The Spectrum of Infections by Fusarium Species on Codiaeum Variegatum (L.) Blume Cultivars as Influenced by Fructose Specific Lectin

This study sought to have an insight into the mechanism of action of diseases responsible for the susceptible Codiaeum variegatum (garden croton) leaves induced by Fusarium species and the clinical importance of the d isease resistant types. The plants were obtained from the Babcock University Germplasm Repository. Therefore, phytopathogenic Fusarium species were isolated from three diseased susceptible cult ivars of C. variegatum (ovalifolium, royal-like and punctatum). Accordingly, lect in was isolated, purified and characterized from the leaves of a resistant garden croton cultivar (royal), and further evaluated for antifungal act ivity using isolates of Fusarium lateritium and F. semitectum obtained from the three diseased cultivars. The heamagglutinating activity of the purified lect in was non selective to type of blood group (A, B, AB and O) and was inhib ited by fructose, sialic acid, and copper sulphate, but was enhanced by galactose, calcium chloride, sodium chloride and magnesium chloride. Optimum heamagglutinating activ ity of the lectin was achieved at 30–40°C and pH 5.0–6.0. The lectin exh ibited antifungal activity against the two Fusarium species in a non-concentration dependent manner. It is therefore concluded that Fusarium species are major phytopathogens of the garden croton plant and their spectrum of pathogenicity is dependent on the presence or absence of lectins. In addition, the resistant cultivar of C. variegatum (cv. royal) used in this study may be a suitable candidate for the prevention and treatment of fungal infections.


Introduction
Lectins are carbo hyd rate-b ind ing p ro teins th at b in d revers ibly and possess the ability to agg lut inate cells or precipitate polysaccharides and glyco-conjugates. They are widely d istributed in animals, plants and microorgan isms and h ave attracted g reat interest due to th eir v arious biological act ivities, such as cell agglutination, anti-tumor, immuno-modulato ry, antifungal, ant iv iral and ant i insect activ ities (1)(2). Recent s tu d ies in g ly co b io lo gy have emp has ized lectins as the p rime to o ls fo r cell-to -cell recognition in interact ions involving nu merous pathogens such as viruses, fungi, bacteria and the pluricellular parasites. The lect ins recogn ize and b ind to the o ligosaccharides exposed by target cells and tissues thereby leading to the establishment of an infection (3 ). Conversely, the pathogen surfaces bear a large number of oligosaccharides that may be bound by specific lect ins which can modulate the host infection (4)(5).
These carbohydrates may be covalently bound, as in glycosylated teichoic acids to peptidoglycan, or non-covalently bound, as in capsular polysaccharides. Every surface-exposed carbohydrate is a potential lectin-reactive site. The ability of lectins to selectively form co mplexes with microbial glyco-conjugates makes them useful investigative tools for host-pathogen interaction. In the natural environment, microorganisms interact with each other to maintain their gro wth, development and stability within ecosystem. The interaction process can only be revealed by understanding the fundamentals of ecological relat ionships of diverse microbial population including pathogens and antagonists. In many instances, physical attachment of microorganis ms is mediated by specific compatib le macro molecules. The specific interaction between microbial populations may play a key ro le for successful establishment, persistence and colonization (6 -7). The role of cell surface macro molecules, potential ligands and receptors of cell surface agglutinins or lectins as recognition factor during attachment of t wo interacting partners, are well known (8)(9).
Previous studies on the Codiaeum variegatum cultivars in the Germp lasm Repository of Babcock University have associated some Fusarium species with the increased disease conditions of this ornamental plant (10)(11). Ho wever, no study has been carried out to investigate the mechanism of disease occurrence in cultivars members of the plant. In the present study, lectin purified fro m a disease resistant croton cultivar in the germplas m repository was partially characterized and evaluated for antifungal activ ity using two Fusarium species isolated from three other diseased susceptible cultivars of the garden croton ornamental p lant.

