**13. Application to antimicrobial activity**

The cell wall of bacteria not only precludes any interaction between the glycoconjugates on their membrane and carbohydrate-binding proteins but also prevents these proteins from

Antimicrobial Activity of Lectins from Plants 163

from *Shigella flexneri* and *Salmonella abortivoequina*. In 1970, other investigators demonstrated that Concanavalin A can be used to detect lipopolysaccharides of various Salmonella strains

In 2010, Petnual et al. reported the antimicrobial activity of *Curcuma longa* lectin, expressed as the minimal inhibitory concentration (MIC), was found to inhibit the growth of all five microbial species tested, the four bacteria, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, plus the yeast *Candida albicans*, at MIC values of ≥ 0.011, 0.005, 0.092, 0.002 and 0.0046 mg/ml, respectively (Petnual et al. 2010). These results demonstrate that the *Curcuma longa* rhizome lectin is likely to be at least one of the, if not the, candidate molecule responsible for the antibacterial action observed in rhizome extracts from this plant. An outstanding feature of the antibacterial activity of the isolated lectin is it is somewhat nonselective against this fairly diverse selection of bacteria. The potentially broad effect of the *Curcuma longa* rhizome lectin on the growth inhibition of several diverse bacterial strains, confirms the important interaction between the lectin and all the strains under consideration. From the tested strains, *Pseudomonas aeruginosa* (lowest MIC) seemed to be most sensitive to the presence of lectin. Previouse studies of the binding of plant lectins to bacterial cell wall peptidoglycans indicate that several lectins of different carbohydrate specificities can recognize most of the components of the bacterial cell wall, such as muramic acid, *N*acetylglucosamine, *N*-acetylmuramic acid and muramyl dipeptide (Ajouba et al., 1994).

Archidendron jiringa seed lectin was selected to test for antimicrobial activity with *Escherichia* coli, Pseudomonas auroginosa, Bacillus subtilis, Staphylococcus aurous, and Candida albican (Charungchitrak et al., 2011). The MIC of Archidendron jiringa seed lectin with Candida albican equal in S. aurous to be 0.0567 mg /ml and in Bacillus subtilis to be 0.2266 mg/ ml. But the MIC with *Escherichia* coli and Pseudomonas auroginosa is not detected, demonstrating stronger antimicrobial activity against gram-positive than gramnegative bacteria. Accordingly, the binding of lectins to muramic acid and N-acetylmuramic acid, carbohydrates present in the bacterial cell wall (mainly in gram-positive bacteria), has been reported (Ajouba et al., 1994). These data suggest that lectins probably play a role in plant defense, not only against phytopathogenic invertebrates, herbivores or fungi, but also against bacteria. The carbohydrate-binding site probably plays a key role in this activity, being responsible for the recognition of bacteria. Almost all microorganisms express surfaceexposed carbohydrates. These carbohydrates may be covalently bound, as in glycosylated teichoic acids to peptidoglycan, or non-covalently bound, as in capsular polysaccharides (Hirmo, et al., 1997; and Caldeon, et al., 1997). Every surface-exposed carbohydrate is a potential lectin-reactive site. The ability of lectins to form complexes with microbial glycoconjugates has made it to be employed as probes and sorbents for whole cells,

The lectin from *Curcuma amarissima* inhibited 4 microbial growth consist of *Bacillus subtilis*, *Candida albicans*, *Escherichia coli*, and *Staphylococcus aureus* at concentration ≥ 0.446, 0.446, 0.223, and 0.892 mg/ml respectively. But can not inhibite *Pseudomonas auroginasa* growth because at the surface of *Pseudomonas auroginosa* cell does not have polysaccharide ligands which can interact with *Curcuma amarissima* lectin. (Kheeree et al., 2011) Similar to Legume lectin from *Trinella foenumgraecum, Trifolium alexandrium, Bauhinia variegata,* and *Delonix regia* had a research that these lectins from sephadex G-150 can agglutinated both gram negative and gram positive bacteria (*Mycobacterium rhodochrous, Bacillus cercur, Bacillus megaterium,* 

as determined by gel diffusion. (Goldstein and Staub, 1970).

mutants, and numerous cellular constituents and metabolites.

penetrating the cytoplasm. Therefore, plant lectins cannot alter the structure and/or permeability of the membrane or disturb the normal intracellular processes of invading microbes. Therefore, if lectins play a role in the plant's defense against bacteria, it must be through an indirect mechanism that is based on interactions with cell wall carbohydrates or extracellular glycans.

