BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
Safa R. Fitouri 1 , Nouri B. Ermeli 2 , Salah M. Bensaber , Mousa I. Jaeda 1, 3 3 , Ibrahim A. Mrema 3 , Anton Hermann 4 and Abdul M. Gbaj 1, 3
1. Department of Genetics, National Medical Research Centre, Zawia, Z16, Libya 2. Department of Natural Products, Faculty of Pharmacy, University of Tripoli, Tripoli, M16, Libya 3. Department of Medicinal Chemistry, Faculty of Pharmacy, University of Tripoli, Tripoli, M16, Libya 4. Department of Cell Biology, Division of Cellular and Molecular Neurobiology, University of Salzburg, Salzburg A-5020, Austria
Received: November 09, 2012 / Accepted: January 05, 2013 / Published: March 30, 2013.
Abstract: The BRCA1 (Breast Cancer Anti-estrogen resistance-1), early-onset gene is expressed in cells of breast and other tissue and helps to repair damaged DNA or destroy cells in cases DNA cannot be repaired. When the BRCA1 gene is damaged, then the DNA is not repaired appropriately and this enhances the risk for cancer. Fluorescence and UV-visible thermal studies were performed for WT (wild type) and MT (mutant type targets) full systems. The target DNAs used were in the form of short oligonucleotides, genomic DNA. The probe system was used for detection of WT and SNP alleles of human BRCA1 [(170-190, G →T) and (290-310,
G →T)]. The Cy5 dye attached to a probe oligonucleotide (10-mer) undergoes a fluorescence intensity change on hybridisation of the probe to the WT compared to MT targets. Our results indicate that the system consisting of the target sequence and the one probe oligonucleotides bearing the Cy5 dye assemble correctly at the specified target. Once the full system (probe and target) is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at a suitable wavelength. Thermal studies also showed significant differences in T m between WT and MT. The results suggest that the differences in the fluorescence intensity at 665 nm and the spectrophotometric T m(s) for the WT and MT can be attributed to the type of binding of the probe to the target. The systems were sensitive to single nucleotide polymorphisms and this may help in high throughput applications in genetic testing and molecular diagnostics.
Key words: DNA, fluorescence probe, BRCA1 gene, melting temperature, anisotropy.
1. Introduction sequences in a complex genome such as that of
humans (the haploid genome contains 3 × 10 9 base Many genetic diseases have been found to be the pairs), methods of hybridisation analysis must be result of a change of a single base pair. These highly sensitive and selective. The probe alterations, termed SNP (single nucleotide oligonucleotide used must be able to discriminate polymorphisms), may cause changes in the amino acid between the desired sequence and all other sequences, sequence of important proteins [1, 2]. Some SNPs on which may differ from the target by only one base pair, the other hand may not cause a change in protein mainly in the detection of SNPs [3]. A probe of 20 expression, but may be close on the chromosome to base pairs has a high probability of occurring only other unknown deleterious mutations, and can thus once in the human genome, and a probe of this length serve as genetic markers. In order to detect specific will be very selective for the desired target. Shorter
sequences have a possibility of occurring more than Corresponding author: Abdul M. Gbaj, Ph.D., assistant
once in the genome and could thereby produce false professor, research fields: genetics and biochemistry. E-mail:
abdulgbaj1@hotmail.com. positive results. Once a suitable probe sequence has
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
been chosen, an appropriate label must be applied, so complex known as the BASC (BRCA1-associated that hybridisation to the target can be detected [4].
genome surveillance complex) [9-11]. The human Breast cancer is the most common carcinoma in
BRCA1 gene is located on the long (q) arm of women. The increasing risk for the disease is 10% up
chromosome 17 at band 21, from base pair 38,449,840 to the age of 80 years. A family history of breast
to base pair 38,530,994 (map). BRCA1 orthologs have cancer and ovarian cancer is an important risk factor.
been identified in most mammals for which complete Some 5%-10% of all cases of breast cancer and
genome data are available [11, 12]. In addition, the 25%-40% of cases in patients under the age of 35
BRCA1 protein (known as RING finger protein 53) years have a hereditary origin. BRCA1/BRCA2
contains two zinc finger domains, the C3HC4 type mutations are responsible for 3%-8% of all cases of
(RING finger) and BRCA1 C terminus (BRCT) breast cancer [5, 6]. One million females worldwide
domain [13].
are diagnosed with breast cancer every year. Some fluorescent dyes (e.g. the cyanine dyes Cy3 Treatment of advanced breast cancer is useless, so the
and Cy5) undergo changes in their fluorescence early diagnosis has a high precedence in medical
spectrum due to changes in their environment. Thus, treatment of the disease [7]. The risk factors of breast
the spectrum of such molecules attached to a probe carcinoma could be genetic, environmental or oligonucleotide may change on hybridisation of the hormonal [5, 6].
probe with the target and thus could be used as Genetic risk factors play role in about 5%-10% of
hybridisation probes [14, 15].
all cases, 90%-95% of them result from genetic The aim of this study was to develop a system that mutation and approximately 5%-10% are inherited as
can be used to detect specific target oligonucleotide dominant breast carcinoma susceptibility genes [5, 8].
sequences. The model system used comprised a target Other genes were found to increase vulnerability to
sequence plus one probe oligonucleotide bearing a cancer and are also associated with familial breast
Cy5 fluorophore. The probes will be Watson-Crick cancer. These genes are BRCA1 (Breast Cancer
complementary to the target sequence. The probe will Anti-estrogen resistance-1), BRCA2 (Breast Cancer
assemble at the target thereby bringing the Cy5 dye Anti-estrogen resistance-2), ATM (Ataxia close to the DNA bases. Telangiectasia Mutant gene), PTEN (Phosphate and
2. Material and Methods
Tensin homology) and P53 (Tumor Protein 53) [8]. There are also other less affecting genes involved with
Reagents of the highest quality available were little importance [5, 8].
