In Vitro Effects of Some Ethanolic Crude Extracts of Medicinal Plants against Colletotrichum gloeospoioides, The Pathogen of Antharacnose Disease in Chilli

The anthracnose disease is one of the major economic diseases in chilli production of Thailand. The present study was aims to test and evaluate the fungicidal activity of the ethanolic crude extracts from thirty-four medicinal plants were tested against Colletotrichum gloeosporioides (the pathogen of anthracnose disease in chilli of Thailand) by poisoned food technique at 0, 2,000, 4,000, 6,000, 8,000 and 10,000 ppm. The inhibition of mycelial growth was evaluated. From the testing, All the used of thirty-four crude extracts showed significant antifungal activity against C. gloeosporioides. The result showed that the Curcuma aromatica, Zingiber zerumbet, Piper betle, Kaempferia galanga, Rosmarinus officinalis and Origanum vulgare crude extracts showed 100% inhibition of mycelial growth at all concentrations, whereas, the Wedelia trilobata and Polygonum odoratum crude extracts at 10,000 ppm gave the lowest inhibition of 70 and 82%, respectively. The study noted that the crude extracts namely C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare showed the completely control of mycelial growth against C. gloeosporioides (the pathogen of anthracnose disease in chilli). These research pointed the oppurtunities for screening and application of some ethanolic crude extracts for a eco-friendly environmental management and exploited method as the biological control in chilli production. Keywods: Fungicidal Activity, Anthracnose Disease, Colletotrichum Gloeosporioides Medicinal Plants, Ethanolic Crude Extracts, Chill


Introduction
Anthracnose disease is one of the major economic disease in chilli production of Thailand and worldwide. Than et al. (2008) reported that the anthracnose disease caused by three pathogens namely C. gloeosporiodes, C. acutatum and C. capsici. The anthracnose disease control in chilli production in Thailand had five methods namely mechanical control, cultural control, biological control, chemical control and integrated control. For the chemical control is the best method for anthracnose disease managements, whereas this method as harmful for environmental condition, product residues and human health .
Although, the management of anthracnose disease with the application of several fungicides. Filoda (2008) reported the effects of three fungicides (Sarfun 500 SC, Amistar 250SC and Gwarant 500 SC) at 0.01 0.20 and 0.40% ihhibited on the colony growth of C. gloeosporiodes and Nagaraju et al. (2020) reported that carbendazim (25 50 75 and 100 µl) inhibited on the mycelial growth of C. gloeosporiodes (the pathogen of anthracnose in mango).
Fungicides can be controlled the anthracnose disease but the toxicity effects on products in human health and environmental issues are studies. Nowadays, the farmers use the biological control for anthracnose disease control in chilli.  noted that the medicinal herb crude extracts for the soil and seed borne pathogen control have attracted wide interest. In general, several researches have been focused on medicinal herb crude extracts to control of plant disease management. (Sawatdikarn, 2011).
Several experiments reported of some plants crude extracts and essential oil for antimicrobial activity. Abera et al. (2011) showed the ethanolic crude extracts of two species (Eucalyptus globules and Eucalyptus citriodera) to inhibit the mycelial growth of Colletotrichum kahawae (the pathogen of berry disease in coffee) for 64-76 %. Sawatdikarn (2011) studied the antifungal activity of crude extracts of six Zingiberaceae species namely Boesenbergia pandurata, Zingiber officinale, Zingiber cassumunar, Amonum xanthioides, Kaempferia galanga and Amonum krervanh against Curvularia sp. (the pathogen of dirty panicle disease in rice), selected crude extracts of B. pandurata at 1,000 ppm showed the highest of mycelail growth inhibition for 57% and the crude extracts of A. Krervanh at 1,000 ppm showed the lowest of mycelail growth inhibition for 43%. Sawatdikarn (2016) noted the crude extract of three medicinal plants namely Curcuma aromatica Syzygium aromaticum and Origanum vulgare showed 100% inhibition on mycelial growth and spore germination of Alternaria sp. (the pathogen od dirty panicle disease in rice at all concentrations (1,000-10,000 ppm) and Palhano et al. (2008) reported that the essential oil of Cymbopogon citratus to inhibited on mycelial growth of C. gloeosporioides.
Jun-Young et al. (2006) noted that the antifungal activity of crude extracts from Curcuma longa against three red pepper anthracnose (Colletotrichum coccodes, C. gloeosporioides and C. acutatum). Rahman et al. (2011) reported that the seed extracts and the pulp extracts from Jatropha curcas had higher antifungal activity than whole fruit extracts against C.
gloeosporioides (the pathogen of anthracnose in papaya). Haron et al. (2013) showed that the fungicidal activity of Allamanda spp. crude extracts against C. gloeosporioides (the pathogen of anthracnose in papaya). Meng et al. (2013) impressed that the antifungal activity of crude extracts from Camellia semiserrata against C. musae and C. gloeosporioides. Marinho et al. (2018) noted that the fungicidal activity of soapberry (Sapindus saponaria) against C.
For the management on some pathogen (C. capsici; the pathogen of anthracnose disease in chilli in Thailand),  showed that the three crude extracts namely Curcuma aromatica, Piper betle and Origanum vulgare showed 100% inhibition of mycelial growth at all concentrations, whereas, the Wedelia trilobata and Polygonum odoratum crude extracts at 10,000 ppm gave the lowest inhibition of 62 and 77%, respectively.
Little information of thirty-four medicinal herb crude extracts on inhibition of mycelial growth of C. gloeosporioides (the pathogen of anthracnose disease in chilli). The objective of this research was to evaluate of thirty-four medicinal herb crude extracts on the mycelial growth of C. gloeosporioides.

