Chapter 7 Antifungal activities -...

Chapter 7 Antifungal activities

Antifungal activities

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SECTION A: ANTIFUNGAL ACTIVITIES 7.1. INTRODUCTION A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds. These organisms are classified as a kingdom, Fungi, which is separated from plants, animals and bacteria. One major difference is that fungal cells have cell walls that contain chitin, unlike the cell walls of plants, which contain cellulose. These and other differences show that the fungi form a single group of related organisms, named the Eumycota (true fungi or Eumycetes), that share a common ancestor (a monophyletic group). The discipline of biology that devoted to the study of fungi is known as mycology, which is often regarded as a branch of botany, even though genetic studies have shown that fungi are more closely related to animals than to plants. Diseases caused or highly suspected of being caused by fungi in the body include: Skin diseases, for example Psoriasis, Eczema, etc., Postpartum Depression, Bladder Diseases, High Cholestorol, Kidney Stones, Vaginitis, Weight Gain, Arthritis, Weakened Immune Systems, Hormone Problems, Mental Fogginess/Dysfunction, Autoimmune Diseases, Diabetes, Cancer, Hair Loss, Allergies, Chronic Sinusitis, Depression, Digestion Problems, such as GERD, IBS and more Diseases of the Respiratory Tract, Heart Problems, Menstrual Cycle and Infertility (both male & female).

7.1.1. Aspergillus niger a) Scientific name: Aspergillus niger. b) Affected Plant Parts: Fruits, inflorescence, leaves, roots, seeds, stems, vegetative organs and whole plant. c) Major hosts: Allium cepa (onion), Allium sativum (garlic), Arachishy pogaea (groundnut), Citrus aurantiifolia (lime), Citrus medica (citron), Citrus reticulata (mandarin), Dioscorea (yam), Dioscorea esculenta (Asiatic yam), Glycine max (soyabean), Lycopersicon esculentum(tomato), Mangifera indica (mango). d) Impact: A. niger is most commonly observed on stored dry-bulb onions, especially when these have been produced in areas with hot, dry climates or grown under


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temperate conditions, but dried using warm air. Black mould has been reported as a major cause of rot of stored yellow onions in Texas, USA between 1974 and 1976, where it caused losses of 22-76% in onions stored for 3 months at 26.7°C and 70% RH [1]. In Japan, losses of 33% in 1979 and 61% in 1980 were recorded for onions stored over the summer. Black mould has been reported on 10% of the total dry onion shipments inspected in the New York market during 1972-84 [2]. The disease has also been reported as the major component of the mycoflora of stored bulbs in the Sudan, where it has been reported that up to 80% of stored bulbs of varieties Wad Ramli (red) and Dongla (white) may be affected with the fungus [3]. e) Plant disease: A. niger causes black mold of onions. Infection of onion seedlings by A. niger can become systemic, manifesting only when conditions are conducive. A. niger causes a common postharvest disease of onions, in which the black conidia can be observed between the scales of the bulb. The fungus also causes disease in peanuts and in grapes.

Figure 7.1. Vegetables contaminated with A.niger

f) Human and animal disease A. niger is less likely to cause human disease than some other Aspergillus species, but, if large amounts of spores are inhaled, a serious lung disease, aspergillosis can occur. Aspergillosis is, in particular, frequent among horticultural workers that inhale peat dust, which can be rich in Aspergillus spores. It has been found on the walls of ancient Egyptian tombs and can be inhaled when the area is disturbed. A. niger is one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss, and, in severe cases, damage to the ear canal and tympanic membrane.


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7.1.2. Aspergillus flavus Aspergillus flavus is a common mold in the environment and can cause storage problems in stored grains. It can also be a human pathogen, associated with aspergillosis of the lungs and sometimes causing corneal, otomycotic, and nasoorbital infections. Many strains produce significant quantities of aflatoxin, a carcinogenic and acutely toxic compound. A. flavus spores are allergenic. A. flavus sometimes causes losses in silkworm hatcheries. a) Scientific name: Aspergillus flavus b) Major hosts: Acacia nilotica (gum arabic tree), Arachis hypogaea (groundnut), Bertholletia excelsa (Brazil nut), Gossypium (cotton), Zea mays (m aize), Ziziphus mauritiana (jujube). c) Impact: A. flavus is a member of a group of fungi known as storage fungi which cause deterioration of grain or seeds of all plant species stored at seed moisture contents in the range 13-20%. It also produces aflatoxin in infected seeds that can cause death or other symptoms of toxicity when ingested by animals or humans. Aflatoxin contamination is of major economic importance in cotton, maize, groundnuts and tree nuts due to invasion of seeds in the field, but it may also develop in seeds and grains of other crop species when they are improperly stored. d) Disease in humans: A. flavus is the second most common agent of aspergillosis, the first being Aspergillus fumigatus. A. flavus may invade arteries of the lung or brain and cause infaction. Neutropenia predisposes to aspergillus infection. A. flavus also produces a toxin (aflatoxin) which is one of the aetiological agents for hepatocellular carcinoma. e) Mold damage: A. flavus is particularly common on corn and peanuts, as well as water damagedcarpets, and is one of several species of mold known to produce aflatoxin which can cause acute hepatitis, immunosuppression, and hepatocellular carcinoma. The absence of any regulation of screening for the fungus in countries which also have a high prevalence of viral hepatitis highly increases the risk of hepatocellular carcinoma.


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(A) (B) Figure 7.2. (A) A young maize kernel infected with A.flavus. (B) Yellow-green

powdery growth of A. flavus on a corn rootworm damaged ear. 7.1.3. Botrytis cinerea a) Scientific name: Botryotinia fuckeliana b) Affected Plant Stages: Flowering stage, fruiting stage, post-harvest, pre-emergence, seedling stage and vegetative growing stage. c) Affected Plant Parts: Leaves and stems. d) Major host plants: Allium cepa (onion), Brassicaceae (cruciferous crops), Chrysanthemum morifolium (chrysanthemum (florists), Cucumissativus (cucumber), Fragaria ananassa (strawberry), Helianthus annuus (sunflower), Lactuca sativa (lettuce), Lycopers iconesculentum(tomato), Malusdomestica(apple), Phaseolus(beans), Pinuspinaster (maritime pine), Pisum sativum(pea), Prunuss alicina (Japanese plum), Pyruscommunis (European pear). e) Impact: B. fuckeliana infects a very wide range of plants including field-grown crops such as grapes and greenhouse-grown vegetables, flowers and fruits. It causes yield losses in the field and during postharvest storage and transport. It is difficult to assess the damage caused by B. fuckeliana. Economic losses of greater than 50% may occur in many crops, depending on the prevailing environmental conditions.


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Figure 7.3. Botrytis cinerea on fruits f) Human disease: Botrytis cinerea mold on grapes may cause “winegrower’s lung”, a rare form of hypersensitivity pneumonitis (a respiratory allergic reaction in predisposed individuals). 7.1.4. Macrophomina phaseolina a) Preferred scientific name: Macrophomina phaseolina (Tassi) Goid b) Affected Plant Stages: Flowering stage, fruiting stage, post-harvest, pre-emergence, seedling stage and vegetative growing stage. c) Affected Plant Parts: Leaves, roots, seeds, stems and whole plant. d) Impact: Charcoal rot, caused by M. phaseolina, is economically important across a broad range of crops throughout the world, particularly in regions that experience hot, dry conditions during the growing time. Annual losses in soyabean were estimated at 5% throughout Missouri, USA, with some growers experiencing 30-50% loss. One report suggested that charcoal rot was responsible for greater losses in soyabean than any other disease from central Mississippi and Alabama to central Illinois and Indiana. The pathogen M. phaseolena generally affects the fibrovascular system of the roots and basal internodes, impends the transport of nutrients and water to the upper parts of the plant. Progressive wilting, premature dying, loss of vigor, and reduced yield are characteristic features of M. phaseolena infection. The pathogen is also responsible for seedling blight, damping off, root rot, basal stem rot and early maturing of sunflower crop. But the characteristic symptoms that appear after flowering are grey black discoloration and shredding of plant tissue at the stem and top of the taproot with


