Appl Biochem Biotechnol (2014) 172:10981105

DOI 10.1007/s12010-013-0600-9

Antifungal Activity of Microgramma vacciniifolia

Rhizome Lectin on Genetically Distinct Fusarium
oxysporum f. sp. lycopersici Races

Lidiane Pereira de Albuquerque &

Giselly Maria de S Santana &
Thiago Henrique Napoleo &
Luana Cassandra Breitenbach Barroso Coelho &
Mrcia Vanusa da Silva & Patrcia Maria Guedes Paiva

Received: 25 July 2013 / Accepted: 9 October 2013 /

Published online: 20 October 2013
# Springer Science+Business Media New York 2013

Abstract Fusarium oxysporum f. sp. lycopersici races 1, 2, and 3 deteriorate tomato crops
since they cause a vascular wilt. Lectins are carbohydrate-binding proteins with hemagglutinating
and antifungal activities. This work reports that Microgramma vacciniifolia rhizome lectin
(MvRL) inhibits F. oxysporum f. sp. lycopersici race 3 growth (61 %) more intensely than of
races 1 (55 %) and 2 (45 %). The hemagglutinating activity of MvRL was inhibited by
glycoprotein preparations from mycelia of races 1, 2, and 3, and these data indicate that lectin
carbohydrate-binding sites recognized glycosylated molecules from races. Inter-simple sequence
repeat (ISSR) marker system showed that race 3 is genetically distinct from races 1 and 2, and
thus the highest sensitiveness of F. oxysporum f. sp. lycopersici race 3 to MvRL may be due to
molecular characteristics of this race.

Keywords Microgramma vacciniifolia . Rhizome lectin . Antifungal activity . Fusarium

oxysporum f. sp. lycopersici . ISSR

Introduction

Lectins are proteins that have the ability to bind to carbohydrates present on different cell
surfaces [1]. Lectins are widely distributed in plant tissues, and it has been suggested that these
proteins could play a protective role against phytopathogenic fungi [24]. Ophiopogon japonicus
rhizome lectin was shown to be a potential fungicide active against Gibberella saubinetii and
Rhizoctonia solani, while the lectin isolated from Curcuma amarissima rhizome inhibited the
growth of Fusarium oxysporum, Exserohilum turicicum, and Colectrotrichum cassiicola [5, 6].
Plant lectins also were antifungal agents against other Fusarium species [2, 3, 7].

L. P. de Albuquerque : G. M. de S Santana : T. H. Napoleo : L. C. B. B. Coelho : M. V. da Silva :

P. M. G. Paiva (*)
Departamento de Bioqumica, Centro de Cincias Biolgicas, Universidade Federal de Pernambuco,
Recife, Pernambuco 50670-420, Brazil
e-mail: ppaivaufpe@yahoo.com.br
Appl Biochem Biotechnol (2014) 172:10981105 1099

The high variability in morphology, colony color, and pathogenicity among Fusarium
species results in a complex classification divided into sections, formae speciales (f. sp.), and
races [8]. Molecular methods like inter-simple sequence repeat (ISSR) marker system have
been used to investigate genetic variability and relationships. ISSR marker was efficient to
detect genetic dissimilarities between isolates of F. oxysporum f. sp. melongenae and
Fusarium poae [9, 10]. In a recent study, a close genetic relationship between isolates of
F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. melongenae was detected using
ISSR, and the authors highlighted that these markers can be used to assess the genetic
divergence of F. oxysporum formae speciales with different hosts [11].
F. oxysporum f. sp. lycopersici has been grouped into three races according to the ability
to infect a set of different cultivars carrying distinct resistance loci: I for resistance to race 1;
I-2 for resistance to race 2; and I-3 for resistance to race 3 [12]. F. oxysporum f. sp.
lycopersici is an aggressive biodeteriorating agent causing a tomato vascular wilt, one of
the most economically important and widespread diseases affecting this crop [13].
In Brazil, race 1 prevails, but race 2 has increased in importance. Although race 3 shows a
more limited geographical distribution, the concern about Fusarium wilt race 3 is increasing,
since cultivars resistant to this race are not widely used [13, 14]. Chemical control of tomato
wilt has not been effective, and the cultivation of resistant strains remains as the most viable
strategy with that end. F. oxysporum f. sp. lycopersici is able to remain in the soil for
decades, so it is highly recommended in raising soil pH, using sterilized soil, promoting crop
rotation, and avoiding the sowing of seed produced from Fusarium-infected plants [15].
Rhizomes of Microgramma vacciniifolia, an epiphytic plant found preferentially at the
tropics, are widely used in folk medicine to treat diarrhea and as balsamic in infections in the
respiratory tract [16]. M. vacciniifolia rhizome contains MvRL, a lectin that hemagglutinates
human and rabbit erythrocytes and showed insecticidal activity against Nasutitermes
corniger workers and soldiers [17, 18]. This paper reports on the effect of this lectin on
the growth of F. oxysporum f. sp. lycopersici races 1, 2, and 3. Also, ISSR markers were
used to evaluate the degree of genetic variability among these races.