Preparation of chilli fruits and Isolation of pathogen
Chilli fruits were obtained from two locations in Central area of Thailand, Phranakhon Si Ayutthaya and Aungthong Province. C. gloeosporioides from the chilli fruits were isolated and maintained on petri dishs containing in Potato dextrose agar (PDA) and incubated at 25 ๐ C. for 3 days before the tests. The preparation of chilli fruits and the isolation of pathogen followed by the methods of Sawatdikarn (2016). Thirty-four medicinal crude extracts used in this study was obtained from four locations in Phranakhon Si Ayutthaya province (Bangban, Wangnoi Bangsai and Bangpa-in) where produce and export of medicinal herb productions. There were washed with tap water and air dried for three days to eliminate surface moisture. Then each part of medicinal plants were packed in to envelop and kept in oven at 80 ๐ C temperature until dried. Dried each parts were grinded separately in an electic grinder to obtain powder which was than kept in plastic bags before the tests (Sawatdikarn, 2016).

Preparation of crude extracts
One hunderd grams of the dried powdered plant were soaked in 1,000 ml of 90% ethanol. These mixtures were refluxed followed by agitation at 200 rpm for 1 hour. The ethanolic extracts were squeezed and filtered by muslin cloth. The crude extracts were placed in to a wide tray to evaporate ethanol and added with water plant extracts (Prasad et al., 2010)

Mycelial growth test ; Food poisoned technique;
Diffusates were added in PDA and poured into petri dishes. PDA medium added only with ethanol and water served as control treatment. Each petri dishes was inoculated with 5 mm plug of pure isolate taken from margins of actively growing culture of pathogen. All petri dishes were incubated at 25 ๐ C.  The screening of crude extracts for fungicidal activity was conducted using the agar dilution method. Different crude extracts were tested using food poisoning technique. Each tested crude extracts was used at different concentrations; 0 (control treament), 2,000, 4,000, 6,000, 8,000 and 10,000 ppm. The petri dishes were incubated in room temperature for 7 days. The efficacy of treament was assessed from all the four plate by mesuring fungal colony development (cm). The mycelial growth inhibition (M) with respect to the control treament was calculated from the formula (Sheng-Yang et al., 2005; Sawatdikarn, 2016) Where A is the colony diameter of the control treament and B is the colony diameter of the treated of crude extracts.