Collection of Plant Materi als
The leaves (diseased and non diseased) of four Codiaeum variegatum (garden croton) cultivars (C. variegatum cv. royal, C. variegatum cv. ovalifolium, C. variegatum cv. royal-like and C. variegatum cv. punctatum) (Fig. 1) were collected fro m the horticultural garden of Babcock University Germp las m Repository (BUGR), Ilishan-Re mo, Nigeria. Field botanical characterizat ion and identification of the cultivars was carried out by one of the authors who is a Plant Scientist and Biotechnologist in charge of the BUGR. Codiaeum variegatum cv. royal was collected as the resistant cultivar wh ile C. variegatum cv. ovalifolium, C. variegatum cv. royal-like and C. variegatum cv. punctatum were obtained as the susceptible cultivars. The cultivars were placed in clean polythene bags and transported to the Microbiology laboratory of Babcock University for further analysis.

Isolati on and Characterizati on of Leaf-B orne Fusarium in Gar den Croton Culti vars
Leaf-borne Fusarium was isolated according to the method described by Dubey & Maheshwari (2006). Diseased leaves of each cultivar of garden croton were surface sterilized for 3 minutes in 2% sodium hypochlorite. The sterilized leaves were rinsed in two changes of sterile distilled water and blotted dry in two fo lds of Whatman No. 1 filter paper. Two 3mm d iscs each were obtained from the visibly infected and uninfected regions of the leaves of a cultivar, and plated out on peptone-pentachloronitrobenzene agar (PPA ), a semi selective mediu m for Fusarium (13). Each set of two discs from the infected region was placed along the diameter line o f the PPA p late at 3cm d istance from each other. This was repeated for the discs fro m the uninfected region of the same cultivar. The set up was replicated thrice for each cult ivar and all the inoculated plates were incubated for 3 days under fluorescent lights on a 12 h day/ night schedule at 22-24℃.
After 3 days, morphologically d istinct colonies of Fusarium species that emerged fro m the leaf d iscs were transferred to ¼ strength potato dextrose agar (PDA) and incubated for 5 days as described above. Single spore was started for each isolate by micro manipulat ion on 2% water agar and the plates were incubated overnight at 22-24℃. The germlings were sub cultured fro m water agar and maintained on a modified Czapek's-Do x comp lete mediu m (CM ). This was stored at 4℃ prior to identification. Each isolate was further sub cultured from CM onto carnation leaf agar (CLA for examination of sporodochia and uniform macro conidia under the Oly mpus BX51 Digital Microscopy, Oly mpus Optical Co., LTD, Japan) and full strength PDA (for pig mentation and colony morphology evaluation). The p lates were incubated as above for 10-14 days. Morphological identification of the Fusarium species was performed as described by Leslie & Su mmere ll (2006).

Pathogenicity Testing of Fusarium Isol ates on Garden Croton Leaves
The pathogenicity of each Fusarium isolate obtained from the diseased parts of the croton leaves was tested by adopting the Koch's postulate as modified and reported by Dubey & Maheshwari (2006). About 20 young uninfected leaves were obtained fro m the ap ical parts of each cultivar and surface sterilized for 3 minutes in 2% sodium hypochlorite. The sterilized leaves were rinsed in two changes of sterile distilled water and blotted dry in two fo lds of Whatman No. 1 filter paper. Mild abrasions along the veins of each young uninfected leaf were induced using a sterile toothpick. Thereafter 0.1ml spore suspension containing 10 6 spores/mL of each corresponding Fusarium isolate was spread over the abrasions using a sterile cotton bud. Each inoculated leaf was placed on a moistened filter paper in a Petri dish which has been under-layed with cotton wool. All Petri dishes were incubated at ambient temperature for 5 days and the leaves were observed daily for sympto ms of the disease on the inoculated part of the leaves. The diseased parts of the inoculated leaves were obtained, surface sterilized and plated on to ¼ strength potato dextrose agar (PDA). After a 5-day incubation period at 22-24 ℃ the associated Fusarium isolates were identified macroscopically and microscopicall y following the procedures reported earlier.