In 1936, a using lectin in clinical microbiology began when Summer and Howell (Summer and Howell, 1936) had a report that Concanavalin A can agglutinated various *Mycobacterium* spp. The interactions between plant lectin and microorganisms have been applied for typing of bacteria, fungi, and protozoa. It is useful for characterizing bacterial cell components and for detecting bacteriophage receptors. (Etzler, 1983; Lis and Sharon, 1986; and Nicolson, 1974). The unique property of lectin to bind non-covalently to simple sugars and polysaccharides has attracted interest in microbial taxonomy. Lectin has a role in the clinical laboratory identification and taxonomic classification of many microorganisms. Because lectins are generally monoclonal proteins and because they possess a spectrum of specificities and molecular weights, they are substantial tools for diagnostic microbiology applications. Recent observations with regard to the binding of plant lectins to components of the bacterial cell wall peptidoglycans (such as muramic acid, *N*-acetylmuramic acid, *N*acetylglucosamine and muramyl dipeptides) revealed that seed lectins from several legume species strongly interact with these bacterial surface carbohydrates (Ajouba et al., 1994). Evidently, the observation that legume seed lectins can recognize and bind to the bacterial cell wall does not imply that such an interaction occurs *in vivo* and certainly does not prove that these lectins are involved in the protection of the seedlings against bacteria.

Lectin has been used for investigating virulence factors, surface structures, and identification of gram-positive bacteria. For example; lectin from *Dolichos biflorus* was used to confirm its specificity for identifying group C streptococci. In another test, its crude extract was coupled to polystyrene particles with a spacer arm to yield an effective lectinlatex reagent that agglutinated group C streptococcal antigens prepared as nitrous acid, autoclave,or enzyme extracts. (Slifkin and Gil, 1984) Group C streptococcal isolates from horses and cattle agglutinated with lectin from *Dolichos biflorus* and *Helix pomatia*. (Schalla et al., 1986). Concanavalin A could be precipitated various bacterial polysaccharides, with interacts specifically with bacterial cell walls containing glycosidic residues associated with teichoic acid. Accordingly, bacteria teichoic acids from cell wall containing α-glucopyranisyl residues,such as *Lactobacillus plantarum*, *Staphylococcus aureus*, and *Bacillus subtilis*. (Archibald and Coapes, 1971; Doyle et al., 1982; and Reeder and Ekstedt, 1971). Lectins from soy bean have been used to assay for detecting *Bacillus anthracis* (Cole et al., 1984). The use of soybean agglutinin (SBA) to detect very low numbers of buffered suspention of *Bacillus anthracis* vegetative cells and spores has been reported (Graham et al., 1984). The strategy was to bind the cells or spores to polystyrene plates and to detect the bound forms with horseradish peroxidase labeled soybean agglutinin (called the lectinosorbent assay).

The contrast of gram-negative bacteria and gram-positive bacteria is the cell wall of gramnegative bacteria contains to lipid but cell wall of gram-positive bacteria does not have the lipid. In 1968, Doyle et al., provided evidence that Concanavalin A reacts with macromolecules that are devoid of terminal glucopyranose or mannopyranose residues (Doyle et al., 1968). Their investigations demonstrated that Concanavalin. A precipitates lipopolysaccharide preparation derived from various strains of *Escherichia coli* as well as

penetrating the cytoplasm. Therefore, plant lectins cannot alter the structure and/or permeability of the membrane or disturb the normal intracellular processes of invading microbes. Therefore, if lectins play a role in the plant's defense against bacteria, it must be through an indirect mechanism that is based on interactions with cell wall carbohydrates or

