Antimicrobial Plant Products for Management of Phytopathogens

Scientific Study, 2012

101 Pages




List of tables

List of figures


2.Literature Survey


4.Results and Discussion

5.Final Comments




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List of Tables

Table 2.1: Class and subclass of natural antimicrobial compounds Table 3.1: Organisms selected from MTCC

Table 3.2: heating and cooling cycles for different solvents during MAE

Table 3.3: Estimation of aflatoxin

Table 4.1: Extraction efficiency and reconstitution efficiency

Table 4.2: Percent inhibition at various concentrations of (EtOH) extract of

A. squamosa seeds

Table 4.3: Percent inhibition at various concentrations of acetone extract of

A. squamosa seeds

Table 4.4: Percent inhibition at various concentrations of acetone extract of

M. zapota seeds

Table 4.5: Percent inhibition at various concentrations of methanol extract of

M. zapota seed

Table 4.6: Percent inhibitions at various concentrations of ethanol extract of

P. sylvestris seeds

Table 4.7 (a): Percent inhibition at various concentrations of methanol extract of

T. indica seeds

Table 4.7 (b): Percent inhibition at various concentrations of methanol extract of

T. indica seeds

Table 4.8: Percent inhibitions at various concentrations of (EtOH) extract of

S. cumini seeds

Table 4.9: Percent inhibition at various concentrations of methanol extract of

S. cumini seeds

Table 4.10: Relation between extraction efficiency and average total activity

Table 4.11: Results of MIC assays of various seed extracts against different


Table 4.12: Results of MBC assays and total activity of various seed extracts

against different

Table 4.13: Time require to kill

Table 4.14: Percent inhibition at various concentrations of curcumin

Table 4.15: Percent inhibition at various concentrations of quercetin

Table 4.16: Percent inhibition at various concentrations of lycopene

Table 4.17: Percent inhibition at various concentrations of gallic acid

Table 4.18: MIC and MBC of pure compounds

Table 4.19 (a): Percent inhibition at various concentrations of antibiotics by

Broth dilution assay

Table 4.19 (b): Disc diffusion assay of antibiotics against phytopathogens

Table 4.20: Percent Inhibition at various concentrations of different extracts

against Aspergillus parasiticus

Table 4.21: Percent inhibition at various concentrations of pure compounds against

Aspergillus parasiticus

Table 4.22: Disc diffusion assay of antifungal against Aaspergillus parasiticus

Table 4.23: Effect of extracts on mycelia growth and aflatoxin production by

A. parasiticus

Table 4.24: Results of synergistic effect

Table 4.25: Results of separation of extracts of T. indica seeds by TLC

(CHCl3: Acetone)

Table 4.26: Disc diffusion assay for fractions separated on TLC plate

Table 6.1: Values of ε and molecular weight of aflatoxin

Table 6.2: Results of effect of plant extracts on mycelial weight and aflatoxin

production by A. flavus and A. parasiticus

List of figures

Fig 1: Correlation between extraction efficiency and average total activity

Fig 2: Potency of extracts on mycelia growth of A. parasiticus

Fig 3: Potency of extracts on aflatoxin production by A. parasiticus

Fig 4: MBC results of methanol extract T. indica seeds against A. tumefaceins

Fig 5: MBC results of methanol extract T. indica seeds against P. syringae

Fig 6: MBC results of curcumin against X. campestris

Fig 7: Time required to kill A. tumefaciens at concentration 625 µg/ml of

T. indica (MeOH) extract

Fig 8: Time required to kill P. syringae at concentration 400 µg/ml of

T. indica (MeOH) extract

Fig 9: Time required to kill X. campestris at concentration 30 µg/ml of curcumin

Fig 10: TLC of T.indica (MeOH) extract in CHCl3: acetone (90:10)

Fig 11: Disc diffusion assay of isolated fractions from TLC of T. indica (MeOH)

extract against P. caratovorum

Fig 12: Study of disease caused by X. campestris on host cabbage (leaf)

Fig 13: Antifungal testing by disc diffusion assay

Fig 14: Decrease in aflatoxin production by M. zapota (acetone) seed extract at

500 μg/mL (365nm)

1 Preamble

“Eat leeks in March and wild garlic in May, and all the year after the physicians may play.”

