Sunday 29 October 2017

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Presenting:

Introduction to 'Mas Cotek'






Literary Research

Antimicrobial

An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicidal, while those that merely inhibit their growth are called biostatic. The use of antimicrobial medicines to treat infection is known as antimicrobial chemotherapy, while the use of antimicrobial medicines to prevent infection is known as antimicrobial prophylaxis

The main classes of antimicrobial agents are:


  • Disinfectants

("nonselective antimicrobials" such as bleach), which kill a wide range of microbes on non-living surfaces to prevent the spread of illness,


  • Antiseptics

(which are applied to living tissue and help reduce infection during surgery),


  • Antibiotics

(which destroy microorganisms within the body). The term "antibiotic" originally described only those formulations derived from living organisms but is now also applied to synthetic antimicrobials, such as the sulphonamides, or fluoroquinolones. The term also used to be restricted to antibacterials (and is often used as a synonym for them by medical professionals and in medical literature), but its context has broadened to include all antimicrobials.

Antibacterial agents can be further subdivided into 


  • Bactericidal agents

which kill bacteria


  • Bacteriostatic agents

which slow down or stall bacterial growth.


In response, further advancements in antimicrobial technologies have resulted in solutions that can go beyond simply inhibiting microbial growth. Instead, certain types of porous media have been developed to kill microbes on contact.



Extract of Ficus deltoidea

Extract

a preparation containing the active ingredient of a substance in concentrated form.

Ficus deltoidea

Ficus deltoidea, commonly known as mistletoe fig (Mas Cotek in Malaysian) is a large shrub or small tree species native to Southeast Asia, and widely naturalized in other parts of the world.


F. deltoidea is a small perennial herb, growing up to about 2 m tall, with thick leaves that are deltoid in shape, rounded at the apex and tapering at the base. The upper surface of the plants' leaves are dark, shining green, while the lower surface is golden yellow with black spots. Male and female plants are physically distinctive, with the leaves of female plants being big and round, while the leaves of male plants are small, round and long. The plant grows wild in eastern peninsular Malaysia (Kelantan, Terengganu) and in Borneo (Sabah, Sarawak and Kalimantan), and is used by the traditional medical practitioners in these regions.


Plant fungal pathogens

Pathogen

In biology, a pathogen in the oldest and broadest sense is anything that can produce disease; the term came into use in the 1880s.Typically the term is used to describe an infectious agent such as:

  • virus
  • bacterium
  • protozoa
  • prion
  • fungus
  • other micro-organism

There are several substrates including pathways where the pathogens can invade a host. The principal pathways have different episodic time frames, but soil contamination has the longest or most persistent potential for harboring a pathogen. Diseases caused by organisms in humans are known as pathogenic diseases.

Pathogenic fungus

Pathogenic fungi are fungi that cause disease in humans or other organisms. The study of fungi pathogenic to humans is called "medical mycology". Although fungi are eukaryotic, many pathogenic fungi are microorganisms. The study of fungi and other organisms pathogenic to plants is called plant pathology.

"Antimicrobial Extract of Ficus deltoidea to control plant fungal pathogens" is about a research to synthesis some kind of bio fungicide from 'Mas cotek'.

Tuesday 24 October 2017

Conclusion

As conclusion, the objectives of the research had been achieved. The antimicrobial compounds in Ficus deltoidea had been extracted and F.deltoidea leaf extract inhibited the mycelial growth of selected plant pathogenic fungi. The hypothesis of this research was accepted where F.deltoidea produced antifungal metabolites that 
can inhibit the mycelial growth of plant pathogenic fungi. 

The leaf extract of F.deltoidea showed antifungal activity against two types of plant pathogenic fungi which were Ganoderma boninense and Rhizoctonia solani. Poisoned agar plates containing F.deltoidea extract showed inhibition of mycelial growth, where percent inhibition of diameter growth (PIDG) of G.boninense was 55.9% at 7th day and R.solani 53.67% at 5th day. Meanwhile, there was 100% inhibition occurred for both G.boninense and R.solani at the concentrations of 10%, 15% and 20% that showed leaf extracted from Ficus deltoidea could fully inhibit the mycelial growth of the pathogens started from 10% concentration till 20% concentrations. Thus, this has been demonstrated that even at lower concentrations, the leaf extract of Ficus deltoidea could control all the pathogenic fungi tested. 