Materials and Methods

Chemicals and Reagents

Acrylamide, agarose, ammonium sulfate, bisacrylamide, bovine serum albumin,

cetyltrimethylammonium bromide, ethylenediaminetetracetic acid (EDTA), glutaraldehyde,
mannose, magnesium chloride, -mercaptoethanol, polyvinylpyrrolidone, potatodextrose agar,
ribonuclease (RNAse), Schiffs reagent, sodium carbonate, sodium chloride, sodium hydroxide,
Sephadex G-25, Taq DNA polymerase, and Tris were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Chloridric acid, chloroform, and ethanol were purchased from Vetec (Rio de Janeiro,
Brazil). Cercobin from Iharabras S.A. was purchased from DuPont do Brasil S.A. (Brazil).

Plant Material

M. vacciniifolia (Langsd. & Fisch) rhizome was collected in Recife City, State of
Pernambuco, northeastern Brazil. Voucher specimen (number 63,291) is deposited at the
herbarium Drdano de Andrade Lima (Instituto Agronmico de Pernambuco, Recife,
Brazil). The rhizomes were washed in tap water, dried for 7 days at 28 C, powdered (40
mesh) and stored at 20 C.
1100 Appl Biochem Biotechnol (2014) 172:10981105

Fungi Cultures and Obtaining Mycelia

F. oxysporum f. sp. lycopersici races 1, 2, and 3 were obtained from Instituto Agronmico de
Pernambuco, Recife, Brazil. Cultures (107 conidia ml1) of races 1, 2, and 3 were grown for
72 h in potatodextrose-agar (PDA) medium at 28 C. The fungal mycelium was harvested
by filtration and washed with distilled water. The mycelia of Metarhizium isolates used as
outgroup in genetic analyses were submitted to the same steps.

Isolation of MvRL

MvRL was isolated according to a protocol previously described [18]. Rhizome crude
extract (10 %, w/v) was treated with 60 % (w/v) ammonium sulfate, and the fraction 0
60 % containing the precipitated protein was collected by centrifugation. The protein
fraction was resuspended in 0.15 M NaCl and dialyzed (10.0 kDa of cutoff membrane)
against 0.15 M NaCl (8 h at 4 C). MvRL was isolated by chromatography of protein
fraction (6.0 mg of proteins) onto a chitin column (10.01.5 cm) equilibrated (20 ml min1
of flow rate) with 0.15 M NaCl. The lectin was recovered by elution with 1.0 M acetic acid
and dialyzed against distilled water (4 h) and 0.15 M NaCl (4 h).

Protein Content

Protein concentration was estimated according to Lowry et al. [19] by using bovine serum
albumin (31.25500 g ml1) as standard.

Hemagglutinating Activity

Hemagglutinating assay was carried out in microtiter plates (Kartell S.P.A., Italy) by using
2.5 % (v/v) suspension of human O-type erythrocytes treated with glutaraldehyde [20]. One unit
(per titer) of hemagglutinating activity was defined as the reciprocal of the highest dilution of
the sample that promotes full agglutination of erythrocytes [21]. Specific hemagglutinating
activity (unit per milligram) was defined as the ratio between the titer and protein concentration.