In 1936, a using lectin in clinical microbiology began when Summer and Howell (Summer and Howell, 1936) had a report that Concanavalin A can agglutinated various *Mycobacterium* spp. The interactions between plant lectin and microorganisms have been applied for typing of bacteria, fungi, and protozoa. It is useful for characterizing bacterial cell components and for detecting bacteriophage receptors. (Etzler, 1983; Lis and Sharon, 1986; and Nicolson, 1974). The unique property of lectin to bind non-covalently to simple sugars and polysaccharides has attracted interest in microbial taxonomy. Lectin has a role in the clinical laboratory identification and taxonomic classification of many microorganisms. Because lectins are generally monoclonal proteins and because they possess a spectrum of specificities and molecular weights, they are substantial tools for diagnostic microbiology applications. Recent observations with regard to the binding of plant lectins to components of the bacterial cell wall peptidoglycans (such as muramic acid, *N*-acetylmuramic acid, *N*acetylglucosamine and muramyl dipeptides) revealed that seed lectins from several legume species strongly interact with these bacterial surface carbohydrates (Ajouba et al., 1994). Evidently, the observation that legume seed lectins can recognize and bind to the bacterial cell wall does not imply that such an interaction occurs *in vivo* and certainly does not prove

that these lectins are involved in the protection of the seedlings against bacteria.

horseradish peroxidase labeled soybean agglutinin (called the lectinosorbent assay).

The contrast of gram-negative bacteria and gram-positive bacteria is the cell wall of gramnegative bacteria contains to lipid but cell wall of gram-positive bacteria does not have the lipid. In 1968, Doyle et al., provided evidence that Concanavalin A reacts with macromolecules that are devoid of terminal glucopyranose or mannopyranose residues (Doyle et al., 1968). Their investigations demonstrated that Concanavalin. A precipitates lipopolysaccharide preparation derived from various strains of *Escherichia coli* as well as

Lectin has been used for investigating virulence factors, surface structures, and identification of gram-positive bacteria. For example; lectin from *Dolichos biflorus* was used to confirm its specificity for identifying group C streptococci. In another test, its crude extract was coupled to polystyrene particles with a spacer arm to yield an effective lectinlatex reagent that agglutinated group C streptococcal antigens prepared as nitrous acid, autoclave,or enzyme extracts. (Slifkin and Gil, 1984) Group C streptococcal isolates from horses and cattle agglutinated with lectin from *Dolichos biflorus* and *Helix pomatia*. (Schalla et al., 1986). Concanavalin A could be precipitated various bacterial polysaccharides, with interacts specifically with bacterial cell walls containing glycosidic residues associated with teichoic acid. Accordingly, bacteria teichoic acids from cell wall containing α-glucopyranisyl residues,such as *Lactobacillus plantarum*, *Staphylococcus aureus*, and *Bacillus subtilis*. (Archibald and Coapes, 1971; Doyle et al., 1982; and Reeder and Ekstedt, 1971). Lectins from soy bean have been used to assay for detecting *Bacillus anthracis* (Cole et al., 1984). The use of soybean agglutinin (SBA) to detect very low numbers of buffered suspention of *Bacillus anthracis* vegetative cells and spores has been reported (Graham et al., 1984). The strategy was to bind the cells or spores to polystyrene plates and to detect the bound forms with

extracellular glycans.

from *Shigella flexneri* and *Salmonella abortivoequina*. In 1970, other investigators demonstrated that Concanavalin A can be used to detect lipopolysaccharides of various Salmonella strains as determined by gel diffusion. (Goldstein and Staub, 1970).

In 2010, Petnual et al. reported the antimicrobial activity of *Curcuma longa* lectin, expressed as the minimal inhibitory concentration (MIC), was found to inhibit the growth of all five microbial species tested, the four bacteria, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, plus the yeast *Candida albicans*, at MIC values of ≥ 0.011, 0.005, 0.092, 0.002 and 0.0046 mg/ml, respectively (Petnual et al. 2010). These results demonstrate that the *Curcuma longa* rhizome lectin is likely to be at least one of the, if not the, candidate molecule responsible for the antibacterial action observed in rhizome extracts from this plant. An outstanding feature of the antibacterial activity of the isolated lectin is it is somewhat nonselective against this fairly diverse selection of bacteria. The potentially broad effect of the *Curcuma longa* rhizome lectin on the growth inhibition of several diverse bacterial strains, confirms the important interaction between the lectin and all the strains under consideration. From the tested strains, *Pseudomonas aeruginosa* (lowest MIC) seemed to be most sensitive to the presence of lectin. Previouse studies of the binding of plant lectins to bacterial cell wall peptidoglycans indicate that several lectins of different carbohydrate specificities can recognize most of the components of the bacterial cell wall, such as muramic acid, *N*acetylglucosamine, *N*-acetylmuramic acid and muramyl dipeptide (Ajouba et al., 1994).