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getting hallowing of the stem. When the epidermis is removed, minute blackmicrosclerotia may be so numerous as to give a grayish black look to the tissues. 7.1.5. Phoma glomerata a) Preferred scientific name: Phoma glomerata b) Affected Plant Stages: Seedling stage and vegetative growing stage. c) Affected Plant Parts: Leaves, roots, vegetative organs and whole plant. d) Major host plants: Abelmoschus esculentus (okra), Chenopodium album (fat hen), Citrus limon (lemon), Cucurbita (pumpkin), Cyclamen persicum (cyclamens), Glycine max (soyabean), Gossypium (cotton), Lablab purpureus (hyacinth bean), Linumusitatissimum (flax), Monstera deliciosa (ceriman), Phaseolus vulgaris (common bean), Phyllanthus emblica (Indian gooseberry), Sechiumedule (chayote), Solanumme longena (aubergine), Solanum tuberosum (potato), Viciafaba (broad bean), Vignamungo (black gram), Vigna radiata (mung bean), Vigna unguiculata (cowpea), Zingiber officinale (ginger). Phoma glomerata is the second most common potato lenticels invader. This species may be pathogenic for man. It causes granuloma of the foot, hand mycosis and domycosis. It is implicated in allergic rhinitis. Chromatographic analyses have shown that it releases trichodermine as well as mycotoxins such as: aflatoxins B1 and B2, kojic acid. e) Pathologies: i) allergies ii) mycosis iii) rhinitis. 7.1.6. Fusarium semitectum F. semitectum is a widespread and common species in the tropic, subtropic and Mediterranean regions and regularly associated with a complex of plant diseases [4]. This cosmopolitan species was included in section Arthrosporiella that was proposed by Wollenweber and Reinking (1935) [5] and has no known sexual stage [6]. a) Pathogenicity and Clinical Significance: As well as being common plant pathogens, Fusarium species are causative agents of superficial and systemic infections in humans. Infections due to Fusarium spp. are collectively referred to as fusariosis. Trauma is the major predisposing factor for development of cutaneous infections due to


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Fusarium strains. Fusarium infections following solid organ transplantation tend to remain local and have a better outcome compared to those that develop in patients with hematological malignancies and bone marrow transplantation patients. Keratitis, endophthalmitis, otitis media, onychomycosis, cutaneous infections particularly of burn wounds, mycetoma, sinusitis, pulmonary infections, endocarditis, peritonitis, central venous catheter infections, septic arthritis, disseminated infections, and fungemia due to Fusarium spp. have been reported.

7.2. REVIEW OF LITERATURE Chohan et al. [7] synthesized and characterized a series of new antibacterial and antifungal Schiff's bases derived from sulphonamides, as well as their transition metal complexes incorporating Co(II), Cu(II), Ni(II) and Zn(II) and also screened for their in-vitro antibacterial activity against six Gram-negative (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Shigella dysentriae) and four Gram-positive (Bacillus cereus, Corynebacterium diphtheriae, Staphylococcus aureous and Streptococcus pyogenes) bacterial strains and for in-vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporumcanis, Fusariums olani and Candida glaberata. The results of these studies show the metal complexes to be more antibacterial and antifungal as compared to the uncomplexed Schiff’s bases. The brine shrimp bioassay was also carried out to study the in-vitro cytotoxic properties of these synthesized ligands and their complexes. Dharmaraj [8] had carried out the reactions of ruthenium(II) complexes, [RuHCl(CO)(PPh3)2(B)] [B=PPh3, pyridine (py) or piperidine (pip)], with bidentate Schiff base ligands derived by condensing salicylaldehyde with aniline, o-, m- or p-toluidine have been carried out. The products were characterized by analytical, IR, electronic, 1H NMR. and 31P-NMR spectral studies and are formulated as [RuCl(CO)(L)(PPh3)(B)] (L=Schiff base anion; B=PPh3, py or pip). An octahedral structure has been tentatively proposed for the new complexes. The Schiff bases and the


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new complexes were tested in vitro to evaluate their activity against the fungus Aspergillus flavus. The synthesis of the Schiff base ligands, 4-[(4-bromo-phenylimino)-methyl]-benzene-1,2,3-triol (A1), 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,2,3-triol (A2), 3-(p-tolylimino-methyl)-benzene-1,2-diol (A3), 3-[(4-bromo-phenylimino)-methyl]-benzene-1,2-diol (A4) and 4-[(3,5-di-tert-butyl-4-hydroxy-phenylimino)-methyl]-benzene-1,3-diol (A5), and their Cd(II) and Cu(II) metal complexes with there stability constants and potentiometric studies was done by Golcu et al. [9]. The structure of the ligands and their complexes was investigated using elemental analysis, FT-IR, UV–Vis, 1H and 13C NMR, mass spectra, magnetic susceptibility and conductance measurements. In the complexes, all the ligands behave as bidentate ligands, the oxygen in the ortho position and azomethine nitrogen atoms of the ligands coordinate to the metal ions. The keto-enoltautomeric forms of the Schiff base ligands A1–A5 have been investigated in polar and non-polar organic solvents. Antimicrobial activity of the ligands and metal complexes were tested using the disc diffusion method and the strains Bacillus megaterium and Candida tropicalis. Protonation constants of the triol and diol Schiff bases and stability constants of their Cu2+ and Cd2+ complexes were determined by potentiometric titration method in 50% DMSO-water media at 25.00 ± 0.02°C under nitrogen atmosphere and ionic strength of 0.1 M sodium perchlorate. It has been observed that all the Schiff base ligands titrated here have two protonation constants. The variation of protonation constant of these compounds was interpreted on the basis of structural effects associated with the substituents. The divalent metal ions of Cu(II) and Cd(II) form stable 1:2 complexes with Schiff bases.The Schiff base complexes of cadmium inhibit the intense chemiluminescence reaction in DMSO solution between luminol and dioxygen in the presence of a strong base. This effect is significantly correlated with the stability constants KCdL of the complexes and the protonation constants KOH of the ligands; it also has a nonsignificant association with antibacterial activity.


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Two new Schiff bases, N-4-hydroxysalicylidene-glycylglycine (K'GGRS'H20), N-O-vanillal-glycylglyeine (K.GGVS.3H20 ) and their Mn(II), Co(II), Ni(II) and Cu(II) complexes have been synthesized and characterized by Zishenet al. [10]with the help of elemental analysis, TGA, molar conductance, IR and UV spectral studies. The 13C NMR spectrum of one of the Schiff base ligands has been recorded. The results showed that the ligand is coordinated to the central metal ion via amide nitrogen, imino nitrogen, phenolic oxygen and carboxyl oxygen to form a Quadridentate complex. Some of the complexes exhibit strong inhibitory action towards Candida albicans and Cryptococcus neoformans. A series of metal complexes of Co(II), Ni(II) and Cu(II) have been synthesized by Bagihalli et al. [11] with newly synthesized biologically active 1,2,4-triazole Schiff bases derived from the condensation of 3-substituted-4-amino-5-mercapto-1,2,4-triazole and 8-formyl-7-hydroxy-4-methylcoumarin,which have been characterized by elemental analyses, spectroscopic measurements (IR, UV-vis, fluorescence, ESR), magnetic measurements and thermal studies. Electrochemical study of the complexes is also reported. All the complexes are soluble to limited extent in common organic solvents but soluble to larger extent in DMF and DMSO and are non-electrolytes in DMF and DMSO. All these Schiff bases and their complexes have also been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Salmonella typhi) and antifungal activities (Aspergillus niger, Aspergillus flavus and Cladosporium) by MIC method. The brine shrimp bioassay was also carried out to study their in vitro cytotoxic properties. Neutral tetradentate chelate complexes of Cu(II), Ni(II), Co(II), Mn(II), Zn(II) and VO(II) have been prepared by Raman et al. [12] in EtOH using Schiff bases derived from acetoacetanilido-4-aminoantipyrine and 2-aminophenol/2-aminothiophenol. Micro analytical data, magnetic susceptibility, IR., UV-Vis, 1H-NMR and ESR spectral techniques were used to confirm the structures of the chelate. Electronic absorption and IR spectra of the complexes suggest a square-planar geometry around the central metal ion, except for VO(II) and Mn(II) complexes which have square-pyramidal and octahedral geometry respectively. The cyclic voltammetric data for the Cu(II) complexes in MeCN show two waves for copper(II) → copper(III) and copper(II) →