Blood Samples
Five milliliter each of 10 hu man b lood samples of different blood groups (A, B, A B and O) was collected fro m Babcock University Medical Laboratory, Ilishan-Re mo, Nigeria. The b lood samples were co llected in heparin ised bottles containing (ethylene diamine tetra acetate, EDTA) to prevent coagulation and kept at 4℃ prior to analysis.
The blood samples were treated as described by Lis et al. (1994). Briefly, 5mL of each sample was centrifuged at 1500 × g for 5 minutes at ambient temperature. The red blood cells obtained were then washed by centrifugation at 1500 × g for 5 minutes at ambient temperature with 0.01M phosphate-buffered saline (PBS, p H 7.2). This was repeated twice and the resulting cells were mixed with 3% formaldehyde in EDTA bottle and allowed to stir gently overnight prior to further centrifugation at 1500 × g for 5 minutes. The centrifuged blood cells were washed again as stated earlier, three times with 0.01M PBS (pH 7.2) and the cells were co llected into a stopped bottle. About 76.8mL of 0.01M PBS was added to dilute the concentration of the cell. This concentration was then stored at 4℃ prior to further analysis.

Isolati on and Purification of Lectin fr om C. Variegatum Le aves
Lectin was isolated and partially purified fro m C. variegatum cv. royal according to the procedures described by Awoyinka & Dada (2011) with slight modification. The leaves were pulverized and defatted using chloroformacetone in ratio 2:1 thereafter the defatted samples were dissolved in distilled water (1:20 v/v). An aliquot of the mixtu re was separated using Whatman filter paper and kept in refrigerator for carbohydrate analysis, while the other part was centrifuged at 1500 × g for 30 minutes. Pellets were obtained and discarded while the supernatant was collected for ammoniu m sulphate precipitation as described by Trowbridge (1974). The precip itated protein were pulled together and dissolved in 240ml of distilled water, the resulting mixtu re was concentrated by ultra-filtration (Millipore, India) at 1500g for 30 minutes before dialyses against 0.15M NaCl -0.01M NaPO 4 buffer for 24 hours. The dialy zed samp le was then collected in an empty bottle and stored at 4℃prior to assay for lectin activ ity.

Assay for Lectin Acti vi ty
Agglutination of red blood cells by the ext ract and the various fractions that were obtained during the purification steps was determined as described by Bing et al. (1967). A serial two-fold dilution of the lectin solution was mixed with 50μL of a 4% suspension of human erythrocytes in PBS (pH 7.2), at ambient temperature. The erythrocytes of human blood group A, B and O were fixed with 3% formaldehyde. The plate was left undisturbed for 60 minutes at ambient temperature in order to allow the agglutination of erythrocytes to take place. The heamagglutination titre of the lectin exp ressed as the reciprocal of the highest dilution exhibit ing visible agglutination of erythrocytes was reckoned as one heam agglutination unit. Specific act ivity was expressed as the number of heamagglutination units per microgram protein (19).

Anti-Fung al Sensitivity Assay
The in vitro sensitivity of the isolated Fusarium species to the partially purified lectin was determined according to a modified disc diffusion method for antibacterial assay (20). Each Fusarium isolate was inoculated at the center of a 9cm Petri dish containing freshly prepared PDA and incubated at 22-24℃ for 3 days under a 12-hour light/darkness schedule. Four pieces of 3mm sterile paper discs were placed on the four cardinal points of the growing culture at distances of 0.5cm away fro m the boundary of the colony so as to give a square. Each of the four d iscs in a Fusarium culture was mo istened with 5µL of the partially purified lectin suspension. This procedure was performed for the 10µL and 20µL treat ments of paper discs in a Fusarium culture with the partially purified lectin. A ll cultures were treated in triplicates for each lectin treat ment. Two sets of triplicate control plates were set up by imp regnating the paper discs with 20µL of 0.15M NaCl in one set and 5µL of sterile distilled water in the other. The p lates were then incubated as stated above for 4 additional days. The zones of inhib ition were measured daily for 4 days.