Antifungal Assay

MvRL (40 g in 0.15 M NaCl) was filtered using a 0.45-m sterile syringe filter
(Minisart). Next, the sample (50 l) was spread on solidified PDA medium in a Petri dish
(9015 mm), and a fungal mycelium disk (0.6 cm in diameter) was placed on the center of
the Petri dish. Negative and positive controls were 0.15 M NaCl and 10 ppm Cercobin,
respectively, and all assays were carried out in triplicate. The plates were incubated at 28 C
for 72 h. Antifungal activity was indicated by reduction in fungal growth zone (diameter),
compared to negative control [2, 22]. The assays were carried out in triplicate.

Effect of Glycoprotein Preparations from F. oxysporum f. sp. lycopersici Races on MvRL

Hemagglutinating Activity

Glycoprotein preparations from F. oxysporum f. sp. lycopersici races were obtained by

maceration of fungal mycelium of race 1 (0.1639 g), race 2 (0.0898 g), and race 3
(0.1149 g) in liquid nitrogen followed by solubilization in 0.15 M NaCl (200 l).
Carbohydrate contents in the solutions were estimated according to phenol-sulfuric acid
Appl Biochem Biotechnol (2014) 172:10981105 1101

method [23] by using mannose as standard. The glycoprotein preparations were submitted to
SDS-PAGE (10 % gel, w/v) [24], and staining was performed using Schiffs reagent [25].
Hemagglutinating activity of MvRL on human O-type erythrocytes was then determined
as described above after previous incubation (45 min) of lectin (50 l, 40 g) with the
glycoprotein preparations from races of F. oxysporum f. sp. lycopersici (50 l, 32 g of
protein).

Extraction of Genomic DNA from F. oxysporum f. sp. lycopersici Races

The cetyltrimethylammonium bromide (CTAB) method [26] was performed with slight
modifications. A mycelia sample (1.0 g) was transferred to a 2-ml sterile reaction tube and
mixed with 1.0 ml of extraction buffer [1 % polyvinylpyrrolidone (PVP), 2 % CTAB (w/v),
1.4 M NaCl, 2 % (v/v) -mercaptoethanol, 20 mM EDTA, and 100 mM TrisHCl, pH 8.0].
After incubation at 65 C for 1 h, with occasional stirring, the suspension was centrifuged
(18,000g, 15 min), and 500 l of the supernatant was extracted with 400 l of chloroform,
stirred, and centrifuged (12,000g, 5 min). The upper phase was transferred to a new tube
(1.5 ml), mixed with 2/3 volume of precipitation solution (5 g l1, 0.04 M NaCl) and
incubated for 1 h at 28 C. After centrifugation (12,000g, 5 min), the supernatant was
discarded, and the precipitate was dissolved in 350 l of 1.2 M NaCl and extracted with
350 l of chloroform. The mixture was centrifuged (12,000g, 10 min), and the pellet was
washed with 1.0 ml of ethanol solution (70 %, v/v) and air-dried at 28 C. After a new
centrifugation, the supernatant was discarded by pipeting, the pellet was dried, and the DNA
was dissolved in 30 l of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing 3 l of
RNAse. DNA quality was checked using 1 % agarose gel electrophoresis.

ISSR Amplifications

Following DNA extraction, a mixture containing sterilized milli-Q water (25 l), PCR
amplification buffer (10 mM TrisHCl pH 8.0, 100 M dNTPs, 0.4 M primer from
Invitrogen, and 2 mM MgCl2), Taq DNA polymerase (1 unit), and 30 ng of genomic
DNA was submitted to PCR amplications. DNA amplifications were carried out using the
thermocycler MJ Research PTC-100 Programmable Thermal Controller (Watetown, USA).
ISSR reactions were carried out with an initial denaturing step at 94 C for 15 min and
followed by 30 cycles with a denaturing step at 94 C for 30 s. Amplified products were
electrophoresed on agarose gels at 2.0 %. Bands were stained with SyBr Gold 1
(Invitrogen, USA) and then photographed under UV light (Vilber Lourmat, France).
Seventeen ISSR primers (University of British Columbia, Canada) were first screened for
PCR amplification. The primers that generated clear and reproducible banding patterns
(UBC 808, 812, 817, 848, 858, 864, 878, 888, and 890) were selected for further statistical
analysis.