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copper(I) couples, whereas the VO(II)complexes in MeCN show two waves for vanadium(IV)→ vanadium(V) and vanadium(IV) → vanadium(III) couples. The ESR spectra of the Cu(II), VO(II) and Mn(II)complexes were recorded in DMSO solution and their salient features reported. The in vitro antimicrobial activity of the investigated compounds was tested against the microorganisms such as Salmonella typhi, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis, Shigella flexneri, Pseudomonas aeruginosa, Aspergillus niger and Rhizoctonia bataicola. Most of the metal chelates have higher antimicrobial activity than thefree ligands. Tarafder et al. [13] have synthesised several new complexes of a tridentate Schiff base derived from the condensation of S-benzyldithiocarbazate with salicylaldehyde and have been characterised by elemental analyses, molar conductivity measurements and by IR and electronic spectra. The Schiff base (HONSH) behaves as a dinegatively charged ligand coordinating through the thio sulphur, the azomethine nitrogen and the hydroxyl oxygen. It forms mono-ligand complexes: [M(ONS)X], [M=Ni(II), Cu(II), Cr(III), Sb(III), Zn(II), Zr(IV) or U(VI) with X=H2O, Cl]. The ligand produced a bis-chelated complex of composition [Th(ONS)2] with Th(IV). Square-planar structures are proposed for the Ni(II) and Cu(II)complexes. Antimicrobial tests indicated that the Schiff base and five of the metal complexes of Cu(II), Ni(II), U(VI), Zn(II) and Sb(III) are strongly active against bacteria. Ni(II) and Sb(III) complexes were the most effective against Pseudomonas aeruginosa (gram negative), while the Cu(II) complex proved to be best against Bacillus cereus (gram positive bacteria). Antifungal activities were also noted with the Schiff base and the UVI complex. These compounds showed positive results against Candida albicans fungi, however, none of them were effective against Aspergillus ochraceous fungi. The Schiff base and its zinc and antimony complexes are strongly active against leukemic cells (CD50=2.3-4.3µg cm3) while the copper, uranium and thorium complexes are moderately active (CD50=6.9-9.5 µg cm3). The nickel, zirconium and chromium complexes were found to be inactive. Alomar et al. [14] investigated the reaction of zinc(II) chloride, cadmium(II) chloride and bromide with 3-thiophene aldehyde thiosemicarbazone leads to the formation of a series of new complexes. They have been characterized by spectroscopic studies: IR, 1H NMR, and electronic spectra. The crystal structures of the compound


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[ZnCl2(3TTSCH)2] and [CdBr2(3TTSCH)2] have been determined by X-ray diffraction methods. For the complexes [ZnCl2(3TTSCH)2] and [CdBr2(3TTSCH)2], the central ion is coordinated through the sulfur and for the complexes [CdCl2(3TTSCH)], [CdBr2(3TTSCH)] the ion is coordinated through the sulfur as well as azomethine nitrogen atom of the thiosemicarbazone. In addition, fungistatic and bacteriostatic activities of both ligand and complexes have been evaluated. Cadmium(II) complexes have shown the most significant activities. Chandra et al. [15] synthesized the Ni(II) complexes having the general composition Ni(L)2X2 [where L: isopropyl methyl ketone semicarbazone (LLA), isopropyl methyl ketone thiosemicarbazone (LLB), 4-aminoacetophenone semicarbazone (LLC) and 4-aminoacetophenone thiosemicarbazone (LLD) and X=Cl−, 1/2SO4

2−]. All the Ni(II)

complexes reported here have been characterized by elemental analyses, magnetic moments, IR, electronic and mass spectral studies. All the complexes were found to have magnetic moments corresponding to two unpaired electrons. The possible geometries of the complexes were assigned on the basis of electronic and infrared spectral studies. Newly synthesized ligand and its Ni(II) complexes have been screened against different bacterial and fungal growth. Abou-melha et al. [16] synthesized a Schiff’s base bis-[4-hydroxycoumerin-3-yl]-1N,5N-thiocarbohydrazone (H2L) by the reaction of 4-hydroxycoumerin-3-carbaldehyde with thiocarbohydrazide in 2:1 molar ratio and its binuclear complexes with Cu(II), Ni(II), Co(II), Mn(II), Fe(III) and Cr(III) ions . The Schiff’s base and its complexes were screened for their antifungal and antibacterial activities against different species of pathogenic fungi (Candida albicans and Fusarium solani) and bacteria (Escherichia coli and Staphylococcus aureus) and their biopotency have been discussed. Several new complexes of diphenylantimony(III) with the bidentatesemicarbazone derived from furan 2- carboxyaldehyde, thiophene-2-carboxyaldehyde, pyridine-2-carboxyaldehyde, indole-3-carboxyaldehyde, 2-acetylfuran, 2-acetylthiophene, 2-acetylpridine and 3-acetylindole had been synthesized by Phor et al. [17]. The synthesized complexes were characterized by elemental analysis, molecular weight determinations, electronic, IR, 1H and 13C NMR spectral studies. The complexes were monomeric and non electrolyte. A probable tetra coordinated environmental around the


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antimony atom has been proposed. The results of fungicidal, bactericidal tests are also reported. Avajiet al. [18] synthesized a series of Co(II) complexes with Schiff’ bases derived from 3-substituted-4-amino-5-hydrazino-1,2,4-triazole and substituted salicyladehydes. Some of the Schiff’s bases and complexes were evaluated for their antimicrobial activities against Staphlococcus aureus and Bacillus cereus (Gram+ve), pseudomonas aeruginosa and Escherichia coli (Gram-ve),Aspergillusnigerand Aspergillus fumigates. A series of new Cu(II), Co(II), Ni(II), Mn(II), Fe(III), and UO(VI) complexes of the Schiff base hydrazone 7-chloro-4-(benzylidenehydrazo)quinoline(HL) were prepared and characterized by Shaalan [19]. The Schiff base behaved as a monobasic bidentate ligand. The complexes with general composition [ML2(Cl)m(H2O)2(OEt)n]·xEtOH (M=Cu(II), Co(II), Ni(II), Mn(II), Fe(III) or UO2(VI);m and n=0-1; x=1-3) were obtained in the presence of Li(OH) as a deprotonating agent. The nature of bonding and the stereochemistry of the complexes have been deduced from elemental analyses, infrared, electronic spectra, magnetic susceptibility and conductivity measurements. An octahedral geometry was suggested for all the complexes except the Cu(II) and UO2(VI) ones. The Cu(II) complex has a square-planar geometry distorted towards tetrahedral, while the UO2(VI) complex displays its favored heptacoordination. The Schiff base ligand, HL, and its complexes were tested against one strain Gram +ve bacteria (Staphylococcus aureus), Gram -ve bacteria (Escherichia coli), and Fungi(Candida albicans). The prepared metal complexes exhibited higher antibacterial activities than the parent ligand and their biopotency was discussed. The design and synthesis of the new amino acid Schiff base, N-(2-hydroxy-1-naphthalidene)phenylglycine (Hhnpg) has been described along with the single crystal X-ray crystallographic studies by Gudasi [20]. Cu(II), Ni(II), Co(II),Mn(II) and Zn(II) complexes of Hhnpg were synthesized for the first time, and were characterized on the basis of elemental analysis, conductivity measurements, spectral (IR, 1H-NMR., UV-vis., EPR), magnetic and thermal studies. The IR spectral studies of all the complexes exhibit a similar feature about the ligating nature of the ligand to the metal ions and reveal that the ligand has coordinated through the carbonyl oxygen, azomethine nitrogen and deprotonated hydroxyl oxygen. The conductance data of the complexes