Archidendron jiringa seed lectin was selected to test for antimicrobial activity with *Escherichia* coli, Pseudomonas auroginosa, Bacillus subtilis, Staphylococcus aurous, and Candida albican (Charungchitrak et al., 2011). The MIC of Archidendron jiringa seed lectin with Candida albican equal in S. aurous to be 0.0567 mg /ml and in Bacillus subtilis to be 0.2266 mg/ ml. But the MIC with *Escherichia* coli and Pseudomonas auroginosa is not detected, demonstrating stronger antimicrobial activity against gram-positive than gramnegative bacteria. Accordingly, the binding of lectins to muramic acid and N-acetylmuramic acid, carbohydrates present in the bacterial cell wall (mainly in gram-positive bacteria), has been reported (Ajouba et al., 1994). These data suggest that lectins probably play a role in plant defense, not only against phytopathogenic invertebrates, herbivores or fungi, but also against bacteria. The carbohydrate-binding site probably plays a key role in this activity, being responsible for the recognition of bacteria. Almost all microorganisms express surfaceexposed carbohydrates. These carbohydrates may be covalently bound, as in glycosylated teichoic acids to peptidoglycan, or non-covalently bound, as in capsular polysaccharides (Hirmo, et al., 1997; and Caldeon, et al., 1997). Every surface-exposed carbohydrate is a potential lectin-reactive site. The ability of lectins to form complexes with microbial glycoconjugates has made it to be employed as probes and sorbents for whole cells, mutants, and numerous cellular constituents and metabolites.

The lectin from *Curcuma amarissima* inhibited 4 microbial growth consist of *Bacillus subtilis*, *Candida albicans*, *Escherichia coli*, and *Staphylococcus aureus* at concentration ≥ 0.446, 0.446, 0.223, and 0.892 mg/ml respectively. But can not inhibite *Pseudomonas auroginasa* growth because at the surface of *Pseudomonas auroginosa* cell does not have polysaccharide ligands which can interact with *Curcuma amarissima* lectin. (Kheeree et al., 2011) Similar to Legume lectin from *Trinella foenumgraecum, Trifolium alexandrium, Bauhinia variegata,* and *Delonix regia* had a research that these lectins from sephadex G-150 can agglutinated both gram negative and gram positive bacteria (*Mycobacterium rhodochrous, Bacillus cercur, Bacillus megaterium,* 

Antimicrobial Activity of Lectins from Plants 165

In 2011, Kheeree et al. purified *Curcuma amarissima* lectin showed *in vitro* antifungal activity against three plant pathogenic fungal species, *Colectrotrichum cassiicola*, *Exserohilum turicicum*, and *Fusarium oxysporum*. It strongly inhibited the growth of *Colectrotrichum cassiicola* at 17.5 µg for *Fusarium oxysporum* and *Exserohilum turicicum*, which were strongly inhibited at the higher concentration of 35 μg (Fig. 6). Antifungal activity has been observed in other lectins where, for example *Astragalus mongholicus* root lectin revealed antifungal activity against various species of phytopathogenic fungi (Yan et al., 2005). Similarly with lectin from *Talisia esculenta* seeds inhibited the growth of *Fusarium oxysporum*, *Colectrotrichum lindemuthianum*, and *Saccharomyces cerevisiae.*(Freire et al., 2002) *In vitro* studies demonstrated that two novel chitin-binding lectins seeds of *Artocarpus integrifolia* inhibited the growth of *Fusarium moniliforme* and *Saccharomyces cerevisae* (Trindade et al., 2006). Many studies of plant lectins have assumed that they are implicated in host defense mechanism as antifungal proteins. However, to date only a small number of lectins have been reported to have actual antifungal activity such as lectin from the rhizomes of *Ophiopogon japonicus* showed antifungal activity against *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008). The purified *Phaseolus coccineus* Lectin (PCL) was devoid of antifungal activity against *Candida albicans* and Penicillium *italicum* (Chen et al., 2009).