Statistical Analysis

The data from antifungal assays were expressed as a mean SD, and significant differences
between fungi were analyzed using the Students t test and ANOVA (significance at p<0.05)
in the Origin 6.0 program. The ISSR bands were scored using the binary scoring system,
which recorded the presence and absence of bands as 1 and 0, respectively. Smeared and
weak bands were excluded. Bands showing the same migration distance were considered
homologous. A pair-wise similarity matrix was computed and analyzed using the GENES
1102 Appl Biochem Biotechnol (2014) 172:10981105

software [27] based on Jaccards similarity coefficients. The resulting matrix of genetic
similarity was used to construct the dendrogram through the unweighted pair group method
with arithmetic mean (unweighted pair group method using arithmetic average, UPGMA).

Results and Discussion

Antifungal activity of MvRL against F. oxysporum f. sp. lycopersici races was investigated,
and the data (Table 1) showed that race 3 was more sensitive to lectin (growth inhibition of
61 %) than races 1 (55 %) and 2 (45 %). The highest sensitiveness of F. oxysporum f. sp.
lycopersici race 3 to MvRL is very interesting, since the effective control of tomato wilt has
only been achieved through the cultivation of strains which usually carry resistance genes
for a single race. In addition, the high antifungal activity on race 3 is singular, since cultivars
resistant to this race are not yet widely available.
Lectins are able to affect fungal growth and development by disturbing the synthesis
and/or deposition of chitin on the cell wall, binding to hyphae and therefore resulting in poor
absorption of nutrients [28]. Antifungal activity has been reported for rhizome lectins, such
as the chitin-binding lectin from Setcreasea purpurea against R. solani, Sclerotinia
sclerotiorum, Penicillium italicum, and Helminthosporium maydis [29], and the mannose-
binding lectin from O. japonicus against G. saubinetii and R. solani [5].
The carbohydrate-binding property of lectins may be relevant to the antifungal
mechanism. In this sense, the hemagglutinating activity of MvRL was assayed in the
presence of glycoprotein preparations from F. oxysporum f. sp. lycopersici mycelia. The
specific hemagglutinating of MvRL (80) in the presence of mycelia preparations from all
Fusarium races was reduced to 20, indicating that the lectin recognized glycosylated
molecules. Linkage of lectin carbohydrate-binding site to monosaccharide or carbohydrate
moiety of glycoprotein avoids binding of lectin to glycoconjugates of erythrocyte surface,
and hemagglutinating activity is reduced or neutralized [1]. Table 1 shows that glycoprotein
preparation from race 2 showed the lowest carbohydrate and protein contents, while the race
3 preparation was the richest in these biomolecules. Glycoproteins from mycelia were
stained with Schiffs reagent on SDS-PAGE (Fig. 1).
Currently, there is an increasing trend to use DNA markers technologies to identify genetic
diversities. ISSR marker is considerably cost-effective, because both afford to analyze a large
number of samples quickly and reliably. The number of amplification products (76) obtained

Table 1 F. oxysporum f. sp. lycopersici growth in the presence of MvRL and characterization of glycoprotein
preparations from races

Race Growth in the presence Growth inhibitiona Carbohydrate Protein contentb

of MvRL (mm) contentb (mg ml1) (mg ml1)

1 24.60.8 A 54.41.6 A
1.660.54 10.01.1
B B
2 29.80.8 44.71.6 1.020.13 7.00.4
3 21.31.9 C 60.53.5 C
2.360.35 10.70.2

Fungal growth in negative control (0.15 M NaCl, 54 mm). Data are presented as mean SD from three
experiments
a
Fungal growth (percent) compared to fungi growth zone (diameter) in negative control. Uppercase letters
indicates significant (p<0.05) differences between antifungal effects
b
Glycoprotein preparations from F. oxysporum f. sp. lycopersici races obtained by maceration of fungal
mycelium in liquid nitrogen followed by solubilization in 0.15 M NaCl
Appl Biochem Biotechnol (2014) 172:10981105 1103

