Antifungal activity of selected medicinal plants against Alternaria species : The pathogen of dirty panicle disease in rice

Antifungal activity of the ethanolic crude extracts from ten plants, namely: Curcuma xanthorhiza, Curcuma aromatica, Kaempferia parviflora, Syzygium aromaticum, Origanum vulgare, Synedrella nodiflora, Sorghum bicolor, Rosmarinus officinalis, Piper longum and Eupatorium odoratum were tested against Alternaria species (the pathogen of dirty panicle disease in rice) by poisoned food technique at 0, 1,000, 2,500, 5,000, 7,500 and 10,000 ppm. The inhibition of mycelial growth and spore germination were evaluated. The results showed C. aromatica, S. aromaticum and O. vulgare crude extracts at 1,000 ppm, the R. officinalis crude extracts at 5,000 ppm, and the C. xanthorrhiza crude extracts at 10,000 ppm showed the highest inhibition of mycelial growth at 100%, whereas S. nodiflora and S. bicolor crude extracts at 10,000 ppm had the inhibition at 56 and 58%, respectively. For inhibition of spore germination, the three plant crude extracts; C. aromatica, S. aromaticum and O. vulgare at 1,000 ppm and C. xanthorrhiza, S. nodiflora, S. bicolor, R. officinalis and P. longum crude extracts at 2,500 ppm reached the highest inhibition of spore germination at 100%, whereas E. odoratum and K. parviflora crude extracts at 10,000 ppm had the inhibition at 20 and 55%, respectively.


INTRODUCTION
Dirty panicle disease is one of the most serious diseases of rice, affecting rice production in the world and rice production grown in Thailand and tropical locations (Ou, 1985).The causal organisms are several pathogens, Fusarium species, Alternaria species, Cercospora species and Curvularia species (Abdelmonem, 2000).The panicle dirty control had several methods, mechanical, cultural, biological and chemical method.Chemical control is the best method for dirty panicle disease, whereas this method is harmful for environmental condition, consumer and user.Nowadays, the farmers use the biological control for dirty panicle control.The medicinal herb crude extracts for the seed borne pathogen control have attracted wide interest, the E-mail: SSanit33@hotmail.comAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License medicinal herbs comprises a rich phytochemicals and biochemicals substances to be used as biopesticide which are more friendly environment than synthetic chemicals.
There are reports that phytochemicals and biochemicals substances of some plants showed fungicidal activity against pathogenic fungi; Syzygium aromaticum crude extract at 1,000 ppm showed 100% inhibition on mycelial growth of F. oxysporum and F. equiseti (Kritzinger et al., 2002) and the S. aromaticum crude extract can be used for Staphylococcus aureus control (Briozzo et al., 1989).Rosemary (Rosmarinus officinalis) crude extracts showed the growth inhibition of three pathagens, namely Leuconostoc mesenteroides, Listeria monocytogenes and S. aureus (Campo et al., 2000).For the some medicinal crude extracts on mycelial growth of some pathogens control including the oregano (Origanum vulgares) crude extract for Bacillus cereus control (Paster et al., 1995); Curcuma xanthorhiza for three pathogen control, namely B. cereus, S. aureus and Escherichia coli (Husein et al., 2009); Curcuma aromatica for S. aureus and E. coli control (Saleem et al., 2011) and Kaempferia parviflora crude extracts for B. cereus and S. aureus control (Butkhup and Samappito, 2011).
No information of ten medicinal herb crude extracts on inhibition of mycelial growth and spore germination of Alternaria spp.(pathogen of dirty panicle disease in rice).The objective of this research was to evaluate ten medicinal herb crude extracts on the mycelial growth and spore germination of Alternaria spp. in Central area, Thailand.(Prasad et al., 2010).

Preparation of rice seeds and isolation of pathogen
Rice seeds were obtained from three location in Central area, Phranakhon Si Ayutthaya, Aungthong and Prathumthani province.Alternaria spp.from the rice seeds were isolated and maintained on petri dishs containing in Potato dextrose agar (PDA) and incubated at 25°C for 3 days before the tests.The preparation of rice seeds and the isolation of pathogen followed by the methods of Sawatdikarn (2011).