purchased from the suppliers indicated. All water used This study concentrates mainly on BRCA1 gene, a
was distilled and purified by ion exchange and human tumor suppressor gene which produces a
charcoal using a MilliQ system (Millipore Ltd, UK). protein called breast cancer type 1 susceptibility
Tris buffer, PBS buffer and TB-buffer etc. were protein. It originates in cells of the breast and other
prepared from analytical reagent grade materials tissues such as the ovaries, where it helps to repair
according to standard procedures. Values of pH were damaged DNA and obliterates cells if DNA cannot be
measured using a Hanna-Instruments HI 9321 repaired. If BRCA1 itself is damaged, the cells will be
microprocessor pH meter, calibrated with standard able to grow and eventually become carcinogen. The
buffers (Sigma, UK) at 20 C.
protein encoded by the BRCA1 gene as well other Ten patients diagnosed with primary breast cancer tumor suppressors, DNA damage sensors and signal
and 10 healthy subjects were included in this study. transducers form a large multi-subunit protein All these patients were treated at Sabratah African
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
Oncology Institute, Libya. DNA was extracted from peripheral blood lymphocytes as indicated in Ref. [16]. Sample purity was checked by agarose gel electrophoresis and the concentration was estimated spectrophotometrically at A260 nm [16]. The ratio OD260/OD280 provides an estimate of the purity of the nucleic acid. Pure preparations of DNA have OD260/OD280 values of 1.8 to 2.0. If there is contamination with protein or phenol, the OD260/OD280 will be significantly less than the values given above. In these cases an accurate quantitation of the amount of nucleic acid will not be possible. DNA samples that deemed not to be sufficiently pure were subjected to re-purification by phenol/chloroform extraction technique.
DNAs (synthetic and genomic) were analysed using 3% agarose gels, which were made by heating a mixture of 1×TAE (Tris-Acetate-EDTA) buffer (50 mL), agarose (1.5 g) and an aliquot (5 µL) of Cyper Gold (1:10000 in DMSO). After cooling the gels, they were cast and loaded in a plastic gel box covered with 1×TAE buffer (200 mL) [16]. Once the gel was set, an aliquot (5 µL) of the DNA was mixed with loading buffer (3 µL) plus water (7 µL) and loaded into the sample holes on the gel. Gels were run at 75 V for 3 h.
A Cary-Eclipse and a UV-Visible 5000 spectrophotometer (Varian, UK) were used to record the fluorescence-absorption spectra and melting
temperature T m in phosphate buffer (0.01 M phosphate,
0.1 M NaCl, at pH 7.4). Absorption and fluorescence spectra were recorded using 2 mL, thermostated, 4-sided quartz cuvettes. The mole ratio of oligonucleotides used was 1:1 (the concentration of each component was 2.5 µM in the cuvette). Most spectra were buffer-corrected for the particular temperature and particular wavelength region used. Melting curves were recorded by starting at a temperature well below the T m and linearly increased the temperature well above the T m (dissociation segment). The temperature was then decreased at the same rate until the starting temperature was reached
again (association segment). T m experiments were directed and controlled by a computer with DNA melting software (Varian, UK). Melting curve experiments were performed spectrophotometrically and spectrofluorimetrically. Spectrophotometry was based on A260 profile, performed using the Cary 5000 UV-visible near IR spectrophotometer by measuring the change in absorbance at 260 nm with temperature. Spectrofluorimetrically determined melting curves were based on monitoring the change in relative fluorescence intensity with temperature at 650 nm (excitation wavelength) and 665 nm (emission wavelength) for the Cy5 at increasing temperature from 5 °C to 100 °C at a rate of 0.25 °C per minute. The sample was cooled at a rate of 0.25 °C per minute and the emission at the same wavelengths was monitored under similar conditions. The melting curve obtained during a cooling cycle was used to determine
T m values. The usual heating rate was 0.15 °C/minute. Values of T m were defined as the maxima of the first-order derivatives of the melting curves and corresponded to within ± 1 °C to the values determined at half of the maximal hyperchromicity after baseline correction [17].
The Cary-Eclipse fluorescence spectrophotometer was used to record the emission/excitation spectra in phosphate buffer (0.01 M phosphate, 0.1 M NaCl, at pH 7.4). Emission spectra were recorded using 2 mL, thermostated, 4-sided quartz cuvettes. The excitation wavelength used was 650 nm and the emission was 665 nm for Cy5. Slit-widths were set from 2.5 nm to
5 nm, depending on the intensity of emission. The automatic shutter-on function was used to minimize photo-bleaching of the sample. All spectra were buffer-corrected for the particular temperature and particular wavelength region used.
Fluorescence anisotropy measurements were made with a Cary Eclipse fluorescence spectrometer. The Cary Eclipse software used for polarization assay was “Advanced Reads”, the fluorescence polarization values being read as anisotropy during the reaction
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
time; the excitation wavelength was 650 nm and the
Table 1 Base sequence of BRCA1 WT and MT alleles
emission was 665 nm for Cy5. Double-grated system (Safa-Cy5 system) using 10-mer uni-probes. The
symbol @ indicates to the deletion (SNP) of adenine (A) and
monochromators were used with a 5 nm slit width for
guanine (G).
maximum intensity at 5 C. The measured anisotropy
Safa-Cy5 system
DNA sequences
represents an average of three measurements. The
nomenclature
3 ′-TTTCTACTGAGGAGTCGTGG-5′ material and the wavelength range was 275-750 nm.