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suggest them to be non electrolytes. The microbial activity of the ligand and the complexes was investigated. El-Wahabet al. [21] reported the synthesis and structural characterization of series of tetra- and hexacoordinate metal chelate complexes of phosphate Schiff base ligands having the general composition LMXn·H2O and L2MXn(L=phosphate Schiff base ligand; M=Ag(I), Mn(II), Cu(II), Zn(II), Cd(II), Hg(II), or Fe(III) and X=NO3

−, Br−or Cl−). The structure of the prepared compounds was investigated using elemental analysis, IR, 1H and 31P NMR, UV–vis, mass spectra, solid reflectance, magnetic susceptibility and conductance measurements as well as conductometric titration. In all the complexes studied, the ligands act as a chelate ligand with coordination involving the phosphate–O-atom and the azomethine-N-atom. IR, solid reflectance spectra and magnetic moment measurement were used to infer the structure and to illustrate the coordination capacity of ligand. IR spectra show the presence of coordinated nitrate and water molecule, the magnetic moments of all complexes show normal magnetic behavior and the electronic spectra of the metal complexes indicate a tetra- and octahedral structure for Mn(II), octahedral structure of Fe(III)and both square-planar and distorted octahedral structure for Cu(II)complexes. Antimicrobial activity of the ligands and their complexes were tested using the disc diffusion method and the chosen strains include Staphylococcus aureus, Pseudomonas aereuguinosa, Klebsiella penumoniae, Escherichia coli, Microsporumcanis, Trichophyton mentagrophyte and Trichophyton rubrum. Some known antibiotics were included for the sake of comparison and the chosen antibiotic were Amikacin, Doxycllin, Augmantin, Sulperazon, Unasyn, Septrin, Cefobid, Ampicillin, Nitrofurantion, Traivid and Erythromycin. Mane [22] has synthesized the solid complexes of Mn(II), Co(II), and Cu(II) with Schiff bases derived from 3-acetyl-6-methyl-(2H)-pyran-2,4(3H)-dione(dehydroacetic acid) and 3-Methoxy aniline and 3-amino toluene and characterized by elemental analysis, conductometry, thermal, magnetic, IR, NMR, UV-Vis spectral and X-ray powder diffraction studies. From the analytical data the stoichiometry of the complexes has been found to be 1:2 (metal:ligand). The low conductance values suggested that the complexes are non electrolytes. IR spectra data suggested that the ligand behaved as a


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bidentate neutral with N, O donor. Thermal studies indicated about the stability of the complexes. The Powder XRD suggested monoclinic crystal system for the complexes. The physic-chemical data indicated octahedral geometry for all the complexes. Complexes have been screened for antifungal activity against A. niger.

7.3. PRESENT WORK The present work is pertaining to screen the antifungal activities of the ligands, 3-bromoacetophenone thiosemicarbazone (L2) and 1-Tetralone thiosemicarbazone (L4) and their Cu(II), Ni(II) complexes. Selection of compounds for activities: Thiosemicarbazones and their complexes are very well known for their pharmacological activities [23]. They have numerous biological activities, e.g. anticarcinogenic, antibacterial [24, 25], anti-HIV [26, 27] anticancer [28], fungicides, antiviral [29, 30], antifungal [31], antitumour etc. [32–35]. Hence in the present work, the thiosemicarbazone based ligand and their complexes have been selected for antimicrobial activities. The antifungal activities of the Ligand and their complexes were carried out using food poison method [36]. a) Fungus Used i) Aspergillus niger and Aspergillus flavus–Culture of these fungi were collected from the Botany department,Delhi University. ii) Botrytis cinerea- Fungal culture of Botrytis cinerea was obtained from Indian Type Culture Collection, Indian Agricultural Research Institute, New Delhi (ITCC No. 6192). iii) Phoma glomerata-Phomaglomerata was isolated from seeds of Impatiens glandulifera received from UK in the Plant Quarantine Division of National Bureau of Plant Genetic Resources, New Delhi for by incubation on blotter. iv) Macrophomina phaseolena- This fungal culture was taken from the NBPGR Pusa, New Delhi. The Pure cultures of all the test fungi were maintained on Potato Dextrose Agar (PDA) medium.


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v) Commercial fungicide used: Bavistin was used as a commercial fungicide b) Preparation of potato dextrose Agar medium: Potato Dextrose Agar (PDA) medium was prepared by dissolving 39 grams of the solid powder from CONDA (Spain) in one litre of distilled water and autoclaving it (Figure 7.4) at 121OC for 15 minutes.

Figure 7.4. Vertical autoclave

c) Stock Solutions: Stock solutions of the test compounds were prepared by dissolving the compounds in DMSO. Appropriate quantities of the compounds dissolved in DMSO were added to PDA in order to get the appropriate concentrations. d) Pouring: The above prepared medium was poured into Petri plates under aseptic conditions in a laminar flow (Figure 7.5).

Figure 7.5. Laminar flow

e) Inoculation: The plates were inoculated using mycelial discs of 0.5 cm in diameter-cut from the periphery of the 7-day old culture. Each treatment was kept in a replication of three. f) Incubation: The Petri plates were incubated at 20 ± 1°C under the alternate cycle of light and darkness (12 hrs. each) for 8 days.


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Figure 7.6. Incubation chamber

g) Measurements: The mycelial growth of fungi (mm) in each Petri plate was measured diametrically and growth inhibition (I) was calculated using the formula: I (%) = (C-T)/C x 100, where I= % Inhibition, C= Radial diameters of the colony in control, T= Radial diameter of the colony in test compound. h) Results, Discussion and mode of action: The antifungal screening data showed that the compounds exhibit antifungal properties and metal complexes (chelate) exhibit more inhibitory effect than the parent ligand. In general, the synthesized metal complexes have higher biological activities compared to the free ligands. The increased inhibition activity of the metal complexes can be explained on the basis of Tweedy's chelation theory [37]. In metal complexes, on chelation the polarity of the metal ion is reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Further, it increases the delocalization of π- electrons over the whole chelate ring. The large ring size of attached two ligand moiety makes the complexes more lipophillic [38]. This increased lipophillicity enhances the penetration of the metal complexes into lipid membranes and block the metal binding sites in the enzymes [39]. Metal complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organisms. The azomethine linkage in the synthesized complexes exhibit extensive biological activities [40, 41] due to increased liposolubility of the molecules in crossing cell membrane of the microorganism. The presence of electron donor group in the complexes also plays a role in enhancing the inhibition activity.


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In the present study all the results of antifungal activities are summarized in the tables 7.1-7.11. Codes for the test compounds used for screening of antifungal activities are given below: Master compound 1 = 3-Bromoacetophenone thiosemicarbazone (L2), Master compound 2 = 1-Tetralone thiosemicarbazone (L4). In the test compounds, compound 1 =[Cu(L2)SO4] complex, Compound 2 =[Cu(L2)(CH3COO)2] complex, Compound 3 =[Ni(L2)SO4] complex Compound 4 =[Ni(L2)(CH3COO)2] complex compound 5 =[Cu(L4)Cl2] complex, compound 6 =[Cu(L4)SO4] complex, compound 7 = [Ni(L4)Cl2] complex and compound 8 =[Ni(L4)SO4] complex. The letter F stands for standard fungicide Bavistein and C is the control. Solvent DMSO served as a control.


274

Table 7.1. Antifungal activities data of master compounds (500 PPM) after 8 days of inoculation

Discussion

With fungus P. glomerata, M. phaseolina, A. flavus and A. Niger order of inhibition was found in following order: Fungicide> Master compound II> Master compound I With fungus B.cinarea order of inhibition was: Master compound I >Master compound II >Fungicide With fungus F.semitactum order of inhibition was: Fungicide > Master compound II > Master compound I The growth of fungi in the plates for master compounds were even more than the control plate so results for % inhibition was in negative.