Collection of and preparation of plants samples
Ten medicinal crude extracts used in this study were obtained from three location in Phranakhon Si Ayutthaya province, Bangban, Wangnoi and Bangpa-in area.Fresh rhizomes of C. xanthorhiza, C. aromatica and K. parviflora were collected from Bangban, fresh leaves of O. vulgare, S. nodiflora, S. bicolor, R. officinalis and E. odoratum were collected from Wangnoi and fresh bud and fruit parts of S. aromaticum and P.longum, respectively were collected from Bangpa-in.They were washed with tap water and air dried for three days to eliminate surface moisture.Then each part of the medicinal plants was packed into envelop and kept in oven at 105°C temperature until it is dried.Dried parts were ground separately in an electic grinder to obtain powder which was than kept in plastic bags before the tests.

Preparation of crude extracts
100 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 h.The ethanolic extracts were squeezed and filtered by muslin cloth.The crude extracts were placed into a wide tray to evaporate ethanol and added with water plant extracts (Prasad et al., 2010).

Mycelial growth test
Food poisoning 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°C.The screening of crude extracts for antifungal 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), 1,000, 2,500, 5,000, 7,500 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 mycelail growth inhibition (M) with respect to the control treament was calculated from the formula (Sheng-Yang et al., 2005).
where A is the colony diameter of the control treament and B is the colony diameter of the treated crude extracts.

Spore germination test
Effect of ten medicinal crude extracts on spore germination of Alternaria spp. was carried out by the spore germination method (Ting-Ting et al., 2011).A spore suspension of Alternaria spp. was prepared by adding 10 ml of sterile water to 15 days old petri plate culture of Alternaria spp.grown at 22°C on PDA.The surface of agar was washed through a double layer of gauze with sterile water.The inoculum was adjusted to a concentration of 1×10 6 ml with haemacytometer.One milliliter of ten crude extract in 20 and 40 mg/ml was mixed with the same amount of Alternaria spp.spore suspension.The mixture was pipetted onto clean concave slips and incubated at room temperature for 24 h.In the control, the sterile water was mixed with spore suspension.Germination rate of spores was observed with microscope after incubation of 12 h.
Each tested crude extracts was used at different concentrations; 0 (control treament), 1,000, 2,500, 5,000, 7,500 and 10,000 ppm.The efficacy of treament was assessed from four slide after treatment by measuring number of spore germination.The spore

Medicinal plant crude extracts
Mycelial growth inhibition degree (%) 1,000 ppm 2,500 ppm 5,000 ppm 7,500 ppm 10,000 ppm Curcuma xanthorrhiza 84 In each column, mean followed by a common letter are not significantly different at the 5% level by DMRT.
germination inhibition (S) with respect to the control treament was calculated from the formula (Sheng-Yang et al., 2005).

S = (A-B) / A ×100
where A is the spore germinarion number of the control treament and B is the spore germination number of the treated crude extracts.

Statistical analysis
For statistical analysis, Duncan Multiple Range Test was used to compare the average.