BRCA1 WT
polarizers used are made of thin film polarizing
(170-190)
3 ′-TTTCTACTGAG@@GTCGTGG-5′ The dimensions of the polarizer are (W × D × H)
BRCA1 MT (170-190)
5 ′-CCTCAGCACC-3′-Cy5 200 mm × 115 mm × 113 mm. The two polarizers can
BRCA1 probe
be set to four possible angles. Vertical (V): The V
3. Results
located at the top of the polarizer indicates vertical or The genomic and the synthetic DNAs purity was 0°; the first notch in a clockwise direction is 35°; the
measured using OD260/OD280 ratio and found to be second notch in an anti-clockwise direction is 55°
1.82 in which the pure preparations of DNA should (magic angle 54.7°). Horizontal (H): the third notch in
have OD260/OD280 values of 1.8 to 2.0. In addition, an anti-clockwise direction is 90° or horizontal.
agarose gel electrophoresis of the synthetic and the Before measurement of sample polarization, the
genomic purified DNA compliments the data from G-factor was determined. This parameter corrects for
absorbance readings.
instrumental polarization artifacts and is calculated Fig. 1 shows the change in the fluorescence automatically as follows:
intensity before and after hybridization of the probe
with the synthetic WT and MT targets. The where Ihv is the intensity with the excitation polarizer
G = Ihv/Ihh
hybridization of the probes with the synthetic WT in the horizontal position and the emission polarizer in
target shows a significant decrease in the fluorescence the vertical position; Ihh is the intensity with both
intensity compared with MT target. excitation and emission polarizer in the horizontal position. 3.1 Melting Curves
Oligonucleotide Probes for Cy5 Systems: The The T m values of the synthetic DNA WT and MT probes and the synthetic targets were purchased from
systems in 0.01 M phosphate, 0.1 M NaCl, pH 7.4 were Sigma-Aldrich, UK. The Safa-Cy5 system (Table 1)
determined at 260 nm (Fig. 2), they were 47.5.0 ± 1.0 was assembled for fluorescence studies by the
ºC and 34.4 ± 1.0 ºC, respectively. For genomic DNA, sequential addition of the components. The full system
the T m of the WT and MT targets were 45.5.0 ± 0.8 ºC was formed by sequential addition of probe and target
and 37.4 ± 1.0 ºC, respectively (data not shown). oligonucleotides. All experiments were carried out in
3.2 Fluorescence Anisotropy Studies of Hybridization
0.01 M phosphate, 0.1 M NaCl, at pH 7.4 at 20 ºC.
of Probes to Their Targets
The sequences of the mismatches were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The human
Fluorescence anisotropy data for probes measured BRCA1 gene is located on the long (q) arm of
in the absence and presence of their complementary chromosome 17 at region 2 band 1, from base pair
oligonucleotide target are summarized in Table 2. 38,429,551 to base pair 38,551,283 (Build
4. Discussion
GRCh37/hg19) [18]. BRCA1 orthologs have been identified in most mammals for which complete
The extent of BRCA1 involvement in the genome data are available [19].
pathogenesis of breast cancer among Libyans is
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
Wavelength (nm)
Fig. 1 Fluorescence spectra of Safa-Cy5 system in phosphate buffer (0.01 M phosphate, 0.1 M NaCl, at pH 7.4). (a) BRCA1 probe and MT; (b) BRCA1 probe alone; (c) BRCA1 probe and WT. The excitation and the emission wavelengths were 650 nm and 665 nm, respectively. Concentration of each oligonucleotide component was 2.5 μM. Spectra were buffer corrected.
0.49 n m 0.48
2 60 0.47 ce at
0.46 b sorban 0.45 A 0.44
Temperature ºC
Fig. 2 Melting curves (T m ) of (a) BRCA1 probe and synthetic WT; (b) BRCA1 probe and synthetic MT in phosphate buffer (0.01 M phosphate, 0.1 M NaCl, at pH 7.4) based on change in absorbance at 260 nm as temperature was ramped at
0.25 °C/min. T m values were determined by the first derivative method. Concentration of each oligonucleotide component was 2.5 μM.
Table 2 Anisotropy results of BRCA1 probe with WT and MT of synthetic and genomic targets in phosphate buffer (0.01 M phosphate, 0.1 M NaCl, at pH 7.4).
Anisotropy of probe alone BRCA1 probe + synthetic target
System used
Anisotropy of probe + MT
Anisotropy of probe + WT
0.20 0.22 0.16 BRCA1 probe + genomic target
unknown. To the best of our knowledge, our study is target with probe/s (Cy5) and mutant target with the first to detect mutations related to the BRCA1 gene.
probe/s (Cy5) indicates that the probe had optimum The hybridization of the probe with the targets was
geometry of hybridization with wild target but not confirmed by the hyperchromic and red shift changes
with the mutant target. The same phenomenon was in the absorption spectra (Fig. 1). The significant
seen also with WT/MT genomic targets. The thermal difference between the spectra of the synthetic wild
curves also indicate the hybridization between the
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
probe and its complementary target. The hybridization of free fluorescein. This shift of emission maximum is of the probe with the targets was also confirmed by
thought to be due to aromatic stacking of fluorescein the fluorescence changes (Fig. 1). When the WT
with the nucleobases in ssDNA, removed on duplex targets (synthetic and genomic) were added to the
formation. In addition, pyrene derivatives attached in probe attached to Cy5, a decrease in the fluorescence
a similar way resulted in much greater changes in the intensity was noticed with a small shift to a longer
emission spectrum on hybridisation of labelled ssDNA wavelength. The thermal curves also indicate the
to its complement, namely a four-fold decrease in hybridization between the probe and its quantum yield on duplex formation. This was thought complementary target.
to be due to pyrene intercalating into the Addition of the WT target to the Cy5 probe resulted
double-stranded structure. Some polyaromatic in immediate fluorescence quenching (Fig. 1). The
compounds such as pyrene can intercalate into β-DNA results obtained in our study for the WT with Cy5 are
between adjacent bases pairs (type I binding). In similar to the results obtained by Ygerabide et al [20].
addition groove binding to the exterior of the duplex is where pyrene attached via ethylenediamine linkers to
also possible for pyrene (type II binding) [22]. Pyrene the N (4) position of cytosine residues in a poly (C)
has also been attached to the 5 ′-terminus of RNA chain (< 5% labeled) showed a four-fold decrease in
probe oligonucleotides [23-26], and on duplex intensity on hybridisation to a poly (I) strand [20].
formation with complementary RNA the fluorescence The change in fluorescence obtained with WT and
intensity was seen to increase significantly. These Cy5 could be related to the amount of base pairing.
