Inhibitory Activity of Crude Extract
Fungus
C. verruculosa has brownish black colonies in the middle and greyish black at the edges (Fig 1). Under the microscope at 10X40 magnification the greyish black mycelia of the isolated fungus was be observed (Fig 2).
P. caninum crude extract showed inhibitory on the fungus
verruculosa with a diameter of inhibitory zone of strong category of 22 mm diameter (Fig 3). The
P. caninum extract has anti-bacterial, antifungal and antioxidants properties due to the present of alkaloid phytochemicals, flavonoids, polyphenols and steroids (Suriani, 2016) and an active compound tetradecane, octadecamethyl cyclononasiloxane and benzenedicarboxylic acid which is an anti-funga.
In previous study,
P. caninum crude extract displayed strong activity against
Pyricularia oryzae (Suriani et al., 2015). Similar result was also observed in the present study against
C. verruculosa as shown in Fig 3. The black spot in the middle is the extract and the white fungi grows to certain limit around the extract creating an inhibition zone. At 4% extract concentration the diameter of the inhibition zone was 22 mm.
It has been reported that the
P. caninum extract contains alkaloid, flavonoids, steroids and polyphenols. Some individually identified chemical components are N-isobutyl-15-(3’,4’-methylenedioxyphenyl)-2E,4E-12E-pentadecatrienamide,N-iosobutyl-(2E,4E,14Z)-eicosatrienamide and six flavonoids; 5-hydroxy-7-methoxyflavone, 4’-hydroxy-5,7-dimethoxyflavone, 4’,5,7-trimethoxyflavone, 5,7-dimethoxyflavone, 2’-hydroxy-4’6’-dimethoxychalcone and 5,7-dimethoxyflavanone. The essential oils in the leaves are safrole, β-caryophyllene, β-pinene and germacrene and linalool have also been reported in previous studies. These essential oils also possess strong antibacterial and antifungal activities
(Salleh et al., 2011, 2015; Suriani, 2016; Appuaka, 2013;
Chouhan et al., 2017; Randrianariveloa, 2009).
Minimum Inhibitory Concentration (MIC)
The knowledge of minimum inhibitory concentration of
P. caninum leaf extract is important in the management of pesticide application. The MIC value is the minimum concentration to see the Efficacy of the biocidal agent. Low concentration is certainly preferred because excessive amount of extract may become toxic to the plant and coulddamage the wax layer on the leaves affecting the efficiency of photosynthesis process and other non-target organisms. The MIC value for the extract against
C. verruculosa fungus was found to be 0.5% similar to keben (
Barringtonia asiatica) seed extract against the same. On the other hand, the MIC value of the flavonoid from the crude extract of
P. caninum against
Escherchia coli, Staphylococcus aureus, Bacillus substilis, Psedomonas areginosa and P. putida bacteria was 125-1000 μg / mL and 500 μg / mL against Aspergilus niger (Salleh), 2015; 2011). Another important information in the formulation of fungicide for field application is the concentration of the agent that can Control the pathogen. Learned by inhibition of fungal colonies by varying the concentration of
P. caninum crude extract at a concentration of 0.5% (the smallest concentration) which can inhibit
C. verruculosa causing brown spots. Inhibition below the concentration of 5% or the same is an effective concentration for biopesticides and is worthy of being recommended for use as biopesticides. The lower the MIC concentration of an extract, the more effective the extract can inhibit a pathogen and the more efficient it is in using a plant resource. The use of high concentrations also causes toxic plants because the phytochemical substances in the extract will damage the wax layer on the leaves of the plant, so the leaves will wither and not be able to photosynthesis (Suprapta, 2014). In previous studies, flavonoid compounds from
P. caninum extract have MIC of 125µg / ml against
Escherchia coli, Staphylococcus aureus, Bacillus substilis, Psedomonas areginosa and P. putida (Salleh, 2015; 2011).
Hyleocerius undatus leaf crude extract with MIC 12 mg / ml against
Fusarium oxysporum f. sp. capsici causes of wilt in parika (Suprapta and Khalimi, 2012). The high alkaloid content of
P. caninum extract is antibacterial, has a MIC of 125 µg / mL against
Bacillus substilis (Wan et al., 2015). Ekstrak Acacia tortilis subsp dapat menekan penyakit Fusarium oxysporum f.sp. albedinis, the cause of Bayoud Disease of the date palm in Southwest Algeria dengan MIC 3 µg/ml
(Mezouari et al., 2019).
Inhibitory test on fungus colonies was carried out to calculate the inhibitory activity of extracts against the pathogen. The inhibition of 1% and 2% extract concentration on the fifteenth days was 27.8% and 35.6%, respectively, which is medium inhibitory (F 1 and F2, Fig 4) The inhibition 54% was displayed by 3% extract concentration (Fig 4, F3). A total inhibition was observed at 4% concentration as shown in Fig 4 F4. One way to calculate the inhibitory activity of extracts against pathogenic fungus is to use the inhibitory test on fungus colonies. At extract concentrations of 3% and 4% classified as very strong inhibitory activity because of more than 75% (Fig 4). All extract concentrations tested were significantly different from controls on the fifteenth days (P <0.05) and thus were also significantly different between treatments (Table 1 and Table 2). All extract concentrations tested under the current study were significantly different against controls (Fig 4). The inhibitory values are shown in Table 1. Good correlation between concentration and inhibition zone parameters was observed (Fig 5). The extract treatment of 1% and 2% on the 15
th days was able to inhibited fungal
C.
verruculosa by 27.8% and 35.6% which is classified as medium inhibitory, 3% extract concentration was able to inhibit 54.4% classified as strong inhibition and 4% extract concentration could inhibit 100% (Table 1) classified as very strong inhibition.