Fig. 6. Inhibitory effect of purified *Curcuma amarissima* lectin on the *in vitro* growth on PDA plates (as an antifungal activity bioassay) of; (A) *Colectrotrichum cassiicola*, (B) *Fusarium oxysporum* and (C) *Exserohilum turicicum*. For each plate, 0.625 cm diameter discs were seeded with 10 μl of TBS (a) alone as the negative control, or containing either (b) 17.5 μg/

A large body of data exists on the interaction of lectins with a relatively broad spectrum of parasites ranging from the protozoa through the metazoa. Although Concanavalin A was used by many investigators as a lectin probe for these organisms, many other lectins have been shown to be of value in the study of cell surfaces and the identification and differentiation of the parasites. In some instances virulence of parasitic protozoa appears to be related to their surface properties, as revealed by interactions with lectins. Thus, several investigators have deemed important the comparison of surface saccharides of parasites known to differ in their virulence traits. It has been conjectured that the virulence of the trophozoite form of *Entamoeba histolytica* may depend, in part, on its surface properties. Data have been presented indicating that only strains isolated from cases of amoebic dysentery agglutinate with Concanavalin A (Martinez-Palomo et al., 1973) strains isolated from asymptomatic cases of amoebic dysentery, however, do not agglutinate with this lectin.

ml or (c) 35 μg/ ml purified *Curcuma amarissima* lectin (Kheeree et al., 2011).

*Bacillus sphaericus, Escherichia coli, Seratia marcescens, Corynebacterium xerosis,* and *Staphylococcus aureus)* (Reda et al., 1992). Inaddition β-galactoside-binding lectin was extracted from the skin of amphibian, *Bufo arenarum*. It had an antimicrobial activity against Gram negative bacteria (*Escherichia coli* K12 4100 and wild strains of *Escherichia coli* and *Proteus morganii*) and Gram positive bacteria (*Enterococcus faecalis*) (Alicia *et al*., 2003).

Several investigators have concluded that lectins are useful reagents for the study of fungal cell surfaces and may also be of value as important aids in the classification of fungi (Barkai and Sharon, 1978). The major components of fungal cell wall is Chitin, a polymer of β-(1, 4)-*N*-acetyl-D-glucosamine (Barkai and Sharon, 1978; and Ebisu et al., 1977). The report of lecin interaction to fungal, such as fluorescein-conjugated wheat germ agglutinin has been shown to be an effective probe to detect chitin on hypha surfaces. (Barkai and Sharon, 1978; Galun et al., 1976; Galun et al., 1981; Mirelman et al., 1975; Molano et al., 1980; Tkacz and Lampson, 1972; Tracz et al., 1971; Tropchin et al., 1981). In 1975 Mirelman et al. was found wheat germ agglutinin (WGA) can be inhibits spore germination and hyphal growth of *Trichoderma viride* and interferes with the synthesis of chitin (Mirelman et al., 1975). A novel mannose-binding lectin was purified from rhizomes of *Ophiopogon japonicus* was showed antifugal activity in three phytopathogenic fungi namely *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008).

In 2010, Petnual et al. purified *Curcuma longa* lectin at a dose of 47 μg and 94 μg/0.3 cm2 disc showed antifungal activity against the three tested phytopathogenic fungal species, *Exserohilum turicicum*, *Fusarium oxysporum* and *Colectrotrichum cassiicola* (Fig. 5). While the lectin dose of 47 μg/0.3 cm2 disc slightly inhibited the growth of these three fungi, that at 94 μg/0.3 cm2 disc showed a higher and significant degree of antifungal activity on all three isolates (Petnual et al. 2010). This effective lectin dose of around 100 μg/ 0.3 cm2 disc is in accord with that reported for the lectin from *Annona muricata* seeds against the growth of *Fusarium oxysporum*, *Fusarium solani* and *Colletotrichum musae* (Damico, et al., 2003), and for the lectin from *Astragalus mongholicus* against *Fusarium oxysporum, Colletorichum* sp. and Drechslera *turcia* (Yan, et al., 2005). Other lectins, such as those from potato (Gomez, et al., 1995) and red kidney beans (Ye, et al. 2001), have also been reported to exhibit antifungal activity. However, novel non-lectin proteins with antifungal activity in plant rhizomes are also known, such as the 32 kDa protein in ginger rhizomes which exhibits antifungal activity toward *Fusarium oxysporum* at a dose of 32-160 μg/ 0.3 cm2 disc of ginger rhizome (Wang and Ng, 2005).