probes were sensitive to mismatches, giving no These changes could be attributed to environmental
enhancement of fluorescence when a mismatch changes resulting from probe hybridisation. It seems
between the target and the probe was present near the that before hybridisation the Cy5 moiety was located
5 ′-pyrene modification site. However, if the mismatch in hydrophobic plaits of the randomly folded poly (C)
was located more than three base pairs away from the chain. On hybridisation Cy5 was expelled from the
position of the pyrene modification, it could not be helix due to steric hindrance into a more hydrophilic
detected.
environment, causing a decrease in the fluorescence Fig. 2 shows differences in the T m between the WT efficiency. When genomic DNA sequences were used
and the MT targets in both cases (synthetic and instead of the synthetic targets, fluorescence was seen
genomics). These results indicate that the ∆G to drop by 50%, which was less than the decrease seen
contribution of a single mismatch and the position of using synthetic WT targets and this is possibly due to
the mismatch are crucial to duplex stability. Evidently, the secondary structure present in the natural DNA.
the different hydrogen bonding and stacking in the Telser et al. [21] reported only a small change in the
mismatches results in different thermodynamic trends. fluorescence intensity of fluorescein-labelled oligos
The fluorescence anisotropy will increase if a probe on hybridisation when fluorescein was attached either
oligonucleotide becomes bound to a target sequence, to thymidine via a 7-atom linker arm attached to the
due to reduced rotational mobility of the probe-target pyrimidine ring at C5 or to a cytosine via a 4-atom
complex. Thus, changes in anisotropy of fluorescent linker to N (4). In this study, where fluorescein was
labeled DNA duplexes can be exploited to monitor attached to single-stranded DNA, fluorescence probe binding to the targets. From the results of the emission was slightly red-shifted compared to that of
Cy5 probe in synthetic and genomic WT and MT free fluorescein. On duplex formation between target
DNAs as shown in Table 2, the fluorescence and probe the emission maximum shifted back to that
anisotropy was higher in WT than in MT, indicating
BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves
that the technique is suitable to detect mutation in
his valuable technical assistance.
genes.
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Clustering and Expression Analysis of Chitinases in Maize and Rice
Kui Xiang 1 , Wei-Tao Li 2 , Xue-Wei Chen 2 , Guang-Sheng Yuan 1 , Wei-Lan Chen 2 , Zhi-Ming Zhang 1 , Ya-Ou Shen 1 ,
1 Hai-Jian Lin 1 and Guang-Tang Pan 1. Maize Research Institute, Sichuan Agricultural University, ChengDu 611130, China
2. Rice Research Institute, Sichuan Agricultural University, ChengDu 611130, China
Received: March 20, 2013 / Accepted: March 21, 2013 / Published: March 30, 2013.
Abstract: Chitinases play an important role in regulating plant growth and development, especially defending themselves from fungal pathogens. It is important to do the biological analyses in crops. In this study, the result showed that the chitinases were distributed into the whole genome in rice, and nearly the whole genome in maize expect for Chromosome 9. The clustering results showed that one out of three chitinases from maize and rice belonged to new groups, which were separated from those in the conformed Classes I-VII. The identification of most amino acid sequences was very low among the chitinases from rice and/or maize. It was inferred that the chitinases with different functions were relatively stable during plant evolution. The relationship of chitinases expression between leaf blade and anther was positively significant in maize, but not significant in rice. Additionally, the ratio of chitinases with up- or down-regulated expression in sensitive maize under Fusarium moniliforme inoculation was different from that in sensitive rice under Magnaporthe grisea inoculation. It might be result from different tissues infected by different fungi. The number of chitinases from resistant maize was less than that from sensitive maize, which inferred that the resistant pathways on F. moniliforme should be not only chitin induced Pathogen-associated molecular PTI (patterns-triggered immunity) pathway, but also might include other PTI pathways that improve tolerance to F. moniliforme. The analysis of expression pattern of chitinases from maize and rice under fungi inoculation will be contributed into further research on the defense mechanism of fungi in crops.
Key words: Chitinases, fungi, clustering, expression, maize, rice.
1. Introduction proteins-3 (PR-3), Class III belongs to PR-8, and Class V to PR-11 [4, 5].
Chitinases (EC3.2.1.14) are present in various Some forms of chitinases are produced organisms [1]. They catalyze the hydrolytic cleavage continuously in vacuole and apoplast of healthy plant of the β-1, 4-glycosidic bond in biopolymers [6, 7]. Chitinases production can be regulated by a N-acetylglucosamine with two different hydrolytic variety of biotic and abiotic stress factors including mechanisms including substrate-assisted catalysis and drought, heavy metals, excessive salinity [1, 6, 8-10]. acid catalysis [1-3]. Additionally, severel phytohormones, such as auxin, On the basis of the protein second structure, jasmonic acid, ethylene, salicylic acid and cytokinin, substrate specificity, mechanisms of catalysis, and can also induce chitinases expression [11]. sensitivity to inhibitors, chitinases are divided into Besides of regulating growth and development by seven classes, I-VII. Chitinases of Classes I, II, IV, VI changing signal molecules [12-14] and programmed and VII belong to Class Pathogenesis-related cell death [15, 16], plant chitinases play the very
important role in active or passive defense against Corresponding author: Guang-Tang Pan, Ph.D, professor, research fields: maize breeding, molecular biology. E-mail:
pathogens [7, 8, 11, 17, 18]. Monocotyledons maize pangt1956@yahoo.com.cn.
(Zea mays L.) and rice (Oryza sativa ssp. Japonica)
Clustering and Expression Analysis of Chitinases in Maize and Rice 245
are the most important crops and the genomes of the development stages and fungal inoculation. The two crops have been (nearly) completely sequenced
results will help to deeply research chitinases and carefully public annotated. Though the structure
mechanisms of development and defense in of rice chitinases was analyzed [18], the analysis of
monocotyledons crops.
expression pattern under different development stages
2. Materials and Methods
and fungal inoculation were not done. In this study, the characteristics of chitinases in both maize and rice
2.1 Identification of Chitinases in Maize and Rice were lucidated by clustering the chitinases and
Twenty confirmed chitinases in the Classes I-VII analyzing the expression pattern under the different
were used as the references (Table 1). On the basis of
Table 1 Details of chitinases from rice and maize including location on chromosomes, sublocation in cell, expression in leaf blade and anther and expression under fungi inoculation. The order of genes was based on the clustering results.