The inhibition of
P. caninum against the fungal colonies of
Pyricularia oryzae ranged from 11.83-37% at concentrations of 0.5-3.5% on PDA media
(Suriani et al., 2015). Higher concentration of
P. caninum extract resulted in higher inhibition of
C. verruculosa fungus (Fig 5). This is because higher extract concentraton contain higher amout of phytochemical content which functions as an antimicrobial
(Nadri et al., 2015). Nasrine et al., (2017) stated that the water plant extract of
Artemisia herb alba can inhibit 40% growth of
Fussarium sp fungal colonies effectively at concentrations 20%. Similarly, bioactive substances in crude extract of
cinnamon leaf can inhibit fusarium fungal colonies causing stem rot disease in tomatoes and dragon fruit (
Darmadi et al., 2016; 2017).
Effect of Extract on Fungal Mycelia
The effect of plant extract on the mycelia of
C. Verrucullosa was observed using scanning electron microscope. Fungus in control treatement showed a clear undamaged mycelia as shown in Fig 6B however Fig 6A shows the mycelia of the treated fungus was damaged and appeared shrink and hollow. Such morphological changes indicate the lysis of the fungus had occured.The observation of fungus mycelia of
C. verrucullosa between control and treatment, using SEM (Scaning Electron microscope). There appeared to be a difference between mycelia control and treatment .
Phytochemical substances include alkaloids, flavonoids, polyphenols and steroids found in extracts
P.caninum can lysemycelia of
C. verruculosa as indicated by small and hollow mycelia. This is because alkaloids can damage the permeability of bacterial and fungal cell walls whilephenol can damage proteins in fungal and bacteria cell walls causing the contents of the cell will go out resulting in lysis (Upadhyay, 2014).
Atuna racemosa Raf. extract contains phytochemicals glycosides, tannins, phenolic and saponins compounds that function to inhibit
Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica ser. Typhimurium, Aspergillus niger, Saccharomyces cerevisiae and
Candida albicans on agar media by the diffusion well method
(Gentallan et al., 2019). Suriani et al., (2015) also stated that
P. caninum extract can lyse the fungi mycelia of
Pyricularia oryzae causing blast disease in rice, seen by using SEM. Crude extract of cinnamon leaf can lyse fusarium fungi that cause wilt on tomato plants, seen using SEM (Darmadi
et al., 2015). Crude extract from
Epicoccum sp leaf can inhibit the formation of apressoria in
P. oryzae fungal seen using SEM
(Sena et al., 2013). Susun et al., (2018) states that
Alamanda cathartica leaf extract in combination with leaf extract
Mansoa alliace can lyse the fungal
Athelia rolfsii causes stem rot disease in peanut plants. Rasu and Hossain (2015) stated that
Allamanda cathartica extract can inhibit fungal
Curvularia lunata causing curvularia spotting disease in rice.
Intensity of curvularia disease
The results showed that
P. caninum leaf extract was highly influential on the intensity of curvularia in Bali red rice. The 3% extract concentration provides the highest obstacle to the intensity of curvularia disease. Table 2 through 6 show that the treatment and control were significantly different. Controls from week 2 to week 6 showed an increase in the intensity of curvularia spotting with 79.12% in week 8 and continued to increase over time to 82.79% at week 12.
According to
Kusai et al., (2015) that
Curvularia sp is a plant pathogen, especially attacking grasses, cereals and also rice plants, resulting in sharply reduced rice yields. Whereas in the treatment with
P. caninum leaf extract the intensity of curvularia disease decreased to an extract concentration of 3%, while the concentration of the extract 4% increase in disease intensity, this is also shown in Fig 7. This proves extracts in excessive concentrations will have toxic effects on plants, because rice plants undergo lysis so that the defense of rice plants decreases and is easily attacked by curvularia.
P. caninum leaf extract can suppress the pathogenic curvularia, although the pathogenic curvularia also has phytotoxic substances against other organisms such as inhibition of bacteria such as Escherichia coli and also has antioxidant activity and inhibition of acetylcholinesterase
(Kaaniche et al., 2019). This is also supported by research by Oraon and Mondal (2020) that the initial growth of
Cicer Arietinum L. was hampered after being given a
Putranjiva roxburghii Wall leaf extract license. at high concentrations (100%), because the extract contains allelopathic substances.
Besides the phytochemical substances in curvulria fungus can also inhibit the fungus
Phytophthora capsici pathogens in vegetables
(Mondol et al., 2017). Although curvuaria fungus has phytotoxic properties,
P. caninum leaf extract can strongly inhibit the development of curvularia fungus. According to Tanin and Soytong (2017), in Cambodia curvuaria spots caused by Curvuaria lunata can be reduced by utilizing the biological agent
Chaetomium cupreum. According to Kithn and Daiho (2014) that the
Millettia pachycarpa root extract can inhibit 55.78% at a concentration of 10% and the extract of Acorus calamus can inhibit 53.40% at a concentration of 10% against the
curvularia lunata spotting disease and the biological agent
Trichoderma harzianum can inhibit 68.85%. Research of
Mohana et al., (2011) in southern India extracts of
Acacia nilotica, Caesalpinia coriaria, Decalepis hamiltonii, Emblica officinalis, Lawsonia inermis and
Mimosops elengi can inhibit several rice diseases including curvularia spotting at concentrations of 3500 µg / ml with inhibitory activity ranging from 62.5%-78%.