Fig. 5. Inhibitory affect of *Curcuma longa* lectin on antifungal protein toward *Exserohilum turicicum* (A), *Fusarium oxysporum* (B) and *Colectrotrichum cassiicola* (C). The negative control is 10 μl of 20 mM Tris-HCl buffer pH 7.4 (a), 47 μg *Curcuma longa* lectin. (b) and 94 μg *Curcuma longa* lectin (c) (Petnual et al., 2010).

*Bacillus sphaericus, Escherichia coli, Seratia marcescens, Corynebacterium xerosis,* and *Staphylococcus aureus)* (Reda et al., 1992). Inaddition β-galactoside-binding lectin was extracted from the skin of amphibian, *Bufo arenarum*. It had an antimicrobial activity against Gram negative bacteria (*Escherichia coli* K12 4100 and wild strains of *Escherichia coli* and

Several investigators have concluded that lectins are useful reagents for the study of fungal cell surfaces and may also be of value as important aids in the classification of fungi (Barkai and Sharon, 1978). The major components of fungal cell wall is Chitin, a polymer of β-(1, 4)-*N*-acetyl-D-glucosamine (Barkai and Sharon, 1978; and Ebisu et al., 1977). The report of lecin interaction to fungal, such as fluorescein-conjugated wheat germ agglutinin has been shown to be an effective probe to detect chitin on hypha surfaces. (Barkai and Sharon, 1978; Galun et al., 1976; Galun et al., 1981; Mirelman et al., 1975; Molano et al., 1980; Tkacz and Lampson, 1972; Tracz et al., 1971; Tropchin et al., 1981). In 1975 Mirelman et al. was found wheat germ agglutinin (WGA) can be inhibits spore germination and hyphal growth of *Trichoderma viride* and interferes with the synthesis of chitin (Mirelman et al., 1975). A novel mannose-binding lectin was purified from rhizomes of *Ophiopogon japonicus* was showed antifugal activity in three phytopathogenic fungi namely *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008).

In 2010, Petnual et al. purified *Curcuma longa* lectin at a dose of 47 μg and 94 μg/0.3 cm2 disc showed antifungal activity against the three tested phytopathogenic fungal species, *Exserohilum turicicum*, *Fusarium oxysporum* and *Colectrotrichum cassiicola* (Fig. 5). While the lectin dose of 47 μg/0.3 cm2 disc slightly inhibited the growth of these three fungi, that at 94 μg/0.3 cm2 disc showed a higher and significant degree of antifungal activity on all three isolates (Petnual et al. 2010). This effective lectin dose of around 100 μg/ 0.3 cm2 disc is in accord with that reported for the lectin from *Annona muricata* seeds against the growth of *Fusarium oxysporum*, *Fusarium solani* and *Colletotrichum musae* (Damico, et al., 2003), and for the lectin from *Astragalus mongholicus* against *Fusarium oxysporum, Colletorichum* sp. and Drechslera *turcia* (Yan, et al., 2005). Other lectins, such as those from potato (Gomez, et al., 1995) and red kidney beans (Ye, et al. 2001), have also been reported to exhibit antifungal activity. However, novel non-lectin proteins with antifungal activity in plant rhizomes are also known, such as the 32 kDa protein in ginger rhizomes which exhibits antifungal activity toward *Fusarium* 

*oxysporum* at a dose of 32-160 μg/ 0.3 cm2 disc of ginger rhizome (Wang and Ng, 2005).

Fig. 5. Inhibitory affect of *Curcuma longa* lectin on antifungal protein toward *Exserohilum turicicum* (A), *Fusarium oxysporum* (B) and *Colectrotrichum cassiicola* (C). The negative control is 10 μl of 20 mM Tris-HCl buffer pH 7.4 (a), 47 μg *Curcuma longa* lectin. (b) and 94 μg

*Curcuma longa* lectin (c) (Petnual et al., 2010).

*Proteus morganii*) and Gram positive bacteria (*Enterococcus faecalis*) (Alicia *et al*., 2003).