Fungus

% Inhibition (radial growth in mm)

Fungicide Master

compound (I)

Master compound

(II) Control plate

Phomaglomerata 76(12.5) 58.7(21.5) 72(21.5) (52.1)

Macrophomina phaseolina 55.9(17.2) -14.9(44.8) 34.6(25.5) (39)

Botrytis cinaria -25(75) 45.8(32.5) 45(33.0) (60)

Aspergillus flavus 46.1(10.5) 27.7(14.1) 58.9(31) (19.5)

Aspergillus niger 70.5(12.1) 23.17(31.5) 44.4(22.8) (41)

Fusariumsemitactum 59.7(08.0) -19.64(47.5) -17.12(46.5) (39.7)


275

Table 7.2. Antifungal activities data of master compounds (500 ppm) after 8 days of inoculation

Discussion With fungus P. glomerata, A. niger and Fusarium semitactum %inhibition was in following order: Fungicide > Master compound II > Master compound I With fungus M. phaseolina and A. flavus, order of inhibition was: Fungicide >Master compound I >Master compoundII With fungus B. cinarea order of inhibition was: Master compound II>Master compoundI>Fungicide.

Fungus % inhibition (Radial growth in mm)

Fungicide Master

Compound ( I)

Master compound ( II)

Control plate

Phoma glomerata 76(18) 68(24) 70.6(22) (75)

Macrophomina phaseolina 85(12) 76.4(20) 74.7(21.5) (85)

Botrytis cinaria 0(85) 54(39) 62.9(31.5) (85)

Aspergillus niger 85(10) 64.7(24.5) 78(15) (68)

Aspergillus flavus 62(10) 55.5(12) 40.7(16) (27)

Fusarium semitactum 90.3(7.5) 21.79(61) 27(55.5) (78)


276

Table 7.3. Antifungal activities data of master compounds (1500 ppm) after 8 days of inoculation

Fungus

% Inhibition (Radial growth in mm)

Fungicide Master

compound (I)

Master compound

(II) Control Plate

Phoma glomerata 90(07) 69(21.5) 72(19.5) (70.00)

Macrophomina phaseolina 87.6(10.5) 70(25) 75.8(20.5) (85)

Botrytis cinarea 0(85) 75.8(20.5) 64(30.5) (85)

Aspergillus flavus 73(17.5) 76(16) 72(18.5) (67)

Aspergillus niger 94(3.5) 53(30.5) 48(33.5) (65.5) Discussion With fungus P. glomerata and M. phaseolina, order of % inhibition was found to be: Fungicide > Master compound II > Master compound I With fungus and A. flavus, order of inhibition was: Master compound I >Master compound II >Fungicide. With fungus B. cinarea, % inhibition was: Master compound II> Master compound I>Fungicide With fungus and A. niger order of inhibition was: Fungicide > Master compound I> Master compound II.


277

Table 7.4. %inhibition and radial growth of master compounds and fungicide with different pathogenic fungi

Discussion Master compound I, At 500 ppm showed maximum inhibition with fungus P. glomerata followed by B.cinerea, A. flavus, A. niger, M. phaseolina and F.semitactum. At 800 ppm the order of %inhibition was: M. phaseolina > P. glomerata > B.cinerea. At 1500 ppm the order of inhibition was: A. flavus >B. cinera >M. phaseolina >P. glomerata > A. niger. Master compound II, At 500 ppm showed maximum inhibition with fungus P. glomerata followed by A. flavus, B.cinerea, A. niger, M. phaseolina and F.semitactum. At 800 ppm the order of inhibition was: M. phaseolina >P. glomerata > B. cinerea. At 1500 ppm the order of inhibition was: M. phaseolina >P. glomerata =A. flavus >B. cinera > A. niger. Fungicide, at 500 ppm showed maximum inhibition with fungus P. glomeratafollowed by A. niger, F.semitactum, M. phaseolina , A. flavus, B. cinerea.

Test Compounds

Concentration (ppm)

% inhibition (Radial growth) P.

glomerata M.

phaseolina B.

cinerea A.

flavus A.

niger F.

semitactum

Master Compound

(I)

500 58.7 (21.5)

-14.9 (44.8)

45.8 (32.5)

27.7 (14.1)

23.17 (31.5)

-19.64 (47.5)

800 68 (24)

76.4 (20).

54 (39) - - -

1500 69 (21.5)

70 (25)

75.8 (20.5)

76 (16)

53 (30.5) -

Master Compound

(II)

500 72 (21.5)

34.6 (25.5)

45 (33.0)

58.9 (31)

44.4 (22.8)

17.12 (46.5)

800 70.6 (22)

74.7 (21.5)

62.9 (31.5) - - -

1500 72 (19.5)

75.8 (20.5)

64 (30.5)

72 (18.5)

48 (33.5) -

Fungicide

500 76 (12.5)

55.9 (17.2)

-25 (75)

46.1 (10.5)

70.5 (12.1)

59.7 (8.0)

800 76 (18)

85 (12)

0 (85) - - -

1500 90 (07)

87.6 (10.5)

0 (85)

73 (17.5)

94 (3.5)

Control 500 (52.1) (39) (60) (19.5) (41) (39.7) 800 (75) (85) (85) - - -

1500 (70) (85) (85) (67) (65.5) -


278

At 800 ppm the order of inhibition was: M. phaseolina >P. glomerata > B. cinerea. At 1500 ppm the order of inhibition was: A. niger >P. glomerata > M. phaseolina > A. flavus > B. cinera. Table 7.5. % Inhibition (radial growth) at 1000 ppm for test compounds with

fungus P. glomerata

P. glomerata 1 2 3 4 5 6 7 8 F

C=73 54.7 (33)

56.2 (32)

78 (16)

78 (16)

51 (36)

77 (16.5)

100 (nil)

90.4 (07)

100 (nil)

After 8

days C=83 42.2

(48) 30.1 (58)

73.5 (22)

65.1 (29)

7.0 (77)

79.0 (17)

82 (15)

88 (10)

100 (nil)

After 15 days

C=83 34.9 (54)

24.1 (63)

78.3 (18)

61.4 (32)

07.2 (77)

81.9 (15)

86.1 (11.5)

91.56 (07)

100 (nil)

After 25 days

Discussion The %inhibition of the test compounds with fungus P. glomerata showed the following order Observation taken after 8 days of inoculation then inhibition was in order: Fungicide > Compound 7 > Compound 8 > Compound 3 = Compound 4 > Compound 6 > Compound 2 > Compound 1 > Compound 5. Observation taken after 15 days of inoculation then inhibition was in order: Fungicide > Compound 8 > Compound 7 > Compound 6 > Compound 3 > Compound 4 > Compound 1 > Compound 2 > Compound 5. Observation taken after 25 days of inoculation then activity was similar than taken after 15 days.


279

Table 7.6. % Inhibition (radial growth) at 1000 ppm for test compounds with fungus B.cinerea

Botrytis cinerea 1 2 3 4 5 6 7 8 F

C=83

71.6 (23.5)

61.4 (32)

76.0 (20)

79.0 (17.5)

72.0 (23.3)

100 (nil)

86.1 (11.5)

100 (nil)

nil (83)

After 8 days

C=83

66.2 (28)

53.6 (38.5)

68.6 (26)

72.2 (23)

70.0 (25)

100 (nil)

70.0 (25)

100 (nil)

nil (83)

After 15 days

C=83

69.8 (25)

67.4 (27)

71.1 (24)

75.9 (20)

71.1 (24)

100 (nil)

75.9 (20)

100 (nil)

nil (83)

After 25 days

Discussion The % inhibition of the test compounds with fungus Botrytis cinerea showed the following order: Observation taken after 8 days of inoculation then the inhibition was in order: Compound 8= Compound 6> Compound 7> Compound 4 > Compound 3> Compound 5> Compound 1 > Compound 2 > Fungicide. Observation taken after 15 days of inoculation then the inhibition was in order: Compound8 = Compound 6> Compound 4> Compound 5 = Compound 7> Compound 3 > Compound 1> Compound 2 > Fungicide. Observation taken after 25 days of inoculation then the inhibition was in order: Compound 8 = Compound 6 > Compound 7 = Compound 4 > Compound 5 = Compound 3 > Compound 1 > Compound 2 > Fungicide.