Statistical analysis
All experiments were done for four replications. Data (inhibition of mycelial growth at 2,000, 4,000, 6,000, 8,000 and 10,000 ppm.) were subjected to analysis using Duncan , s Multiple Range Tests (DMRT).

Results and discussion
The thiry-four medicinal plant crude extracts showed inhibition on mycelial growth of C. gloeosporioides at different concentrations ( Table 1). The crude extracts of C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare showed 100% inhibition of mycelial growth at all concentrations, whereas, the W. trilobata and P. odoratum crude extracts at 10,000 ppm gave the lowest inhibition of 70 and 82%, respectively.
For the C. aromatica crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1) can be used crude extracts of these species for C. gloeosporioides management (the pathogen of anthracnose disease in chilli) (1,000-10,000 ppm). These results are in agreement with that the researches of  noted that the C. aromatica crude extracts showed 100% inhibition on mycelial growth at 5,000-10,000 ppm for F. semitectum control (the pathogen of dirty panicle disease in rice) and related to the data of C. aromatica crude extracts showed 100% inhibition on mycelial growth at 5,000-10,000 ppm for C. lunata control (the pathogen of dirty panicle disease in rice) ) and the C. aromatica crude extracts showed 100% inhibition on mycelial growth at 1,000-10,000 ppm for C. capsici control (the pathogen of anthracnose disease in chilli)  The C. aromatica crude extracts showed the inhibition on mycelial growth of C. gloeosporioides, the results are in agreement with two researches, Saleem et al.
(2011) reported the crude extracts of C. aromatica at 0.4% showed the completely inhibition on mycelial growth of three pathogen namely Staphylococcus aureus, Enterococus faecalis and Pseudomonas aeruginosa and Harit et al. (2013) impressed that the ethanolic extract of C. aromatica was found to have both antibacterial activity (S. aureus and Bacillus subtilis) and antifungal activity (Candida albicans and Aspergillus flavus).
For the Z. zerumbet crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1) can be used crude extracts of these species for C. gloeosporioides control (the pathogen of anthracnose disease in chilli) (1,000-10,000 ppm). These results are in agreement with that Sawatdikarn (2016) noted that the plants species (The Zingiberaceae species), the two crude extracts namely Z. zerumbet and C. longa showed 100% inhibition on mycelial growth at 5,000-10,000 ppm for F. semitectum control (the pathogen of dirty panicle disease in rice).  For the P. betle crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1) can be used crude extracts of these species for C. gloeospoioides control (the pathogen of anthracnose disease in chilli) (1,000-10,000 ppm). These results are in agreement with that the researches of Sawatdikarn (2016) impressed that the P. betle crude extracts showed 100% inhibition on mycelial growth at 2,500-10,000 ppm for F. semitectum control (the pathogen of dirty panicle disease in rice) and related to the researchs of P. betle crude extracts showed 100% inhibition on mycelial growth at 2,500-10,000 ppm for Curvularia lunata control (the pathogen of dirty panicle disease in rice) ( For the K. galanga crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1)  For the R. officinalis crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1) can be used crude extracts of these species for C. gloeospoioides control (the pathogen of anthracnose disease in chilli) (1,000-10,000 ppm). These results are in agreement with that the researches of  impressed that the R. officinalis crude extracts showed 100% inhibition on mycelial growth at 2,500-10,000 ppm for F. semitectum control (the pathogen of dirty panicle disease in rice) and these data related to the researchs of R. officinalis inhibited of some pathogens, Centeno et al. (2010) noted that the crude extracts of R. officinalis showed 100% inhibition on mycelial growth at all concentrations (0.004-0.4%) for two pathogens control (Aspergillus flavus and A. ochraceus) and Matsuzaki et al. (2013) reported that the essentail oil from R. officinalis had an effect on mycelail growth of Candiada albicans.