Gene ID
Chr SL a RC b ERM c ESM d ESR e ClassIV-AAO10093
Chr SL a RC b ERM c ESM d ESR e Gene ID
1 LOC_Os04g41620 4 S 1 *+ LOC_Os01g18400
3 ClassIII-CAA77656 S 1 LOC_Os08g41100
3 *+ ClassIII-CAA77657 S 1 GRMZM2G092621 2 S 3
4 GRMZM2G017186 1 S 2
ClassII-CAB99486
1 *+ GRMZM2G172043 5 S 1
GRMZM2G162359
5 GRMZM2G328171
GRMZM2G389582
2 ClassIII-AAN37391 S 1 GRMZM2G064360
1 GRMZM2G083292 8 S 2 ClassIV-BAD77932
1 ClassI-AAC24807
1 ClassIII-CAA76203
4 *+ GRMZM2G005633
1 GRMZM2G133781
2 GRMZM2G057093 1 S 1
10 S
3 GRMZM2G168364
1 *+ GRMZM2G837822 3 S 4
LOC_Os09g32080
2 Class IV-CAA74930
GRMZM2G052175
3 Class I-AAA62421 M 5 GRMZM2G129189 5 S 1 *+ LOC_Os03g30470 3 S 1 LOC_Os02g39330 2 S 1 *+ Class-II-AAX83262
1 GRMZM2G400497
2 ClassIV-AAM95447 S 1 LOC_Os03g04060
4 *+ GRMZM2G051921 2 S 1 ClassV-AAM18075 _ 3
GRMZM2G051943 2 S 1 ClassIII-AAM08773 _4
GRMZM2G373106 8 S 4 LOC_Os10g28080 10 _4
*+ LOC_Os06g51050 6 S 2 *+ LOC_Os10g28120 10 _4
3 *+ GRMZM2G057766 5 _ 3
LOC_Os04g30770
2 GRMZM2G358153 4 _ 3
LOC_Os11g27400
11 S
2 LOC_Os07g23850
GRMZM2G062974
3 *+ GRMZM2G074781
2 LOC_Os06g51060
4 S 2 *- Class VII-AAP80801
1 AC211652.4_FGT003
2 GRMZM2G141456
2 *- GRMZM2G148167 6 S 4 GRMZM2G090441 10 S 4 GRMZM2G430936 3 S 1 GRMZM2G403475 3 S 1 GRMZM2G453805 3 S 1 GRMZM2G412577 6 S 5
LOC_Os04g27980 4 S 4 LOC_Os10g39680 10 S 2 *+ GRMZM2G447795 6 S 1
2 Class III-AAO47731
GRMZM2G037694
2 GRMZM2G343144
10 S
2 GRMZM2G430942 3 S 1 GRMZM2G122708 2 _ 4 ClassIII-BAC65326 S 2 GRMZM2G145518 6 _ 4
Clustering and Expression Analysis of Chitinases in Maize and Rice
(Table 1 continued) Gene ID
Chr SL a RC b ERM c ESM d ESR e GRMZM2G080547 7 S 1 LOC_Os12g13610 12 _ 2 GRMZM2G130686 7 S 1
Chr SL a RC b ERM c ESM d ESR e Gene ID
4 GRMZM2G160265 7 S 1
GRMZM2G099454
5 ClassI-AAD04295
LOC_Os05g04690
4 LOC_Os04g41680
2 LOC_Os10g39700
LOC_Os05g33130
2 GRMZM2G145461
2 LOC_Os05g33150
2 GRMZM2G023650 3 S 5 LOC_Os05g33140
1 LOC_Os01g43220 1 M5 *- GRMZM2G103668 1 _ 4
GRMZM2G162505 1 S 1 *+ *+ GRMZM2G147422 7 S 2 LOC_Os10g28050 10 S 1 ClassII-AAC36359
a C: Chloroplast; M: Mitochondrion; S: Secretory pathway; _: Any other location; b RC: Reliability class, from 1 to 5, where 1 indicates the strongest prediction. RC is a measure of the size of the difference between
the highest (winning) and the second highest output scores. There are five reliability classes, defined as follows: 1: diff > 0.800; 2: 0.800 > diff > 0.600; 3: 0.600 > diff > 0.400; 4: 0.400 > diff > 0.200; 5: 0.200 > diff;
c ERM: Expression in resistant maize under F. moniliforme inoculation; *+: up-expression; *-: down-expression; d ESM: Expression in sensitive maize under F. moniliforme inoculation; *+: up-expression; *-: down-expression;
e ESR: Expression in sensitive rice under M. grisea inoculation; *+: up-expression; *-: down-expression.
the typical domain (Glyco_18, ChtBD1, Glyco_ whereas Magnaporthe grisea infecting rice was hydro_19 and Glyco_hydro18) of chitinases, the
selected in this study. The microarray data were amino acid sequences of chitinases in maize and rice
downloaded from websites (http://www.plexdb.org/ were downloaded from websites plex.php?database=Corn) and (http://www.ricearray. http://www.maizesequence.org/index.html and org/expression/expression.php), respectively. The http://rice.plantbiology.msu.edu/, respectively.
expression view of chitinases under fungi inoculation
2.2 Phylogenic Analysis of Chitinases was carried out using the Cluster 3.0 version. The alignment of multiple amino acid sequences
3. Results
were carried out using Clustal W. The phylogenic tree
3.1 Distribution on Chromosomes was constructed with the neighbor-joining method
using MEGA 5 (molecular evolutionary genetics Forty-six and 25 chitinases were identified in maize analysis) [19]. The bootstrap was 1,000 replicates, and
and rice on the basis of typical domain, respectively. the Gaps/Missing Data treatment was completed
The number of chitinases from maize was further deletion.
more than that from rice. The chitinases genes were distributed into the whole genome except for Chr
2.3 Bioinformatics Analysis (Chromosome) 9 in maize (Fig. 1a), the number of
TargetP1.1 (http://www.cbs.dtu.dk/services/TargetP) chitinases in Chr were from one to five, the highest was used for identification of the subcellular location.
number (eight) of chitinase was located on Chr 8. The expression in different tissues of maize and rice
Meanwhile, the chitinases genes were distributed into was downloaded from websites (http://bar.utoronto.ca/
the whole genome in rice (Fig. 1b), the number of efp_maize/cgi-bin/efpWeb.cgi?dataSource=Sekhon_et
chitinases in Chr was from three to eight, and the _al) and (http://ricexpro.dna.affrc.go.jp/). The selected
highest number (five) of chitinases genes was on fungus infecting maize was Fusarium moniliforme,
Chr10.