Further investigations are needed to isolate and distinguish the bioactive compounds of F.deltoidea that has potential to be used as bio fungicide.


Discussion



The first objective of this study, which was to extract the antimicrobial compounds from Ficus deltoidea was achieved. Bioactive compounds were effectively obtained from plant extraction and this was depended on the type of solvent used in the extraction procedure. To choose which type of solvent to use to get a good extraction, the solvent must be easy to evaporate at low toxicity, easy to evaporate at low heat, have preservative action and the inability to cause the extract to complex or dissociate (Tiwari et al, 2011).

In this experiment, methanol was used as the solvent for the extraction process because it has characteristic of all good solvent. Since all identified compounds from plant against microorganisms are aromatic or saturated organic compounds, they are most easily obtained through methanol extraction
(Cowan, 1999) 

Then the second objective was to determine the effect of F.deltoidea leaf extract on mycelial growth of selected plant pathogenic fungi. The extract from F.deltoidea leaves showed antifungal activity against the two tested fungal pathogens. The leaf extract inhibited the mycelial growth of Ganoderma boninense at 55.9% compared to Rhizoctonia solani inhibited at 53.67% at 5% concentration. Statistical analysis of percent of inhibition of Ganoderma boninense and Rhizoctonia solani showed no significant difference. 

Different concentrations of F.deltoidea leaf extract used in this experiment also influenced the mycelial growth of the pathogens. During the tests, the leaf extract of F.deltoidea was observed to inhibit the mycelial growth for both fungal pathogens at the minimum concentration of 5%. It shows that even at 5% concentration of F.deltoidea leaf extract could inhibit the mycelial growth of both fungal pathogens even though the percentage of inhibition is different. Besides, the other reasons are related to the types of secondary metabolites existing and the composition of these secondary metabolites differs in plants from species to species. However, the other concentrations for both fungal pathogens showed a good percentage of inhibition. 

Most of the studies of the Ficus species revealed the presence of phenolic compounds as major components from different parts (leaves, stem wood, branches, stem bark, roots, root bark, fruits, and seeds)
(Abdel-Hameed, 2009). 

Besides, the other reasons are related to the types of secondary metabolites existing and the composition of these secondary metabolites differs in plants from species to species. The methanol leaf extract of F.deltoidea exhibited good antibacterial and antifungal activities against the test organisms.
(Salem et.al., 2013).

The presence of alkaloids, flavonoids and cardiac glycosides in the leaves of these Ficus species may have conferred the antimicrobial properties on these species. (Salem et.al., 2013). 

Thus additional study is recommended to classify the chemical constituents in F.deltoidea leaf extract that inhibit the growth of fungi and to study concentrations of leaf extract below 10% as this study showed that 10% concentration of the leaf extract could fully inhibit the two tested plant pathogenic fungi. 

In addition, more study should be done to test more on plant pathogenic fungi. Moreover, other parts of F.deltoidea plant could be tested to determine their effectiveness on plant pathogenic fungi.

Result

Leaf extraction from Ficus deltoidea

About one kilogram fresh plant materials was used in this experiment and about 50 grams of leaf powder were obtained. Then after extraction, about five grams of dried extract were obtained with a modification of the procedures where the extraction was done by methanol.

The effect of Ficus deltoidea extract on Ganoderma boninense 

From the result, plates containing F. deltoidea extract showed antifungal activities against G. boninense. The mycelial growth of G. boninense was inhibited 55.9% at 5% concentration and 100% inhibited at 10,15 and 20% concentrations.

Percentage inhibition of diameter growth (PIDG) of F.deltoidea extract against G. boninense at 7 days after incubation. Data represents results of 3 replicates. Values are the means of 3 replicates. Means with the same letter are not significantly different at P = 0.05. 