280

Table 7.7. % Inhibition (radial growth) at 1000 ppm for test compounds with fungus M. phaseolina

M.p

1 2 3 4 5 6 7 8 F

C=83

05.0 (79)

59.6 (33.5)

79.5 (17)

73.5 (22)

37.3 (52)

80.1 (16.5)

90.36 (08)

100 (nil)

91.5 (07)

After 8 days

C=83

nil (83)

03.0 (80)

66.3 (28)

63.2 (30.25)

14.4 (71)

82.5 (14.5)

87.3 (10.5)

100 (nil)

85.0 (12.2)

After 15 days

C=83

nil (83)

24.1 (63)

66.3 (28)

68.07 (26.5)

14.4 (71)

80.7 (16)

83.1 (14)

100 (nil)

89.1 (09)

After 25 days

Discussion The activities of the test compounds with fungus Macrophomina phaseolinashowed the order: Observation taken after 8 days of inoculation then inhibition was in order: Compound 8 > Fungicide > Compound 7 > Compound 6 > Compound 3 > Compound 4> Compound 2> Compound 5 > Compound 1. Observation taken after 15 days of inoculation then inhibition was inorder: Compound8 > Compound 7> Fungicide > Compound 6 > Compound 3>Compound4> Compound 5> Compound 2 > Compound 1. Observation taken after 25 of inoculation then inhibition was in order: Compound 8 > Fungicide > Compound 7 > Compound 6 > Compound 4 > Compound 3 > Compound 2 > Compound 5 > Compound 1.


281

Table 7.8. Antifungal activities data with different fungi at (1500 ppm) for test compounds

Fungus % Inhibition (radial growth)

1 2 3 4 F

P. glomerata C=81

90.74 (7.5)

80 (15)

100 (nil)

100 (nil)

100 (nil)

After 8 days

C=83 86 .7 (11)

81.9 (15)

100 (nil)

100 (nil)

100 (nil)

After 15 days

B. cinerea C=83

100 (nil)

100 (nil)

100 (nil)

100 (nil) nil After

8 days

C=83 100 (nil)

100 (nil)

100 (nil)

100 (nil) nil After

15 days M.

phaseolina C=83

67 (27)

76 (20)

100 (nil)

100 (nil)

100 (nil)

After 8 days

C=83 62.6 (31)

82 (15)

100 (nil)

100 (nil)

100 (nil)

After 15 days

Discussion With fungus P. glomerata at 1500 ppm the test compounds 1, 2, 3, 4 and Fungicide (F) showed the good inhibition and After 8 days of inoculation the activity order was: Fungicide = Compound 4 = Compound 3 > Compound 1>Compound 2. The order of activity was same when noted after 15 days but the % inhibition was more. With fungus B. cinerea at 1500 ppm the test compounds 1, 2, 3 and 4 showed the 100% inhibition while the commercial fungicide did not show any inhibition. With fungus M. phaseolina at 1500 ppm the test compounds 3, 4 and Fungicide showed the 100 % inhibition. After 8 days of inoculation the activity order was: Fungicide = Compound 4 = Compound 3 > Compound 2 > Compound 1. The order of activity when noted after 15 days was different as activity of compound 2 increased while decreased in case of compound 1. Hence the order was: Fungicide = Compound 4 = Compound 3 >Compound 1 >Compound 2.


282

Table 7.9. Antifungal activities data of different test compounds with different fungi at different concentrations

Test Compounds

Concentration (ppm)

% inhibition (Radial growth)

P. glomerata M. phaseolina B. cinerea

1 1000 54.7

(33) 5

(79) 71.6 (23.5)

1500 90.74 (7.5)

67 (27)

100 (nil)

2 1000 56.2

(32) 59.6 (33.5)

61.4 (32)

1500 80 (15)

76 (20)

100 (nil)

3 1000 78

(16) 79.5 (17)

76 (20)

1500 100 (nil)

100 (nil)

100 (nil)

4 1000 78

(16) 73.5 (22)

79 (17.5)

1500 100 (nil)

100 (nil)

100 (nil)

Fungicide 1000 100

(nil) 91.5 (07)

Nil (83)

1500 100 (nil)

100 (nil)

(nil) 100

Control 1000 73 83 83

1500 81 83 83


283

Table 7.10. Antifungal activities data (800 ppm) for test compounds at different intervals of time

Fungus

% Inhibition (radial growth)

6 7 8 F P.glomerata C=81

100 (nil)

91.3 (07)

100 (nil)

100 (nil) After 8 days

C=83

84.0 (13)

85.5 (12)

100 (nil)


B. cinerea C=83

100 (nil)

90.3 (08)

100 (nil)

Nil (83) After 8 days

C=83

100 (nil)

90.3 (08)

100 (nil)

Nil (83) After 15 days

M. phaseolina C=83

27.7 (60)

92.2 (6.5)

100 (nil)


C=83

100 (nil)

90.3 (08)

100 (nil)


Discussion At 800 ppm test compounds 6, 7, 8 and fungicide showed good activity. After 8 days of inoculation compound 6, 8 and fungicide gave 100% inhibition while compound 7 showed 91.3%. Compound 6 and 7 showed decreases in activity with increase in time. With fungus B. cinerea at 800 ppm the test compounds 6, 8 showed the 100% inhibition and compound 7 showed 90 % inhibition while the commercial fungicide did not show any inhibition.The activities remained same after 15 days. With fungus M. phaseolin aat 800 ppm the test compound 8 and Fungicide showed the 100 % inhibition. After 8 days of inoculation the activity order was: Fungicide = Compound 4 = Compound 7 > Compound 6. The order of activity when noted after 15 days was different as activity of compound 6 increased while decreased in case of compound 7. Hence the order was: Fungicide = Compound 8 = Compound 6 >Compound 7.


284

Table 7.11. Comparison of Antifungal activities of different test compounds with different fungi.

Test Compounds

Concentration (ppm)

% inhibition (Radial growth) P. glomerata M. phaseolina B. cinerea

5 800 - - - 1000 51(36) 37.3(52) 72(23.3)

6 800 100(nil) 27.7(60) 100(nil) 1000 77(16.5) 80.1(16.5) 100(nil)

7 800 91.3(07) 92.2(6.5) 90.3(08) 1000 100(nil) 90.36(08) 86.1(11.5)

8 800 100(nil) 100(nil) 100(nil) 1000 90.4(07) 100(nil) 100(nil)

Fungicide 800 100(nil) 100(nil) Nil(83) 1000 100(nil) 91.5(07) Nil(83)

Control 800 81 83 83 1000 73 83 83

Discussion The activity of compound 5 was checked at 800 ppm as it did not show activity at lower concentration. However it is not always true that on increase the concentration activity increases as the compound 6 and 8 showed decrease in activity with increase in concentration against fungus P. glomerata and compound 7 decreased in activity against fungus M. phaseolina and B. cinerea. Similarily fungicide also showed decrease in activity with increase in concentration against the fungus M. phaseolina.


285

7.4. CHARTS

Figure 7.7. % inhibition of test fungi by master compounds 1 and 2 and fungicide (500 ppm)

Figure 7.8. % inhibition of test fungi by master compounds 1 and 2 and fungicide

(800 ppm)

-80-60-40-20

020406080

P. g. M.p. B.c A.f. A .n F. s% in

hibi

tion

test fungus

fungicideMaster compound 1master compound 2

0102030405060708090

100

P.g M.p B.c A.f. A.n F. s.

% in

hibi

tion

test fungus

fungicideMaster compound 1master compound 2


286

Figure 7.9. % inhibition of test fungi by master compound 1 and 2 and fungicide

(1500 ppm).