Fig. 1 SDS-PAGE (10 %, w/v)

of glycoprotein preparations
from mycelia of F. oxysporum f.
sp. lycopersici races detected by
periodic acid Schiff (PAS).
Numbers 1, 2, and 3 represent
the races

here with ISSR primers was adequate for analysis of genetic diversity. Oliveira and Costa [8]
showed that 7 to 30 primers yielding between 50 and 200 polymorphic amplicons were
sufficient to characterize diversity among pathogenic and non-pathogenic isolates of Fusarium
solani and to determine genetic relationships among formae speciales.
Eight of the 17 ISSR primers yielded good amplification products and an appropriate degree
of reproducibility. A total of 76 amplicons were produced, with a mean of eight amplicons per
oligonucleotide. Amplicon sizes ranged between 150 and 2,000 bp. Electrophoresis profile

Fig. 2 Assessment of genetic diversity among F. oxysporum f. sp. lycopersici races 1, 2, and 3. a ISSR
profiles of DNA from F. oxysporum f. sp. lycopersici races using the primer UBC-808. Numbers 1, 2, and 3
represent the races. M: 1 kb of molecular weight ladder. b Dendrograms of F. oxysporum f. sp. lycopersici
races generated by UPGMA analyses to the data obtained with ISSR primers. Genetic similarity relationships
were analyzed based on Jaccards similarity coefficients
1104 Appl Biochem Biotechnol (2014) 172:10981105

obtained with the primer UBC-808 is shown in Fig. 2a. UPGMA-based cluster analysis
(Fig. 2b) grouped Metarhizium into one main group (extern group) and F. oxyporum f. sp.
lycopersici races into other group subdivided in genetically distinct subgroup 1 (races 1 and 2)
and subgroup 2 (race 3). Jaccards similarity coefficient between subgroups 1 and 2 (group 2)
was 0.38 (38 %).
The Jaccards coefficients obtained here showed the genetic proximity among isolates
from races 1 and 2 and prove that race 3 is highly genetically distinct from the other races.
Similarly, Mes et al. [30] reported that isolates from races 1 and 2 were not distinguished by
random amplified polymorphic DNA (RAPD) analysis. In another study, among 16 isolates
of F. oxysporum f. sp. lycopersici, only one was not grouped in the same branch in a
dendogram obtained by Baysal et al. [11] by using ISSR markers. This finding was similar to
our results since only isolates from race 3 were separated in a distinct branch by analysis
using ISSR.
Different races of fungi possess different transcriptome profiles which account for distinct
virulence levels and colonization patterns [31]. The genetic diversity among races and the
race-specific differential expression of genes probably result in the presence of different
glycoconjugates in F. oxysporum f. sp. lycopersici races 1, 2, and 3.
The present work reveals that antifungal activity of MvRL on F. oxysporum f. sp.
lycopersici race 3 was higher than that on races 1 and 2. The inhibition of hemagglutinating
activity of MvRL by glycoproteins from races indicates that carbohydrate-binding sites of
lectin may be involved in its antifungal activity. The race 3 was genetically distinct from
races 1 and 2, and thus the highest sensibility of race 3 to MvRL can be due to its molecular
characteristics.

Acknowledgments The authors express their gratitude to the Conselho Nacional de Desenvolvimento
Cientfico e Tecnolgico (CNPq) for research grants and fellowships (LCBBC and PMGP), Coordenao
de Aperfeioamento de Pessoal de Nvel Superior (CAPES), Fundao de Amparo Cincia e Tecnologia do
Estado de Pernambuco (FACEPE), and Ministrio da Cincia, Tecnologia e Inovao (MCTI) for research
grants. L.P. Albuquerque would like to thank CAPES and the Brazilian Ministry of Education (Programa de
Reestruturao e Expanso das Universidades Federais - REUNI) for graduate scholarship. G.M.S. Santana
would like to thank FACEPE for graduate scholarship. The authors are also deeply grateful to Maria Barbosa
Reis da Silva for the technical assistance and Felix Nonnenmacher for English editing.

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