In 2011, Kheeree et al. purified *Curcuma amarissima* lectin showed *in vitro* antifungal activity against three plant pathogenic fungal species, *Colectrotrichum cassiicola*, *Exserohilum turicicum*, and *Fusarium oxysporum*. It strongly inhibited the growth of *Colectrotrichum cassiicola* at 17.5 µg for *Fusarium oxysporum* and *Exserohilum turicicum*, which were strongly inhibited at the higher concentration of 35 μg (Fig. 6). Antifungal activity has been observed in other lectins where, for example *Astragalus mongholicus* root lectin revealed antifungal activity against various species of phytopathogenic fungi (Yan et al., 2005). Similarly with lectin from *Talisia esculenta* seeds inhibited the growth of *Fusarium oxysporum*, *Colectrotrichum lindemuthianum*, and *Saccharomyces cerevisiae.*(Freire et al., 2002) *In vitro* studies demonstrated that two novel chitin-binding lectins seeds of *Artocarpus integrifolia* inhibited the growth of *Fusarium moniliforme* and *Saccharomyces cerevisae* (Trindade et al., 2006). Many studies of plant lectins have assumed that they are implicated in host defense mechanism as antifungal proteins. However, to date only a small number of lectins have been reported to have actual antifungal activity such as lectin from the rhizomes of *Ophiopogon japonicus* showed antifungal activity against *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008). The purified *Phaseolus coccineus* Lectin (PCL) was devoid of antifungal activity against *Candida albicans* and Penicillium *italicum* (Chen et al., 2009).

Fig. 6. Inhibitory effect of purified *Curcuma amarissima* lectin on the *in vitro* growth on PDA plates (as an antifungal activity bioassay) of; (A) *Colectrotrichum cassiicola*, (B) *Fusarium oxysporum* and (C) *Exserohilum turicicum*. For each plate, 0.625 cm diameter discs were seeded with 10 μl of TBS (a) alone as the negative control, or containing either (b) 17.5 μg/ ml or (c) 35 μg/ ml purified *Curcuma amarissima* lectin (Kheeree et al., 2011).

A large body of data exists on the interaction of lectins with a relatively broad spectrum of parasites ranging from the protozoa through the metazoa. Although Concanavalin A was used by many investigators as a lectin probe for these organisms, many other lectins have been shown to be of value in the study of cell surfaces and the identification and differentiation of the parasites. In some instances virulence of parasitic protozoa appears to be related to their surface properties, as revealed by interactions with lectins. Thus, several investigators have deemed important the comparison of surface saccharides of parasites known to differ in their virulence traits. It has been conjectured that the virulence of the trophozoite form of *Entamoeba histolytica* may depend, in part, on its surface properties. Data have been presented indicating that only strains isolated from cases of amoebic dysentery agglutinate with Concanavalin A (Martinez-Palomo et al., 1973) strains isolated from asymptomatic cases of amoebic dysentery, however, do not agglutinate with this lectin.

Antimicrobial Activity of Lectins from Plants 167

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The unique property of lectins to bind noncovalently to simple sugars and therefore to polysaccharides and glycoconjugates has attracted the interest of virologists. In virology, lectins have been used for detection of viral glycoproteins in purified and infected cells, as well as for viral purification. Lectin studies have revealed information about the structure of viral glycoproteins, structures important in their pathogenicity. A significant contribution of lectin use in virology has been in the development of unique diagnostic methods that yield specific identification of viral agents. Purified influenza virus yields macroscopically visible flocculation when mixed with Concanavalin A. (Klenk et al., 1984) When influenza virus is treated with a proteolytic enzyme, the glycoprotein spikes of the virus are released. These treated viral particles no longer agglutinate with this lectin, but will flocculate in the presence of *N*-acetylgalactosamine-associated lectins, such as *Dolichos biflorus* or *Helix pomatia*. Other viruses, including arboviruses, vesicular stomatitis virus, paramyxoviruses, leukoviruses, and hepatitis B virus, also agglutinate with Concanavalin A. Concanavalin A was shown to block specifically adsorption of the bacteriophage binding sites of *Bacillus subtilis* possessing αglucosylated teichoic acids in the cell walls associated with teichoic acids. It was suggested that the application of this lectin might be useful as a means to correlated bacteriophage and serologic typing of staphylococci. (Archibald and Coapes, 1972).