RESULTS AND DISCUSSION
The ten medicinal herb crude extracts showed inhibition on mycelial growth of Alternaria spp.different concentrations (Table 1).The crude extracts of C. aromatica (Figure 1), S. aromaticum and O. vulgare at all concentrations, the R. officinalis crude extracts at 5,000 ppm and the Curcuma xanthorrhiza crude extracts at 10,000 ppm (Figure 2) showed 100% inhibition on mycelial growth, whereas the S. nodiflora (Figure 3) and S. bicolor (Figure 4) crude extracts at 10,000 ppm gave the highest inhibition of 56 and 58%, respectively.C. aromatica, S. aromaticum and O. vulgare crude extracts showed that 100% inhibition on spore germination (Table 2) at all concentrations can use crude extracts of these species for Alternaria spp.control (the pathogen of dirty panicle disease in rice) at all concentration (1,000 to 10,000 ppm).
O. vulgare crude extracts showed 100% inhibition on mycelial growth and spore germination of Alternaria spp. at all concentration (1,000 to 10,000 ppm); this result agrees with the O. vulgare crude extracts inhibited the growth of Bacillus cereus (Paster et al., 1995).For the three crude extracts showed the inhibited mycelial growth in Alternaria spp., because phytochemical effects for antimicrobial activity, curcumin from the rhizome of C. aromatica (Saleem et al., 2011), phenolic compound and eugenol from the bud or fruit of S. aromaticum (Briozzo et al., 1989;Maarse, 1991) and carvacrol from the leaves of O. vulgare (Ultee et al., 2000;Chaudhry et al., 2007).
The two crude extracts, R. officinalis (5,000 to 10,000 ppm) and C. xanthorrhiza (10,000 ppm) showed 100% of inhibition on mycelial growth of Alternaria spp.(Table 1).This result agrees with the R. officinalis crude extracts showed the growth inhibition of Leuconostoc mesenteroides, Listeria monocytogenes and S. aureus (Campo et al., 2000) and the result indicated that the C. xanthorrhiza crude extracts showed the inhibition for three pathogen control, namely B. cereus, S. aureus and E. coli (Husein et al., 2009).
The three crude extracts, namely C. xanthorrhiza, R. officinalis and P. longum at 2,500 ppm reached the highest inhibition of spore germination at 100% (Table 2).For the R. officinalis and C. xanthorrhiza crude extracts showed the inhibition of mycelial growth and spore germination of Alternaria spp., because of phytochemical potential for antimicrobial activity, carnosic acid and     In each column, mean followed by a common letter are not significantly different at the 5% level by DMRT.
rosmarinic acid from the leaves of R. officinalis (Frankel et al., 1996) and curcumin from the rhizome of C. xanthorrhiza (Husein et al., 2009).The three crude extracts, K. parviflora, P. longum and E. odoratum at 10,000 ppm had the inhibition on mycelial growth at 75, 75 and 71%, respectively (Table 1).This result agrees with the K. parviflora, P. longum and E. odoratum crude extracts inhibited the growth of some pathogens; K. parviflora crude extracts for B. cereus and S. aureus control (Butkhup and Samappito, 2011) P. longum crude extracts for Bacillus subtilis control (Kumar et al., 2010) and E. odoratum showed the growth inhibition of two pathogen, namely B. cereus and Aspergillus niger (Owalabi et al., 2010).The phytochemical compounds in each species showed the inhibition on growth of pathogen; methoxyflavone from the rhizome of K. parviflora (Kummee et al., 2008), pipenonaline from the fruit of P. longum (Lee et al., 2001) and alpha-pinene and beta-pinene from the leaves of E. odoratum (Owalabi et al., 2010).
S. nodiflora and S. bicolor crude extracts at 10,000 ppm gave the highest inhibition of mycelail growth at 56 and 58%, respectively (Table 1).S. nodiflora and S. bicolor crude extracts at 2,500 ppm showed the highest inhibition of spore germination at 100% (Table 2).This result agrees with the S. nodiflora crude extracts inhibited on growth in five pathogens, namely; B. subtilis, S. aureus, E. coli, Candida albicans and Aspergillus flavus (Bhogaonkar et al., 2010) and S. bicolor crude extracts inhibited on growth in three pathogens, namely; Fusarium moniliforme, Curvularia lunata and A. flavus (Seetharaman et al., 1997).The phytochemical compounds of S. nodiflora and S. bicolor crude extracts showed the inhibition on growth of some pathogens; alkaloids and flavanoids from the leaves of S. nodiflora (Bhogaonkar et al., 2010) and saponins and tannins from the leaves of S. bicolor (Soetan et al., 2006).
In total, this study indicated that the ten crude extracts can be used for Alternaria spp.control and three plant can be used for crude extracts of dirty panicle control.The crude extracts of three species, namely, C. aromatica, S. aromaticum and O. vulgare showed 100% inhibition on mycelial growth and spore germination of Alternaria spp. at all concentrations (1,000 to 10,000 ppm).

Conclusion
C. aromatica, S. aromaticum and O. vulgare crude extracts at 1,000 ppm, the R. officinalis crude extracts at 5,000 ppm and the C. xanthorrhiza crude extracts at 10,000 ppm showed the highest inhibition of mycelial growth at 100%, whereas the S. nodiflora and S. bicolor crude extracts at 10,000 ppm had the inhibition at 56 and 58%, respectively.For inhibition of spore germination, the three plant crude extracts, C. aromatica, S. aromaticum and O. vulgare at 1,000 ppm and the C. xanthorrhiza, Synedrella nodiflora, S. bicolor, R. officinalis and P. longum crude extracts at 2,500 ppm reached the highest inhibition of spore germination at 100%, whereas the E. odoratum and K. parviflora crude extracts at 10,000 ppm had the inhibition at 20 and 55%, respectively.

Figure 4 .
Figure 4. Effect of Sorghum bicolor crude extract on the mycelial growth of Alternaria sp. at different concentrations (Control treatment, 1,000 2,500 5,000 7,500 and 10,000 ppm).

Table 1 .
Efficacy of different concentration of some medicinal plant crude extracts on mycelial growth inhibition of Alternaria spp.

Table 2 .
Efficacy of different concentration of some medicinal plants crude extracts on spore germination inhibition of Alternaria spp.