-Traditional Welsh rhyme

A large proportion of crops are lost due to plant pathogens each year, there is currently much interest in developing strategies to control plant pests. Major crop losses occur due to diseases and insects demanding serious attention towards food protection. Xanthomonas campestris causes 30% loss in rice production in Southeast Asia [Beattie, 2006]. International trade of fruits and vegetables has grown greatly in the past 20 years and is presently a multi-billion dollar business [Golan and Paster, 2008]. Callasobruchus chinensis L is devastating pests of various storage pulses throughout the world, causes 32.64% of damages to stored pulses as compared with vegetables and oil seeds (3%) [Kumar et al., 2011]. Fungal contamination and subsequent production of aflatoxin can occur in crops in the field, during harvest, postharvest operations and in storage. Food and Agricultural Organization (FAO) estimated 25% loss of world food crops, affected by mycotoxins [Dubey et al., 2011]. Fungal deterioration of storage seeds and grains is a major problem in the Indian storage system due to hot humid climate. Aspergillus sp. is most common fungal species that can produce mycotoxins in food and feed stuffs. Among all mycotoxins, particularly aflatoxin B1 (AFB1) is the most toxic form for mammals and cause damage as toxic hepatitis, hemorrhage, immunosuppression, hepatic carcinoma, etc [Reddy et al., 2009]. AFB1 has been classified as a class II human carcinogen by the International Agency for Research on Cancer [IARC, 1993].

In past few decades, a worldwide increase in reports of multi-drug resistant microbial strains, increased usage of chemical pesticides to control plant infections, has added new aspects to be focused. “India ranks fourth in Asia and tenth in the world in plant diversity” said by N.N. Singh (vice chancellor, Birsa Agricultural University) []. The usage of synthetic compounds to control pests has caused several problems such as, contamination of soil and ground water, toxicity towards non target species including humans, disturbance of ecosystem, etc. Biochemical pesticides include plant extract, pheromones, plant hormones, natural plant derived regulators, enzymes, etc. [Chunxue et al., 2010].

The research on natural products and compounds derived from natural products has accelerated in recent years due to their importance in drug discovery. Natural products from plant source may be used directly or considered as a precursor for developing better molecules. From centuries, the use of phytochemicals in food preservation and improvement of qualities of certain traditional foods has been in practice. Isolation of several phytochemicals is successfully achieved with advance in separation technology. Different compounds isolated from plants such as dimethyl pyrrole, hydroxydihydrocornin-aglycones, indole derivatives, etc., are reported to have antifungal activities [Arif et al., 2009]. Oil as plant component has been used for post harvest protection of crop [Davidson and Naidu, 2000]. The isoquinoline alkaloid emetine obtained from Cephaelis ipecacuanha and related species, has been used for many years as amoebicidal drug and also for the treatment of abscesses due to the spread of Escherichia histolytica infections [Ciocan and Bara, 2007].

Therefore, alternative disease management using natural compounds and other resistance types needs to be considered to inhibit the growth of plant pathogens.


1.To screen certain plant extracts/pure phytochemicals for their activity against various plant pathogenic microbes.
2.To determine Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC), and Minimum fungicidal concentration (MFC) of potential extracts against susceptible microbes.
3.To study the effects of plants extracts on aflatoxin production by Aspergillus parasiticus.

2. Literature Survey

2.1. Synthetic pesticides and its challenges

The usage of synthetic pesticides has increased for the control of plant disease due to its effectiveness in controlling phytopathogens. The unrestrained use of these chemicals, under the adage, “if little is effective, a lot more will be powerful” has played ravage with human and other life forms, environment, etc. Due to non-biodegradable nature of chemical pesticides they get accumulated at each trophic level of food chain. Since humans occupy the top level in any food chain, so the maximum amount of harmful chemical pesticides gets accumulated in our bodies. Thus accumulation of such chemicals in living bodies at each trophic level of food chain is called biomagnifications. [].