Clustering and Expression Analysis of Chitinases in Maize and Rice 247
3.2 Subcellular Location of the Chitinases III-AAO47731, GRMZM2G430942, ClassIII-BAC65326,
GRMZM2G080547, In maize, it was deduced that only two chitinases GRMZM2G130686 and GRMZM2G160265. (GRMZM2G162359 and GRMZM2G133781) were
located inside mitochondrion, eight 3.4 Expression Pattern in Leaf Blade and Anther (GRMZM2G057766, GRMZM2G358153,
The expression level in the two tissues (leaf blade GRMZM2G103668, GRMZM2G389582, and anther) of maize and rice were both detected in GRMZM2G062974, GRMZM2G037694, public microarray databases. Therefore, the GRMZM2G122708 and GRMZM2G145518) were expressions just in leaf blade and anther were unknown, and the others existed in secretory analyzed in this study. The expression level ranged pathway (Table 1). Meanwhile, in rice, it was deduced from 32.16 to 14,931.63 with an average of 679.82 in that four chitinases (LOC_Os03g04060, maize leaf blade, and from 34.93 to 6,923.56 with an LOC_Os06g51060, LOC_Os05g04690 and average of 637.62 in maize anther (Fig. 3). The LOC_Os01g43220) were located inside relationship of expression level of chitinases between mitochondrion, four (LOC_Os10g28080, leaf blade and anther was positively significant in LOC_Os10g28120, LOC_Os07g23850 and maize (r = 0.825, P = 1.73E-12). The expression level LOC_Os12g13610) were unknown, and others existed ranged from 4.25 to 4,878.50 with an average of in secretory pathway (Table 1). 648.25 in rice leaf blade, and from 3.24 to 123,590
3.3 Phylogeny of the Chitinases with an average 6,149.52 in rice anther (Fig. 3). The relationship of expression level of chitinases between
The phylogenic tree of amino acid sequences of the leaf blade and anther was not negatively significant in
reference chitinases and the chitinases from maize and rice (r = -0.05, P = 0.81). The expression level in leaf
rice was shown in Fig. 2. The chitinases from maize blade and anther of maize and rice had not similar
and rice were classed into two parts. One was grouped tendency in the same class (Fig. 3).
with the reference chitinases together, the others were
separated from the reference chitinases. Among the
(a)
chitinases grouped with references, those from maize and rice were distributed into different classes except for Classes V and VII. There was no chitinases from maize and rice in Class V, and no chitinases from rice was found in Class VII. As for the chitinases from maize and rice separated from references, they were classed into nine groups with the frequency of more than zero. Except for Groups 1, 3 and 6, the chitinases
(b)
from maize and rice were both presented in Groups 2,
4, 5, 7, 8 and 9. Additionally, the results showed that the chitinases in two reference classes were tightly clustered into together by comparing to others. One included Class III-AAM08773, LOC_Os10g28080, LOC_Os10g28120, GRMZM2G057766 and GRMZM2G358153, the other included
Fig. 1 Distribution of chitinases from maize and rice on LOC_Os04g27980, GRMZM2G447795, Class chromosomes.
Clustering and Expression Analysis of Chitinases in Maize and Rice
Fig. 2 Phylogenic tree of the chitinases from maize and rice and references chitinases. Hollow round means the chitinases from maize, solid round means the chitinases from rice.
3.5 Expression of Chitinases under Fungi Inoculation chitinases, GRMZM2G162505 was up-regulated under F. moniliforme inoculation in both resistant and
The expression data of 12/46 maize chitinases sensitive maize. In rice, all the data were from
genes under F. moniliforme inoculation were sensitive rice. It was shown that there were
downloaded from database, whereas the expression
12 chitinases genes with significant regulation data of 20/25 rice chitinases genes under M. grisea
including 11 up-regulated genes and one inoculation were obtained from database (Fig. 3). The
down-regulated gene (Table 1).
chitinases from maize and rice with significant regulation under fungus inoculation were not clustered
4. Discussion
into together based on the multi-sequences alignment, Plant chitinases participated in many biological but nearly distributed into all the classes (Table 1).
processes [11-16]. Distribution of the chitinases on There were one and five un-regulated genes in the
chromosomes in maize and rice was scattered, which resistant and sensitive maize, respectively. There were
inferred that the functions of chitinases were two down-regulated genes in the resistant maize and
performed by the whole genome but not single or none in the sensitive maize (Table 1). Among the
several chromosomes. The results of subcellular
Clustering and Expression Analysis of Chitinases in Maize and Rice 249
Fig. 3 Expression level of chitinases in tissues and under fungi inoculation. CK means control, FI means fungus inoculation (maize infected by F. moniliforme, rice infected by M. grisea), R means resistant maize, S means sensitive maize and rice, respectively. 1 means the first repeat, 2 means the second repeat. The value of expression under fungi inoculation is exchanged by log2.
Clustering and Expression Analysis of Chitinases in Maize and Rice
location of chitinases showed that most of chitinases genes exist in secretory pathway. It should be related with the main function of degrading exo-or endo-chitin [11].