Figure 7.10. % inhibition of test fungi by test compounds and fungicide (800 ppm).

-60-40-20

020406080

100

P.g M.p B.c A.f A.n F.s

% in

hibi

tion

Test fungus

fungicideMaster compound 1master commpound 2

0102030405060708090

100

6 7 8 Fungicide Control

% in

hibi

tion

Test compounds

P.glomerataM. phaseolenaB. cineria


287

Figure 7.11. % inhibition of test fungi by test compounds and test fungicide (1000 ppm)

Figure 7.12. % inhibition of test fungi by test compounds and fungicide (1500 ppm)

0102030405060708090

100

1 2 3 4 5 6 7 8Fu

ngici

de

Contr

ol

% in

hibi

tion

Test compounds

Phoma glomerataMacrophomina phaseolena

0102030405060708090

100

% in

hibi

tion

Test compounds

Phoma glomerataMacrophomina phaseolenaBotrytis cineria


288

7.5. PHOTOGRAPHS C= Control, F= fungicide, I= Master compound 1, II= Master compound 2

Figure 7.13. Master compounds I, II and fungicide with fungus Phoma glomerata (500 ppm). A=Control, B=Fungicide, C=Master compound 1, D=Master Compound I.

Figure 7.14. Master compounds I, II and fungicide with fungus Phoma glomerata (800 ppm). A=Control, B=Fungicide, C=Master compound 1, D=Master compound II.


289

Figure 7.15. Master compounds I, II and fungicide with fungus M. phaseolena (500 ppm). A=Control, B=Fungicide, C=Master compound 1, D = Master compound.

Figure 7.16. Master compounds I, II and fungicide with fungus M. phaseolena (800 ppm). A = Control, B = Fungicide, C = Master compound, D = Master compound II.


290

Figure 7.17. Master compounds I, II and fungicide with fungus fusarium semitactum

(500 ppm). A = Control, B = Fungicide, C = Master compound 1, D = Master compound II

Figure 7.18. Master compounds I, II and fungicide with fungus F. semitactum (800 ppm). A = Control, B = Fungicide, C = Master compound 1, D = Master compound II.


291

Figure 7.19. Master compounds I, II and fungicide with fungus Botrytis cinerea (500 ppm). A = Control, B=Fungicide, C = Master compound 1, D = Master compound II

Figure 7.20. Master compounds I, II and fungicide with fungus Botrytis cinerea (800 ppm). A = Control, B = Fungicide, C = Master compound 1, D = Master compound II


292

Figure 7.21. Test compounds 1, 2, 3, 4, fungicide and control plate with Phoma glomerata (1000 ppm).

A=Test compound 1, B=control, C=Test compound III, D=Test compound II, E= Fungicide, F = Test compound IV.

Figure 7.22. Test compounds 5, 6, 7, 8 fungicide and control with test fungus Botrytis

cinerea (1000 ppm). A = Test compound 5, B = Control, C = Test compound 7, D = Test compound 6, E = Fungicide, F = Test compound 8.


293

Figure 7.23. Compound 1, control plate and fungicide with fungi, P. glomerata,

B.cinerea, M.phaseolena (1000 ppm). A=Control (P. glomerata), B=Control (M. phaseolena), C=Control (B. cinerea), D=Fungicide with P. glomerata, E=Fungicide with M.phaseolena, F=Fungicide with B.cinerea, G=Test compound 1 with P. glomerata H=Test compound 1 with M. phaseolena I = Test compound 1 with B.cinerea.

Figure 7.24. Compound 2, control plate and fungicide with fungi, P. glomerata, B.cinerea, M.phaseolena (1000 ppm). A=Control with P. glomerata, B=Control withM.phaseolena,C=Control with B.cinerea, D = Fungicide with P. glomerata, E = Fungicide with M. phaseolena,F = Fungicide with B. cinerea, G = Test compound II with P. glomerata, H = Test compound II with M.phaseolena, I = Test compound II with B.cinerea.


294


B.cinerea, M.phaseolena (1000 ppm). A=Control with P. glomerata, B=Control with M.phaseolena, C=Control with B. cinerea, D=Fungicide with P. glomerata, E=Fungicide with M. phaseolena, F=Fungicide with B.cinerea, G=Test compound III with P. glomerata, H=Test compound III with M. phaseolena, I=Test compound III with B.cinerea.


B.cinerea, M.phaseolena (1000 ppm). A=Control with P.glomerata, B=Control with M. phaseolena, C=Control with B.cinerea, D=Fungicide with P. glomerata, E=Fungicide with M.phaseolena, F=Fungicide with B. cinerea, G=Test compound IV with P. glomerata, H=Test compound IV with M. phaseolena, I = Test compound IV with B.cinerea.


295

Figure 7.27. Compound 6, control plate and fungicide with fungi, P. glomerata, B.cinerea, M.phaseolena (1000 ppm). A=Control with P. glomerata, B=Control with M.phaseolena, C=Control with B.cinerea, D = Fungicide with P. glomerata, E=Fungicide with M.phaseolena, F=Fungicide with B.cinerea, G=Test compound VI with P. glomerat, H=Test compound VI with M.phaseolena, I = Test compound VI with B.cinerea


B.cinerea, M.phaseolena (1000 ppm). A=Control with P. glomerata, B=Control with M. phaseolena, C=Control with B. cinerea, D=Fungicide with P. glomerata, E=Fungicide with M. phaseolena, F=Fungicide with B. cinerea, G = Test compound VII with P. glomerata, H=Test compound VII with M. phaseolena, I=Test compound VII with B. cinerea.


296


B.cinerea, M.phaseolena (1000 ppm). A=Control with P. glomerata, B=Control with M. phaseolena, C=Control with B.cinerea, D=Fungicide with P. glomerata, E=Fungicide with M. phaseolena, F=Fungicide with B. cinerea, G = Test compound VIII with P. glomerata, H=Test compound VIII with M. phaseolena, I=Test compound VIII with B.cinerea.


297

SECTION B. IN-VIVO SEED TREATMENT This section includes the treatment of most effective antifungal compound on the seed to check the In-Vivo treatment of compound on the seeds or crop. 7.6. INTRODUCTION a. Definition: Seed treatment refers to the application of fungicide, insecticide, or a combination of both, to seeds so as to disinfect and disinfect them from seed-borne or soil-borne pathogenic organisms and storage insects. It also refers to the subjecting of seeds to solar energy exposure, immersion in conditioned water, etc. The seed treatment is done to achieve the following benefits. b. Benefits of Seed Treatment: 1) Prevents spread of plant diseases 2) Protects seed from seed rot and seedling blights 3) Improves germination 4) Provides protection from storage insects 5) Controls soil insects. c. Types of Seed Treatment: 1) Seed disinfection: Seed disinfection refers to the eradication of fungal spores that

have become established within the seed coat, or i more deep-seated tissues. For effective control, the fungicidal treatment must actually penetrate the seed in order to kill the fungus that is present.

2) Seed disinfestation: Seed disinfestation refers to the destruction of surface-borne organisms that have contaminated the seed surface but not infected the seed surface. Chemical dips, soaks, fungicides applied as dust, slurry or liquid have been found successful.

3) Seed Protection: The purpose of seed protection is to protect the seed and young seedling from organisms in the soil which might otherwise cause decay of the seed before germination.


298

d. Conditions under which seed must be treated: 1) Injured Seeds: Any break in the seed coat of a seed affords an excellent opportunity

for 2) fungi to enter the seed and either kill it, or awaken the seedling that will be

produced from it. Seeds suffer mechanical injury during combining and threshing operations, or from being dropped from excessive heights. They may also be injured by weather or improper storage.

3) Diseased seed: Seed may be infected by disease organisms even at the time of harvest, or may become infected during processing, if processed on contaminated machinery or if stored in contaminated containers or warehouses.