The effect of Ficus deltoidea extract on Rhizoctonia solani 

From the result plates containing F.deltoidea extract showed antifungal activities against R.solani. The mycelial growth of R.solani was inhibited 53.67% at 5% concentration and 100% inhibited at 10,15 and 20° 0 concentrations.

Percentage inhibition of diameter growth (PIDG) of F.deltoidea extract against R.solani at 5 days after incubation. Data represents results of 3 replicates. Values are the means of 3 replicates. Means with the same letter are not significantly different at P = 0.05. 

The effect of Ficus deltoidea extract based on concentration

 From the result, plates containing F.deltoidea extract showed slightly higher inhibition on G. boninense compared to R.solani. However, for 10, 15 and 20% concentrations, the treatments inhibited 100% on both pathogens. The percentage of inhibition of G. boninense was 55,9% with R solani 53.67% at 5% concentration.

Therefore, this experiment showed that 10, 15 and 20% concentrations of F.deltoidea leaf extract could effectively stop the mycelial growth of both pathogens. Thus, 10% concentration of F. deltoidea leaf extract is recommended to be used for inhibition 
both fungal pathogens.

Comparison of percentage inhibition of diameter growth (PIDG) of Ficus deltoidea extract against Ganoderma boninense and Rhizoctonia solani based on concentration G, Ganoderma boninense; R, Rhizoctonia solani .Values are the means of 3 replicates. Means witfh the same letter are not significantly ditferent at P = 0.05


Materials and methods



Plant material
This study was conducted in the laboratory of the  Department of  Plant Protection, Faculty of  Agriculture, Universiti Putra Malaysia.
The leaves of Ficus deltoidea were collected from Taman Herba Unit Taman Pertanian Universiti, Universiti Putra Malaysia. About one kilogram fresh plant materials were washed under running tap water, oven dry for four days and finally stored until further use 

Microorganisms 

The fungal stock cultures were obtained from the Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia. Two fungal species were used in this experiment which was Ganoderma boninense, and Rhizoctonia solani. R. solani was maintained on Potato Dextrose Agar (PDA) while G.boninense was cultured on Malt Extract Agar (MEA) and stored in the culture chamber under laboratory conditions.

Preparation of plant extract 

The leaves of F. deltoidea were thoroughly washed, oven dried and crushed by using a commercial blender
(Retsch Model SK 100).

Leaf powder of 50g was soaked in 300 ml of methanol in a 500ml conical flask and loaded onto an orbital shaker at a speed of 120 rpm for 24 h. The mixture was filtered using Whatman No-l filter
paper and the filtrate concentrated using rotary evaporator
(Buchi Model R215W).

Dried extract was collected in a conical flask covered with aluminiums foil, sealed with parafilm and stored at 4°C. 

Screening of antifungal activity

By using poison agar technique that involved the cultivation of pathogen plugs on a culture medium containing F. deltoidea leaf extract, and then the percent inhibition of diameter growth (PIDG) was determined by measuring the diameter of mycelial growth daily until a week for G.boninense and five days for R.solani, F.deltoidea leaf extract with different concentrations as treatments were incorporated and mixed well with respective culture medium and poured into culture plates. 

In this experiment four different concentrations of leaf extract were used which were 5%, 10%, 15% and 20% and compared with control (0%). Stock solution was prepared by diluting the extract with methanol. The obtained crude extract was added to 100 ml methanol and left it for 24 h in the laminar air flow. To make up other concentrations, for example, 5%, 1 m1 of stock solution was taken and added into a Petri dish and was added with 19 ml agar solution.

For each treatment, three replicates were done. Agar plugs of G. boninense and R. solani were placed at the center of Petri dishes containing MEA and PDA respectively, with extracts at the defined concentrations.