In agreement with this research, Alemu et al. (2014) noted that the methanol extract of R. officinalis has focused fungicidal activity against C. gloeosporioides (the pathogen of anthracnose disease in mango).
For the O. vulgare crude extracts showed 100% inhibition on mycelial growth at all concentration (Table 1)  For the E. camaldulensis crude extracts at 2,000-8,000 ppm showed the inhibition on mycelial growth for 65-82% (Table 1). This agreed with the results of Abera et al (2011) showed the ethanolic crude extracts of Eucalyptus globules and Eucalyptus citriodera to inhibit the mycelial growth of Colletotrichum kahawae (the pathogen of berry disease in coffee) for 64-76 %.
In the present study, extrect from Moringa oleifera (2,000-8,000 ppm) showed the inhibition on mycelial growth for 58-75% (Table 1) The ethanolic crude extract from lemon grass (C. citratus) showed the highest antifungal activity (100% inhibition of mycelial growth) against C. gloeosporioides (Table 1). This data corresponds with research done by Perez-Cordero et al. (2017) who reported that extract of C. citratus have antifungal activity and inhibit the growth of C. gloeosporioides (the pathogen of anthracnose disease in yam) and this agree with the data of Palhano et al. (2004) exhibited the essential oil from lemon grass (C.
For the two crude extracts (W. trilobata and P. odoratum) at 10,000 ppm concentration gave the lowest inhibition of 70 and 82%, respectively (Table  1). These results are in agreement with that the researches of Sawatdikarn (2016) noted the crude extract of W. trilobata and P. odoratum showed 62 and 77% inhibition, respectively on mycelial growth of C. capsici (the pathogen of anthracnose disease in Chilli) at 10,000 ppm concentration. Inaddition, Biju and Praveena (2018) reported that the crude extract of W. chinensis showed 17-33 inhibition on mycelial growth of C. gloeospoioides (the pathogen of anthracnose disease of black pepper) at 2.5 5 10 and 20% concentrations.
The goal of this study was to screening of the thirtyfour crude extracts on the mycelial growth of C. gloeospoioides.The management of all crude extract was the best for C. gloeospoioides control due to their harmless on enviromental condition, to user and to consumer. The study that the related to the several researcher have noted the antifungal activity of crude extracts and essential oils including, the researchs of    (Umar et al., 2011) and carvacrol and p-cymene from the leaves of O. vulgare (Papajani et al., 2015) This study noted that the thirty-four crude extracts can be use for C. gloeospoioides management and can be used the six plants crude extracts for anthracnose disease control. The six crude extracts (C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare) showed 100% inhibition of mycelial growth of C. gloeospoioides (the pathogen of anthracnose disease in chilli) at all concentrations.
The study noted that the six crude extracts (C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare) gave the completely control of mycelial growth. In addition, the six crude extracts namely C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare can be used for anthracnose disease management in chilli production as strongest inhibition crude extract and the two crude extract (W. trilobata and P. odoratum) as weakest inhibition crude extract.
Data of the research pointed the oppurtunities for screening and application of some ethanolic crude extracts for a eco-friendly environmental management and exploited method the biological control of chilli production in Thailand. In the same column, mean followed by a common letter are not significantly different at the 5% level by DMRT.

Conclusion
All the used of thirty-four crude extracts showed significant antifungal activity against C. gloeospoioides. The result showed that the C. aromatica, Z. zerumbet, P. betle, K. galanga, R. officinalis and O. vulgare crude extracts showed 100% inhibition of mycelial growth at all concentrations, whereas, the W. trilobata and P. odoratum crude extracts at 10,000 ppm gave the lowest inhibition of 70 and 82%, respectively.