4) Undesirable soil conditions: Seeds are sometimes planted under unfavourable soil conditions such as cold and damp soils, or extremely dry soils. Such unfavourable soil conditions may be favourable to the growth and development of certain fungi spores enabling them to attack and damage the seeds.

5) Disease-free seed: Seeds are invariably infected, by disease organisms ranging from no economic consequence to severe economic consequences. Seed treatment provides a good insurance against diseases, soil-borne organisms and thus affords protection to weak seeds enabling them to germinate and produce seedlings.

e. Precautions in Seed Treatment: Most products used in the treatment of seeds are harmful to humans, but they can also be harmful to seeds. Extreme care is required to ensure that treated seed is never used as human or animal food. To minimise this possibility, treated seed should be clearly labelled as being dangerous, if consumed. The temptation to use unsold treated seed for human or animal feed can be avoided if care is taken to treat only the quantity for which sales are assured. Care must also be taken to treat seed at the correct dosage rate; applying too much or too little material can be as damaging as never treating at all. Seed with a very high moisture content is very susceptible to injury when treated with some of the concentrated liquid products. If the seeds are to be treated with bacterial cultures also, the order in which seed treatments should be done shall be as follows


299

i) fungicide ii) bacterial cultures. Most seed treatment products are fungicides or insecticides applied to seed before planting. Fungicides are used to control diseases of seeds and seedlings; insecticides are used to control insect pests. Some seed treatment products are sold as combinations of fungicide and insecticide. Fungicidal seed treatments are used for three reasons: 1) to control soil-borne fungal disease organisms (pathogens) that cause seed rots,

damping-off, seedling blights and root rot; 2) to control fungal pathogens that are surface-borne on the seed, such as those that

cause covered smuts of barley and oats, bunt of wheat, black point of cereal grains, and seed-borne safflower rust; and

3) to control internally seed-borne fungal pathogens such as the loose smut fungi of cereals (Figure 7.30).

Figure 7.30. Reasons for seed treatment.

Most fungicidal seed treatments do not control bacterial pathogens and most will not control all types of fungal diseases, so it is important to carefully choose the treatment that provides the best control of the disease organisms present on the seed or potentially present in the soil. The degree of control will vary with product, rate, environmental conditions and disease organisms present. Some systemic fungicidal seed treatments may also provide protection against early-season infection by leaf diseases.


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Hence it is it is highly required to synthesize a compound which can be applied safely on seeds to enhance the growth and to control the diseases. In our experiment, we applied the most effective antifungal compound on the seeds to check the afficacy of compound on the seed also. And one experiment is also performed in which the most effective compound is mixed with the natural oil and then applied on the seeds. For comparison, the natural oil alone is also treated with the seed. All experiments are performed at the same time under similar conditions to avoid the effect of temperature, pressure and light etc.

7.7. FUNGUS SELECTED a. Grey Mould (Botrytis) Scientific Name: Botrytis cinerea • Main symptoms on fruit are ghost spots- a pale halo or ring with a brown to black

pinpoint spot in the centre. Occasionally fruit will rot • The fungus can appear as a grey, velvety covering of spores on leaves, stems, dying

flowers, or fruit • Infection appears first on leaves in contact with the soil, damaged leaves, or flowers • Infection on stems may girdle the plant.


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b. Pictorial representation of the symptoms of fungus Botrytis cinerea

(A) (B) (C)

(D) (E) (F)

(G) (H) (I) Figure 7.31. Grey mould symptoms of fungus Botrytis cinerea on various

plants. (A) on tomato plant, (B) on rose flower, (C) on tomato fruits, (D)on Dahlia leaves, (E) on raspberry fruits, (F) on orchid flower, (G) on wine grapes, (H) in Pears, (I) on flowers.

.


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7.8. PROCEDURE FOR SEED TREATMENT In seed treatment with the synthesized complex we also compare the antifungal activities with natural oil i.e. Geranium oil on seeds. The detailed procedure for the experiment can be given in the following points: 1. Collection of seeds: A sample of mung bean (Phaseolus aureus L.) seed was

procured from the “National Seeds Corporation Ltd ”, New Delhi. Two thousand and four hundred (2400) seeds were collected for the experiment.

2. Selection of seeds: For seed treatment, broken and damaged seeds were removed and good quality of seeds was selected manually.

3. Surface sterilization of seeds: Surface sterilization of seeds was done by treating the seeds with alcohol by soaking them in alcohol for one minute and then washing with distilled water.

4. Drying: Seeds were kept overnight for drying on the blotting paper. 5. Treatment:A set of 400 seed was separated for control. Rest of the seed was rolled

in eight day old culture of Botrytis cinerea. Infected seeds were examined under the stereo-binocular microscope to verify the inoculum load on the seed. Five sets of four hundred each of infected seed were prepared and used for seed treatments comprising of 0.03% bavistin (Carbendazim 50 wp) , 0.1% Geranium oil, 0.1% [Ni(L)2(SO4)] and 0.1% Geranium oil +0.1% [Ni(L)2(SO4)]. Tween-80 was added in each set for proper dispersion.

6. Incubation: Four hundred seed from each treatment and control were incubated by placing 20 seed each in plastic Petri dish with three layers of wet blotter. These plates were then incubated at 20 ± 1OC for 7 days under alternate cycle of 12 hr of light and darkness.

7. Observations and results: The observation was recorded on eighth day with the parameters viz. fungal infection, seed germination, and average shoot length and is given in Table 7.12.


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Table 7.12. Effect of the [Ni(L)2(SO4)], Bavistin and Geranium oil on seed germination

It is evident from the data of the seed treatment presented in Table 7.12, that the infection of tested fungus Botrytis cinerea was maximum with Bavistin followed by Geranium oil, Geranium oil + [Ni(L)2(SO4)] and [Ni(L)2(SO4)]. The seed germination was appreciable with [Ni(L)2(SO4)] followed by Geranium oil + [Ni(L)2(SO4)], Geranium oil and least with Bavistin. The seed germination of [Ni(L)2(SO4)] was almost equal with healthy control (Figure 7.32). The average shoot length was also maximum in the case of [Ni(L)2(SO4)]. On this bases we can say that our complex would be even more growth promoter than the standard. Hence [Ni(L)2(SO4)] was effective against Botrytis cinerea but it was not effective against Aspergillus niger.

Treatment % Infection

%Seed Germination

Average shoot length (mm)

Remarks

Healthy control nil 98.5 29..4 5%

Aspergillus niger

Infected control 21.5 91 34.5

[Ni(L)2(SO4)] 4.5 98 39.5 6%

Aspergillus niger

Bavistin (standard) 16.5 92 25

Geranium oil 10.5 96 29.5

Geranium oil + [Ni(L)2(SO4)] 10.5 96 29.5 Poor growth


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Figure 7.32. Effects of different treatments on the seed.

Treatments

Seed Treatment

Average shoot length

% Seed Germination

% Infection


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7.9. PHOTOGRAPHS OF PLATES SHOWING SEED TREATMENT

Figure 7.33. Plates (A) and (B) showing infection of B. cinerea on seeds


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Figure 7.34. Plates having (A) chemical treated seeds and (B) infected control seeds.

In chemical treated seeds no infection of fungus B.cinerea can be seen.

Figure 7.35. Plates having (A) chemical treated seeds (B) and oil treated seeds.


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Figure 7.36. Plates having (A) Chemical treated seeds, (B) Infected control seeds and

(C) Fungicide (Bavistein) treated seeds. It may be noted that in case of fungicide, the treated seeds neither grew nor fungus of the infected seeds killed.

Figure 7.37. Plates showing comparison of different treatments (A) chemical Treated

seeds, (B) healthy control (C) Fungicide treated seeds (D) Infected control.


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It must be noted that chemical treated seeds showed growth similar to the healthy control. In chemical treated seeds no sign of fungal infection is seen while in fungicide treated seeds fungal infection is clearly visible.

7.10. CONCLUSION Synthesized complex,[Ni(L)2(SO4)] can safely be applied on the seeds against the fungus Botrytis cinerea.


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