Inhi bi tion of Lecti n-induced Heamagglutinati on by Various Carbohydr ates
The ability of various carbohydrates to inhibit lectin-induced heamagglutination was investigated using a procedure that is analogous to the heamagglutination test described by Kuku et al. (2009). The sugars used were glucose, galactose, maltose, fructose, sucrose, lactose, raffinose, trehalose and sialic acid. Serial two-fold dilutions of each sugar sample were prepared in PBS. A ll the dilutions were mixed with an equal volume (50μL) of the lectin solution of known heamagglutination units. The mixture was allo wed to stand for 60 minutes at ambient temperature and then mixed with 50μL of a 4% hu man erythrocyte suspension. The heamagglutination titres obtained were compared with a non-sugar containing blank. The minimu m concentration of the sugar in the final reaction mixture which completely inhibited heamagglutination units of the lectin sample were obtained (19).

Effect of salts on Heamagglutinating Acti vity of Lectin
Nine salts [calciu m chloride, CaCl 2 ; iron (III) sulphate, Fe 2 (SO 4 ) 3 ; ferric chloride, FeCl 3 ; sodium sulphate, Na 2 SO 4 ; copper sulphate, CuSO 4 ; magnesium ch loride, Mg Cl 2 ; sodium chloride, NaCl; potassium chloride, KCl; potassium phosphate, KH 2 PO 4 ] were evaluated for their inhibitory ability against heamagglutination induced by lectin. Serial two-fold d ilutions of salt samp les were prepared in PBS. A ll the dilutions were mixed with an equal volume (50μL) of the lectin solution of known heamagglutination units. The mixtu re was allowed to stand for 60 minutes at ambient temperature and then homogenized in 50μL of a 4% hu man erythrocyte suspension. The heamagglutination titres obtained were compared with a non-salt containing blank. The minimu m concentration of the salt in the final reaction mixtu re wh ich co mpletely inhibited heamagglutination units of the lectin sample were obtained (19).

Effect of Te mperature on Heamagglutinating Acti vi ty of Lectin
The method of Patrick et al. (2007) was adopted for testing the effect of various temperature reg imes on the agglutinating activity of lectin obtained from croton. The purified lectin was incubated in a water bath for 30 minutes at various temperatures: -10, -4, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100℃, and then cooled to 20℃. Heamagglutination assay was carried out as previously described.

Effect of pH on Heamagglutinating Acti vity of Lectin
The effect of different pH regimes (2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) on the activity of lectin obtained fro m croton was determined by incubating the lectin in the buffers of different pH values. The pH values of 0.15M NaCl -0.01M NaPO4 buffer were altered using concentrated HCl and 1M NaOH, and assaying for heamagglutinating activ ity. The control values were the agglutination titre of the lectin in PBS (pH 7.2).