2.1.1 Effect of pesticides

The credits of pesticides include enhanced economic potential in terms of increased production of food & fibre, amelioration of vector-borne diseases and then their debits have resulted in serious health implications to man & his environment [Aktar et al., 2009]. The World Health Organization (WHO) estimated that 200,000 people are killed worldwide, every year due to pesticide poisoning [Dubey et al., 2011]. During pesticide spraying, it can enter to body through skin contact and inhalation of aerosols. Among side effects of pesticide are hormonal disruption, cancer, neurotoxicity, birth defects, etc.

2.1.2 Risk associated to pesticides

Acephate, dithiocarbamates, DDT, endosulfan, thiabendazole, triazophos, methidathion, etc are common pesticides used to spray on agricultural field. Over 100 people died after consuming wheat flour contaminated with parathion (herbicide), this was first report of poisoning due to pesticide in India (1958). Herbicides are formulated to kill particular plant but few herbicides get volatilized off treated plants and cause sub-lethal effects on non-target plants like phenoxy herbicides,2,4-D, glyphosate can severely reduce seed quality [Aktar et al., 2009]. Leakage of pesticides from soil to water leads to contamination in rivers, lakes, aquatic vegetation, etc. Also over spraying of pesticides on plants causes severe reduction of normal flora of soil and cause impact on fertility of soil. Majority population affected includes agricultural farm workers, formulators, workers, sprayers, etc.

2.1.3. Advantage of plant products over chemical pesticides

The plant product used as pesticide is referring to biochemical pesticide. They are generally less toxic to the user and to non-target species, making them desirable and sustainable tools for disease management. They are much cheaper than chemical pesticide & ecofreindly.

2.2. Major Phytochemicals

In late 1990s the use of plant products as therapeutic agent was in high popularity. In 1996 there was increase in sales of botanical medicines by 37% over 1995. Phytochemicals serve as a plant defense mechanism against infection by microorganisms, insects, etc [Cowan, 1999]. Different parts of plant contain active compounds like, roots of ginseng plants contain saponins, eucalyptus leaves has tannins [Sharma and Arora, 2006].

2.2.1. Phenols and Polyphenols

Majority of plants have ability to synthesize aromatic substances like phenols, oxygen-substituted derivatives. A phenolic compound has a single substituted phenolic ring, C3 side chain at a lower level of oxidation. Few common phenolic compounds are caffeic acid from tarragon and thyme, cinnamic acid from brassica oil seeds, coumarin from spices, sesamol from sesame oil, quinnones, tannins, flavonoids, etc [Davidson and Naidu, 2000]. The hydroxylated phenols, catechol and pyrogallol are toxic to microorganisms, as catechol has two OH- groups and pyrogallol has three [Sharma and Arora, 2006]. Amentoflavone from Selaginella tamariscina have antifungal activity with IC50 18.3 µg/mL [Arif et al., 2009]. Quercetin is the most abundant of the flavonoids found in lotus leaves as a component that may be a potential antibacterial agent [Mingyu and Zhuting, 2008].

2.2.2. Terpenoids

The fragrance of plants is carried in essential oil fraction. Terpenoids share origins with fatty acids, synthesized from acetate units [Cowan, 1999]. An antimicrobial diterpene 8 from Alpinia galanga synergistically enhanced the antifungal activity of quercetin and chalcone against Candida albicans [Arif et al., 2009]. These compounds are based on isoprene structure, they occur as diterpenes, triterpenes, and tetraterpenes (C20, C30, and C40) and sesquiterpenes (C15). The sesquiterpene isolated from dichloromethane extract from the roots of Vernonanthura tweedieana was effective against Trichophyton mentagrophytes. Clerodane diterpenes isolated from fruit pulp extract of Detarium microcarpum showed inhibition of growth of the plant pathogenic fungus Cladosporium cucumerinum [Abad et al., 2007].

2.2.3. Essential oils

The fragrance of plants is carried in essential oil fraction. Antifungal activity of essential oil isolated from Eucalyptus against phytopathogenic fungi Pythium ultimum, Rhizoctonia solani and Bipolaris sorokinian [Katooli et al., 2011]. Essential oil from oregano showed antibacterial activity against phytopathogens P. marginalis, P. syrinagae and Xanthomonas vesicatoria [Vasinauskiene et al., 2006]. The antifungal activities of the essential oil from Agastache rugosa and its main component, estragole, combined with ketoconazole, were reported to have significant synergistic effects [Arif et al., 2009].