The formula used to calculate the percent of inhibition was:

{[diameter of mycelial growth of fungal pathogen in control plate- Diameter of mycelial growth of fungal pathogen in treatment plate]  /Diameter of mycelial growth of funga1 pathogen in control plate} x 100

Experimental design and statistical analysis

Recorded data was analyzed with SAS Software (SAS Institute, North Carolina, State University, Version 9.1, 2004). The effects and the interactions between treatments and phatogens were tested by ANOVA using CRD (Completely Randomized Design) where there were five treatments with three replications for each treatment and the means of significant results were compared using Least Significance Difference (LSD) at 5% probability level.

Literature Review

Control Methods of Plant Disease 

Chemical Control 

Chemical control is one of the methods that are broadly used because of favorable and quicker effect. Chemicals used to protect plant diseases that caused by fungal pathogens are known as fungicides. Fungicides kill the pathogens by injuring cell membranes, disabling critical enzymes or proteins and interfering with key metabolic process (Mueller, 2006). Contact fungicides do not go deeper and just remain on the surface where it is applied. This type of fungicide need frequent application to protect new growth and to substitute material that has been washed up by irrigation or rain (Mueller, 2006). Systemic fungicides immersed into plant tissues and move throughout the tissues. Every fungicide has a part in protection such as when a fungicide is present on the plant as a protective barrier before the pathogen arrives or begins to develop so that it prevents infection from occurring. Eradication is the ability to stop disease development after symptoms had developed. Very few fungicides have this capability, and growers must not rely on this as a mean of disease control (Mueller, 2006). Then, growers must know the specific mode of action each fungicide has, so that they know how the fungicide works and apply the fungicide in a correct way in order to fight the disease from spreading.

Biological Control

Biological control is the lessening of pest populations by using natural enemies, to help reduce its impact. By using biological control instead of chemical control, this can avoid the pathogens from building resistance towards pesticides (Linker et.at.,2009). Most of the farmers do not like to use biological control because of expensive cost and less knowledge about the application of biological control agents. Other important steps before applying biological control agents, it must be tested first to make sure they will not be pest themselves and will not post a danger to non-target species. Example of well-known bio control agents is Bacillus thuringiensis that are used as bio pesticide. This bacterium produces delta-endotoxin that affects a variety of species from the orders Coleoptera (beetles), Lepidoptera (moths and butterflies), and Diptera (flies and mosquitoes) and this Bt’s toxin are very specific to certain harmful insects. (Neppl,2000)

Cultural Control

Cultural control is one of the methods to suppress pest population. The strategies of cultural controls include the modifying the relationships between a pest population and its natural environment. In easy way, it is about interrupting an insect’s life cycle. The common cultural controls would be tillage, strategic planting dates, or crop rotation. All of these practices that in some way interrupt an insect pest's life cycle (Storey,2014).

Genetic resistance

One of the pest control methods is by using genetic resistance host plant. There are two types of resistance which are vertical and horizontal resistance. Vertical resistance is when a plant variety exhibits a high degree of resistance to a single race or strain of a pathogen (Saldana.2011). Vertical resistance is also known as specific, qualitative, monogenic, and non-durable and this type of resistance hardly exhibits symptoms (Saldana, 2011). It results in the death of the infected cells, restricting the establishment of the pathogen (Saldana, 2011). Horizontal resistance, on the other hand, protects the plant against several strains of a pathogen, although the protection is not complete, it involves more resistance genes than the vertical resistance and this type of horizontal resistance, in contrast, is equivalent to non-specific, quantitative, multigenic, durable, and field resistance, sometimes infection and disease onset is possible, but this type of resistance protects from many virulent races of the pathogen. (Saldana, 2011). One of the widely used genetic resistance crops is Bacillus thuringiensis (Bt) corn that has been genetically modified through the addition of a small amount of genetic material from other organisms through molecular techniques. (Ric Bessin,2004) 

Physical control

Physical control refers to methods that physically keep the pest from reaching their host. There are several methods of physical control one that are widely used is traps, the methods is by using yellow sticky traps. They are covered in a substance that attracts insects, but are actually very sticky or poisonous. These traps are commonly used for fruit flies or leafhoppers. (Meyer, 2003). 