Investigations of the evolution of gene families were important for knowing about the relationship between evolutionary and functional changes [20]. The clustering of chitinases showed that there were some new classes by comparing to references and dramatically different modes of sequence divergence in both maize and rice, which may be good for degrading different chitin polymers in vivo or in fungi to help plant regulate growth and development [12, 13]and improve tolerance to fungi [11]. There were two classes that were tightly clustered into together. It was inferred that the chitinases with different functions were a little stable during plant evolution, and less duplication occurred. It was also indicated that chitin secreted by each planta and each fungus should be relatively stable during fungi evolution.
In this study, the expression levels of chitinases in the leaf blade and anther of maize was different from those from rice in the same class. The relationships of chitinases expression levels in different tissues (leaf blade and anther) were not same between maize and rice (significant in maize, not in rice). The results should be related with different chitin in vivo of maize and rice, and several different fungi inoculation. NBS (nucleotide-binding site) gene family is a very large family in plant disease resistance genes families [21]. It was proved that some of NBS genes belonged to ETI (effector-triggered immunity) pathway, whereas some of chitinases genes belonged to the Pathogen-associated molecular PTI (patterns-triggered immunity) pathway [22]. The number of NBS genes in rice was more than that in maize [21], whereas the number and classes of chitinases from rice were both less than those from maize in this study, which supported that there was cooperation between PTI and ETI, which was in agreement with Zigzag model [23].
The development and functioning of an organism
require cellular response to a variety of internal and external signals [20]. It is of great significance to study the expression patterns and regulative mechanisms of chitinases in biotic stress. Though the number of chitinases from maize was more than that from rice, more chitinases from the sensitive rice for M. grisea inoculation were significantly regulated by comparing to those from the sensitive maize for F. moniliforme inoculation. It might be related with that different tissues was infected by different fungi in different plants. The number of chitinases from the resistant maize was less than that from the sensitive maize for F. moniliforme inoculation, which inferred that the resistant pathways on F. moniliforme should
be not only chitin induced PTI pathway, and might include other PTI pathways that improve tolerance to
F. moniliforme .
5. Conclusion
Chitinases are distributed into the whole genome in maize or rice. New groups including chitinases are found in maize and rice based on the amino acid sequences alignment. The chitinases from rice are less than those from maize. However, the chitinases from maize and rice are both present in nearly each group. The expression patterns in leaf blade and anther of maize are different from those of rice. The relationship of chitinases expression between leaf blade and anther is positively significant in maize, but not significant in rice. Though the chitinases expression pattern under M. grisea in rice inoculation is different from that under F. moniliforme in maize, this difference of chitinases expression pattern under fungi inoculation between maize and rice can be understood based on the analysis of both PTI and ETI (NBS-LRR family).
Acknowledgments
This work was supported by Specialized Research Fund for New Teachers of Doctoral Program in the University of China (No. 20125103120011) and the
Clustering and Expression Analysis of Chitinases in Maize and Rice 251
Natural National Science Foundation of China (No. [12] S. Goormachtig, S. Lievens, W. Van de Velde, M. Van Montagu, M. Holsters, Srchi13, a novel early nodulin
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Mar. 2013, Vol. 7, No. 3, pp. 252-258 Journal of Life Sciences, ISSN 1934-7391, USA
Simulation of Spread of Infectious Diseases and Population Mobility in a Deterministic Epidemic Patch Model
Ariel Félix Gualtieri and Juan Pedro Hecht Department of Biophysics, Faculty of Dentistry, University of Buenos Aires, Buenos Aires C1122AAH, Argentina
Received: November 22, 2012 / Accepted: January 10, 2012 / Published: March 30, 2013.
Abstract: Computer simulation models are widely applied in various areas of the health care sector, including the spread of infectious diseases. Patch models involve explicit movements of people between distinct locations. The aim of the present work has been designed and explored a patch model with population mobility between different patches and between each patch and an external population. The authors considered a SIR (susceptible-infected-recovered) scheme. The model was explored by computer simulations. The results show how endemic levels are reached in all patches of the system. Furthermore, the performed explorations suggest that the people mobility between patches, the immigration from outside the system and the infection rate in each patch, are factors that may influence the dynamics of epidemics and should be considered in health policy planning.
Key words: Simulation, spread of infectious diseases, population mobility, epidemic patch model, SIR model.
1. Introduction represent the transition of individuals between these categories over a period of time. Computer simulation
Epidemic models are useful tools for the study of models have been widely applied in various areas of the spread of infectious diseases [1-4]. Basic the health care sector, including the spread of theoretical designs, such as the model presented in the infectious diseases [6]. By means of simulations, in present article, provide a framework for organizing the context of an epidemic model, it is possible to our knowledge about the dynamics of epidemics; investigate different problems that are ethically or these serve as a point of departure for adding realistic logistically unfeasible to study in the real world [4]. In complications step by step and help to suggest what recent years there has been a growing interest in kinds of data need to be sought in order to design designs that allow incorporating spatial aspects of the programmes of control [1]. In an epidemic model, spread of infectious diseases [7]. The patch models individuals of the population are usually classified which are also called metapopulation models involve into different categories corresponding to possible explicit movements of the individuals between distinct states for the disease under study. For example, in a locations (patches) [8]. Patch models have been used SIR (susceptible-infected-recovered) type model, for studying diseases of important public health individuals can be in one of three states: susceptible concern, such as influenza [9] and HIV [10]. (S), infected (I) or recovered and immune (R) [5]. Simulations and numerical methods are common in According to this grouping, parameters are defined to the investigation of patch models [10-12]. In a
previous work, the authors developed a computer Corresponding author: Ariel Félix Gualtieri, Ph.D., research fields: applied mathematics, epidemiology, program with the purpose of implementing the biostatistics. E-mail: agualtieri@odon.uba.ar.