2.2.4. Alkaloids

A natural compound isolated from medicinal plants. It is a heterocyclic nitrogen compounds. Morphine isolated from opium poppy Papaver somniferum in 1805, was first medically useful example of alkaloid. A quinolinone alkaloid from leaves of Melochia odorata, were reported to exhibit antifungal activities against a broad spectrum of pathogenic fungi [Arif et al., 2009]. Pyrrolizidine alkaloids from Heliotropium subulatum extracts showed antimicrobial activity against both fungal and bacterial species [Craig, 1998].

2.2.5. Saponins

These are glycosylated compounds. It is stored in plant cells as inactive forms but in presence of pathogen it gets converted in to biologically active antibiotics by enzymes. It is divided into three major groups, a triterpenoid, a steroid, or a steroidal glycoalkaloid [Arif et al., 2009]. Triterpenoid saponins are found primarily in dicotyledonous plants but also in some monocots, whereas steroid saponins occur mainly in monocots, steroidal glycoalkaloids are found primarily in members of the family Solanaceae, which includes potato and tomato, but also in the Liliaceae [Osbourn, 1996].

2.2.6. Peptides and proteins

In 1942, it was first reported that peptides can be inhibitory to microorganisms. The vulgarinin purified from the seeds of Phaseolus vulgaris L displayed antifungal property against few plant pathogenic fungi Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola and Botrytis cinerea [Abad et al., 2007].

Table 2.1: Class and subclass of natural antimicrobial compounds

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2.3. Plant Material

Medicinal herbs are significant source of synthetic and herbal drugs. Isolated active compounds are used for applied research. Herbs like turmeric, fenugreek, ginger, garlic, holy basil, etc are integral part of ayurvedic formulations [Ahmed, 2010]. Medicinal herbs are considered to be natural factory producing natural products having antimicrobial, antiviral activities, etc. Plants comprise several active components as described in section 2.2. Different parts of plant like leaves, roots, bark, fruit and seeds contain different active ingredients, few may be toxic, others may be harmless. For example fruit capsules of P. somniferum produce powerful drugs while seeds do not contain alkaloids [Wyk and Wink, 2004]. Phytochemicals have astronomical usage like therapeutic materials for humans, animals, plants ailments, treatment of challenging diseases like cancer, asthma, diabetes, fungal infections, biopesticides, preservatives, etc. Failure of chemical pesticides in controlling pest and increasing resistant strain of microorganisms has coerced to search novel source of natural compounds. In India and Africa the development of biopesticides is specially advocated to develop their own natural resources in crop protection [Agrawal and Pandey, 2011]. Along with research on emerging natural plant products, phytochemicals industry is also growing with tremendous pace. Antibiotics may be more useful than synthetic chemicals in the control of plant diseases due to following reasons: applied selectively in low concentrations, easily broken down by soil microorganisms, etc. But application of antibiotics on fields in uncontrolled manner might develop resistance in organisms. Different antibiotics used to control plant pests are blasticidin, mildomycn, polyoxin, prumycin, cycloheximide, kasugamycin, validamycin and tetranactin. Streptomycin is used to combat plant disease caused by Pseudomonas sp. and Xanthomonas oryzae [Crueger and Crueger, 1989].

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The information regarding selected seeds is given below.

2.3.1. Tamarindus indica

Family: Leguminosae

Common name: Imli

The plant is extensively grown in all over the Bangladesh, is widely used all over tropical Africa, Sudan, India, Pakistan for different purposes. Different parts of this plant are used in the indigenous systems of medicine for the treatment of a variety of human ailments. Ara and Islam, (2009), reported the presence of alkaloids, glycosides, flavonoids and saponins in ethanolic extract of T. indica seeds and its antibacterial activity against Shigella dysentriae and Staphylococcus aureus. Methanolic extract of tamarind seeds has high concentration of flavonoids, tannins, and steroids. Tamarind fruit pulp is used for seasoning, as a food component, to flavor confections, curries and sauces and is a main component in juices and certain beverages. The major industrial product of tamarind seed is the tamarind kernel powder (TKP) which is an important sizing material used in the textile, paper and jute industries. Tamarind seed kernels have a relatively high antioxidant activity and phenolic content [Caluwe et al., 2010].