Temperature control is one of the physical control, the methods is by placing produce inside of cold storage containers lengthens how long the produce lasts while also hindering the growth of insects inside of them. Another method to use is to heat, as it will kill the insect larvae in certain types of produce. An example would be with mangoes, where they are placed into a hot water bath in order to kill any eggs and larvae. (Meyer, 2003).

Another method called barriers is useful for keeping insects out of one‘s plants. This method known as row covers and they are made out of either plastic or polyester. They are made thin and light to allow plants to still absorb sunshine and water from the air. 
(Mahr et. al.,1993) 

Regulatory control

Regulatory pest management is to prevent the introduction or spread of pests through the application of various pest management techniques such as pest exclusion, detection, eradication, mitigation, and public education. (Jenkins et.aI.,2006).

When developing a regulatory control program, many factors should be considered including population densities, reproduction and mortality rates, age distributions, the pest’s vigor, the potential for growth and spread of the pest given the new environmental conditions. (Jenkins et.al.,2006).

Regulatory pest management programs use several different strategies which may include identification of risk prevention of entry, survey and detection, eradication, retardation of spread, mitigation of losses. 
(Jenkins et.al.,2006).

Ficus deltoidea

Taxonomic Classification

Commonly known as Mas cotek. Ficus deltoidea is the kingdom of plant, a division of Magnoliophyta, class of Magnoliopsida, order of Rosales, family of Moraceae, genus of Ficus and species of deltoidea

Description

Is an evergreen shrub, F.deltoidea can reach a height of two meters, with whitish grey bark. Leaves of F.deltoidea are broadly spoon-shaped to obovate. Above the leaves has bright green colored whereas beneath the leaves has rust-red to olive brown color.



'Mas cotek'


The average leaf length is between 4 to 8 cm. Mas cotek produces figs that are spherical to round, with a width of 1.0 cm to 1.5 cm.(Figure 2.2.2) (Bunawan et.al.,2014)

Anti-fungal and Antibacterial Activity of Ficus deltoidea

Ficus species is a rich source of polyphenolic compounds, flavonoids which are responsible for strong antioxidant properties. (Sirisha et. al.,2010).

An antibacterial compound known as lupeol (C30H500) was also isolated from the leaves of F. deltoidea and exhibited toxicity against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli (Bunawan et.al.,2014).

Two bioactive constituents known as vitexin and isovitexin have been isolated, identified, and evaluated to show alpha-glucosidase inhibition. (Bunawan et.al.,2014).

Qualitative Phytochemical Studies of Ficus deltoidea

Studies showed Ficus deltoidea consists high phenolic acid, pholiphenol, tannin, saponin and flavonoids.(Amiera et.al., 2014). Phenolics play a variety of important roles in the plant. Majority of phenolic substances have important effects on defense against herbivores and pathogens. Many simple phenolic compounds have important roles in plants as defenses against insects, herbivores and fungi. (Ozeker, 1999)

Rhizoctonia solani

Rhizoctonia solani forms colonies on potato dextrose agar (FDA) that range in colour from buff to black and do not produce asexual spores but grow by producing thin, vegetative strands called hyphae. This fungal pathogen will cause sheath blight disease in host like paddy (Naipictuasdharwad,2009) 

The symptoms can be seen when the hosts were infected by this fungal pathogen and appear as one or more relatively large, oblong or irregularly elongated lesions on the leaf sheath in advanced stages while the center of the lesion becomes bleached with an irregular purple brown border. Initially these lesions are white in colour, later they turn to dark brown at severe condition (drying of leaves). (Naipictuasdharwad,2009)

Symptoms shows by paddy leaves. Grey spots are formed, as the spots enlarge the centre becomes grayish white. Lesion on the upper parts of plants extend rapidly coalescing with each other to cover entire tillers from the water line to the flag leaf.

Check it out!

Let's watch the video as a kickstart: Presenting: Introduction to 'Mas Cotek'