Results
As a result of init ial field observations carried out so as to determine the magnitude and severity of the infection by the Fusarium species, the cultivars of the garden croton have been described and categorized into THREE: namely, heavily susceptible (A), resistant (B) and tolerant (C) cultivars. In the category "A" the pathogen kills aborts the leaves and kills the twigs; whilst in the category "B" no signs of pathogenicity was observed. However, in the category "C" only necrotic leaf spots which do not spread or coalesce was the case. The leaves of Codiaeum variegatum cv. royal showed no growth of Fusarium while the diseased leaf discs of C. variegatum cv. ovalifolium, C. variegatum cv. royal-like and C. variegatum cv. punctatum showed growths of Fusarium species (Fig. 2). The isolates obtained from C. variegatum cv. ovalifolium were identified as F. semitectum and F. lateritium while those from C. variegatum cv. royal-like and C. variegatum cv. punctatum were all F. lateritium.
Lectin fro m C. variegatum cv. royal was found to agglutinate human erythrocytes and showed an activity that was non-selective to type of blood group (A, B, A B and O). The three concentrations of lectin exh ibited antifungal activity towards all Fusarium isolates by inhib iting hyphal growth of the test isolates while the controls (NaCl and sterile distilled water) had no inhibitory effect on the isolates (Fig. 4). The data obtained from the studies for the inhibitory effects of different sugar concentrations on lectin activity of C. variegatum cv. royal lectin are presented in Tab le 1. It was observed that all concentrations of maltose, lactose and sucrose had no inhibitory or stimulatory effect on heamagglutinating activity of the lectin. The 100 and 200 mM/ L glucose concentrations enhanced agglutination of human erythrocytes more than other concentrations of the sugar while the highest concentration of 800mM/ L resulted in a complete loss of heamagglutination activity. In the presence of very low (25mM/L) and very high (400 and 800mM/ L) galactose concentrations, we observed an increased binding of the lectin to human erythrocytes. On the other hand, fructose, trehalose and sialic acid d id not inhib it or stimulate heamagglutination at very low concentration of 25mM/L, but showed a complete inhib ition of the lectin's heamagglutination activity at higher concentrations of 100-800mM/ L sugar. At concentrations of 50-200mM/L raffinose, heamagglutinating activity of the lectin was completely lost while an increase of the concentration of raffinose to 400mM/ L enhanced the lectin's potential to bind to the red blood cells. Results similar to those observed in our study were reported by Perez (1995), who purified lectin fro m the seeds of Erythrina costarisencis.
The characterizat ion and inhibition studies to define the salt specificit ies of lectin purified fro m C. variegatum cv. royal ( Table 2) showed that all concentrations of Fe 2 (SO 4 ) 3 and FeCl 3 did not inhib it or enhance the heamagglutinating potential o f the lectin. On the contrary, the presence of 100-800mM/ L CuSO 4 co mp letely inhib ited heamagglutinat ion wh ile the lower concentrations had no stimulatory effect either. The heamagglutinating potential of the lectin was at its peak in the presence of lower concentrations of CaCl 2 and NaCl, and as the concentrations reached 200mM/ L for CaCl 2 and 400mM/ L for NaCl the activity reduced until no significant influence of the salt on lectin -binding activ ity was observed at 400-800mM/ L CaCl 2 and 800mM/ L NaCl. Contrariwise is the higher stimulatory effect of increased concentrations of MgCl 2 (200-800mM/ L) and KCl (400-800mM/ L) towards the agglutination of hu man erythrocytes by lectins. Similar results were reported on lectin ext racted fro m the seeds of Cissus populnea (16). Figure 5 shows the heamagglutinating activity of lectin obtained from C. variegatum cv. royal under different temperature regimes. It was observed that there was no heamagglutination activity at incubation temperature of 20 ℃ and below. However, a rapid increase in heamagglutination and consequent peak level of activity was reached at incubation temperature of 30-40℃; this formed a plateau between the regimes. When the mix (lectin -erythrocytes) was incubated at higher temperatures (50 ℃ and above), a drastic reduction in activity was observed until the activity was comp letely lost at 80℃ and above. This therefore indicates that ambient temperature is best suitable for the b inding activity of this C. variegatum cv. royal lectin. This finding is in contrast to that of the fructose-binding lectin purified fro m the gill of Aristichthys nobilis (24), where the lect in incubated at 50℃ for 30mins showed agglutination that was fourfold stronger than when it was incubated at ambient temperature.
Data on the effect of pH on lect in activity are p resented in Fig. 6. Lect in activity was stable at two pH ranges of 5.0-6.0 (acidic) and 9.5-10.5 (alkaline). However, optimu m act ivity was recorded at pH range of 5.0-6.0. This result may therefore suggest that the protein has two binding sites; one site more act ive at a slightly acid ic p H range of 5.0-6.0 and the other at basic pH of 10.0. Th is finding is however, in contrast to that reported in which Manila clam lectin activity was stable between pH 6 and pH 9, and was temperature-dependent (25). Our findings is also in contrast to other reports where A. nobilis lectin showed a decrease in agglutination activity on incubation at pH 6 for 30 mins therefore indicating its unstable nature under such conditions (24).