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2.3.2 . Syzgium cumini

Family: Myrtaceae

Common name: Jamun

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Different parts of this plant, such as seeds, bark, fruit, and leaves are used to treat diabetes mellitus in many countries [Oliveira et al., 2007]. Fruit of S. cumini contains mallic acid, a small quantity of oxalic acid, gallic acid and tannins account for astringency of the fruit. Seeds contain flavonoid such as rutin, quercetin and 11ß-sitosterol. Stem of S. cumini tree contains betulinic acid, ß-sitosterol, friedelin, epi-friedelanol and eugenin. The plant possess antidiabetic, anti-inflammatory, antiallergic, gastroprotective, antiviral, antibacterial activity, etc [Jadhav et al., 2009]. Escherichia coli and Vibrio cholera were inhibited by methanol extract of S. cumini seeds at 1100 µg/mL and its ethanol extract at 2500 µg/mL. The HPLC and TLC of S. cumini (MeOH) seed extract confirmed the presence of quercetin and gallic acid [Kothari et al., 2011].

2.3.3. Phoenix sylvestris

Family: Arecaceae

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Common name: Dates

Phoenix sylvestris Roxb. is gregarious in many parts of India. It is an ornamental tree and can also be used as an avenue plant The fruit is cooling, oleaginous, cardiotonic, fattening, constipative, good in heart complaints, abdominal complaints, fevers, vomiting and loss of consciousness. The juice obtained from the tree is considered to be a cooling beverage. The roots are used to stop toothache. The fruit pounded and mixed with almonds, quince seeds, pistachio nuts and sugar, form a restorative remedy. The central tender part of

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the plant is used in gonorrhea [Parmar and Kaushal, 1982]. Ethanolic extract of P. sylvestris showed antibacterial activity against Salmonella paratyphii A and Staphylococcus epidermidis [Kothari, 2011].

2.3.4. Manilkara zapota

Family: Sapotaceae

Common name: Sapodilla, cheeku

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M. zapota is a species of the lowland rainforest. Trees grow well in a wide range of climatic conditions from wet tropics to dry cool subtropical areas. The seed kernel (50% of the whole seed) contains 1% saponin and 0.08% sapotinin. Immature sapodillas are rich in tannin (proanthocyanadins) and very astringent. Ripening eliminates the tannin except for a low level remaining in the skin. It is highly drought-resistant, can stand salt spray, and approaches the date palm in its tolerance of soil salinity. The antimicrobial potential of M. zapota has been reported against different pathogenic bacteria and fungi eg., Salmonella typhi, S. dysenteriae, Shigella sonnei, Shigella shiga, Aspergillus flavus, Fusarium spp , Aspergillus fumigatius, C. albicans, Vasianfactum sp. This plant has antioxidative property and its fruit is preventive against biliousness and attacks of fever where as seeds are diuretic [Osman et al., 2011]. Acetone extract of M. zapota seeds was found to have significant antibacterial activity against V. cholera and Pseudomonas oleovorans, this extract showed positive results for alkaloids, phenols and flavonoids tests [Kothari and Seshadri, 2010].

2.3.5. Annona squamosa

Family: Annonaceae

Common name: Custard apple

The fruit is juicy and creamy white, it may contain up to 40 black seeds. These seeds are poisonous. The peelings and pulp contain oil that is useful in flavouring, the bark and leaves contain annonaine, an alkaloid. In tropical America, a decoction of the leaves is used as a cold remedy and to clarify urine, root is used in treatment of dysentery []. The plant contains glycoside, alkaloids, saponins, flavonoids, tannins, phenolic compounds, phytosterols. The ethanolic extract of leaves and stem is reported to have anticancer activity, flavonoids


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Antimicrobial Plant Products for Management of Phytopathogens
Nirma University  (Institute of Science)
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Vijay Kothari (Author)Binjal Darji (Author)Megha Doshi (Author)Jaydeep Ratani (Author), 2012, Antimicrobial Plant Products for Management of Phytopathogens, Munich, GRIN Verlag,


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