Discussion
In this study, the inhibitory activity of the lectin was not concentration dependent because there was no significant difference in the zones of inhibition caused by the lectin concentrations against the isolates although the zones produced by the 20µL lectin concentration were higher than those of the lower concentrations (data not shown). This finding supports the report of Awoyinka & Dada (2011) and the wider inhibit ion zone may be due to an increased concentration translating into a higher antifungal activity. It has been suggested that plant lectins may have important roles according to their abundance, including in the immune defense, and also that lectins have been co-opted for several functions during evolution (24). The role of lectin in defense mechanis m of plants may have evolved fro m the ability of the lectins to agglutinate and immob ilize microorganisms. The supporting evidence for this proposed role in defense against pathogens falls into two categories: the presence of lectins at potential sites of invasion by infectious agents and the binding of lectins to various fungi and their ability to inhibit fungal gro wth and germination (26). The ability of C. variegatum lectin to inhib it fungal gro wth suggests that it may play an important role in immobilizing invading micro organism particu larly fungal infections. The inhibit ion of fungal growth may have occurred through lectin binding to hyphae resulting in poor absorption of nutrients as well as by interference on spore germination process. This by interpretation means that resistance occurs as soon as the pathogen arrives on the leaves, a phenomenon referred to as a Systemic Acquired Resistance (SAR) due to the production of phytoallexins (27). When plants respond to attacks by either other plants especially lower plants called cryptogams, as pathogens or animals (herb ivores) plants counter these threats with inherent defence systems that deter herbivory and or prevent infections or combat pathogens that infect. Defense systems could be physical, genetic, chemical or biochemical, that is, by ability of the plant to recognize invading pathogen; especially if unsuccessful. Occasionally, a kind of co mpro mise might have been evolved between the plants and pathogens. Under such cases, the pathogen simp ly ensures its own survival without severely damaging or killing the plant who lly or partially (27). The relationship between plant lect ins and phyloallexins though not studied in the investigation it might be another classical examp le of the fact that in plants, there are usually more alternative biochemical pathways evolved to regulate or control processes that ensure survival when plants are under stress, unlike what obtains in animals.
The isolation of Fusarium species fro m the diseased leaves of susceptible cultivars of C. variegatum investigated in this study (Fig. 3) is in line with a previous report (11). This study has shown that Fusarium species are major phytopathogens of the garden croton plant and their spectrum of pathogenicity is dependent on the presence or absence of lectins. The ability of lectin fro m disease resistant C. variegatum to inhib it the Fusarium species suggests that it may play an important role in immob ilizing invading microorganis ms particularly fungal in fection. In addit ion, the carbohydrate-binding site of C. variegatum lectin may be vital in this activ ity, being responsible for the recognition of the fungi. The resistant cultivar of C. variegatum used in this study may be a suitable candidate for the p revention and treatment of fungal infect ions.

Conclusions
This study has shown that Fusarium species are major phytopathogens of the garden croton plant and their spectrum of pathogenicity is dependent on their Fructose binding specific lectins and their clin ical importance. In addition, the resistant cultivar of C. variegatu m (cv. royal) used in this study may be a suitable candidate for the prevention and treatment of these fungal infections, through graftage and or hybridization. Although this study is not a comprehensive exercise on plant defenses against pathogens, the "lectins concept" is probably just one of these defenses.