Simulation of Spread of Infectious Diseases and Population
Mobility in a Deterministic Epidemic Patch Model
Gillespie algorithm in stochastic models of spread of studies, in the present work the authors have designed infectious diseases in metapopulations [13].
a deterministic simulation model with the aim of Understanding how the arrival of people from
exploring, by numerical methods, the dynamics of outside affects the spread of infectious in a population
epidemics in a patch system that presents exchange of is of high importance for public health. Brauer and
individuals with an external population.
van den Driessche developed deterministic theoretical models for transmission of disease with immigration
2. Materials and Methods
of infected individuals into a spatially homogeneous
2.1 The Model
population [14]. Li et al. extended this work and included immigration of susceptible and recovered
The authors considered a individuals [15]. Later Hsieh et al. [16] and Yang et al.
susceptible-infected-recovered SIR scheme in a [17] designed epidemic patch models with recruitment
scenario of n patches, which obeys the following of susceptible people. Inspired by these previous
system of equations:
dS n i
μ (N ) (1 p q )[a (N )] i i i i i i (m S ) ji j (m S ) a S ij i ii βSIμS iii ii
dt
dI i
p [a (N )] i i i (m I ) ji j (m I ) a I ij i ii βSIγIμI iii ii ii
(1) dt
dR i
q [a (N )] i i i (m R ) ji j (m R ) ij i aR i i γIμR ii i i
dt
In each patch i the population is divided into each patch, the outflow to the external population compartments of susceptible, infected and recovered
equals the inflow from it. That is, the value of the total individuals, for i = 1, 2, . . . , n. Thus, S i (t), I i (t) and
outflow for same patch i is also a i N i (t). Thus, from the R i (t) are the numbers of the susceptible, infected and
assumptions made so far, it follows that the total recovered individuals at time t in the patch i,
population size of the patch system remains constant. respectively. Then, N i (t) is the total population size at
Individuals travel from a patch i to another patch j at a time t in the patch i, N i (t) = S i (t) + I i (t) + R i (t). Births
constant rate m ij . Following conventional lines, the and deaths are included. In each patch, birth and death
authors assumed that the net rate at which infections rates have the same value μ i . So, the net input of
are acquired in each patch is proportional to the susceptible individuals into the patch i by births is
number of encounters between susceptible and equal to the net mortality μ i N i (t). Deaths caused by the
infected individuals, β i S i I i , where β i is a transmission disease are neglected. There is mobility of individuals
coefficient. Infected individuals in patch i recover at a between each patch and an external population, and
constant rate γ i .
also between different patches. It was assumed that
2.2 Numerical Simulations
individuals do not change their disease state during the travel. The total flow of people from the external
The dynamics of the model was explored by population to same patch i is a proportion a i of N i (t).
computer simulations, which were performed with the In turn, p i and q i are the fractions of infected and
program Dynamics Solver 1.87 [18]. Particular cases recovered individuals, respectively, in that flow. For
of four and two patches were studied. Since frequently
Simulation of Spread of Infectious Diseases and Population
Mobility in a Deterministic Epidemic Patch Model
in theoretical epidemic models (for example, Ref. [19]
P4 and Ref. [20]), arbitrary values were established for
u al 0.18 id
the initial conditions and the parameters. The time and P3
0.12 ← ← the parameters are expressed in adimensional units WS
on of iv
ti
P2 (au). The authors established a benchmark case for a
or d ind
P ecte 0.06 P1 patch i where we set the following values for the
initial conditions and the parameters: S i (0) = 100, I i (0)
0 40 80 120 160 200 = 0, R i (0) = 0, N i (0) = 100, μ i = 3 × 10 -5 ,a i = 0.02, p i = Time (au)
0.3, q i = 0.1, m ij = 0.05 (for all j), β i = 0.005, γ i = 0.3.
Fig. 1 Evolution of the prevalence in the model for a
The authors have explored the sensitivity of the
system of four patches. These differ from one another by the value of the parameter a i (a 1 = 0.02, a 2 = 0.05, a 3 = 0.08,
system to the parameters m, a and β. As was indicated
a 4 = 0.11). P1: patch 1, P2: patch 2, P3: patch 3, P4: patch 4,
in the description of the model, the first two are
WS: whole system. The initial conditions and the rest of the
related to the mobility of the population, and the third
parameters are as in the benchmark case. Time expressed
to the infection rate. These parameters may be
in adimensional units (au).
affected by public health measures in real scenarios. the benchmark case ( β 1 = β 2 = β 3 = 0.005), while in For example, the limitation of movement of people is
patch 4 the value of β was reduced to 20% (β 4 =
a strategy that could be adopted to mitigate an 0.001). In the four patches, the values of the initial influenza pandemic during phase 4 [21]. Also, some
conditions and of the rest of the parameters were kept measures implemented during an influenza epidemic
as in the benchmark case. Simulations show that as can cause a direct reduction in the rate of infection of
the mobility increases, the peak prevalence decreases the disease, such as antiviral prophylaxis or promotion
in the whole system (Fig. 2a) and in the three patches of hand and respiratory hygiene [21, 22].
that have the highest value of β (Fig. 2b), while that
3. Results
increases in patch 4 (Fig. 2c).
The authors have also evaluated the effects of The typical evolution of prevalence in the model is
reducing the parameters a and β. In order to do it, a shown in a system of four patches (Fig. 1). In all of
system of two patches was explored, where these them, the initial conditions and the parameters have
parameters were varied one by one, keeping the rest of the same values as the benchmark case; except the
the parameters and the initial conditions as in the parameter a i , which differs between the different
benchmark case. In particular, to study the reduction
of the parameter a, the authors compared three can be observed that in each patch and in the whole
patches (a 1 = 0.02, a 2 = 0.05, a 3 = 0.08, a 4 = 0.11). It
situations: a control case (a 1 =a 2 = 0.020), a uniform system, the proportion of infected individuals reduction of 45% in both patches (a 1 = a 2 = 0.011),
increases exponentially to a maximum value and and a localized reduction of 90 percent only in patch 1 decreases subsequently to stabilize at a plateau. The
(a 1 = 0.002, a 2 = 0.020). Fig. 3 shows the results of the prevalence in each patch depends on the value of its
performed simulations. In the whole system and in corresponding parameter a.
patch 1, the localized reduction of a produced a In order to study the influence of the travel rates
greater decreased of peak prevalence than the uniform between patches (m ij ), the authors explored a case of
measure (Fig. 3a and 3b, respectively). The reverse four patches (Fig. 2). Three different values of m ij (for
was true in patch 2 (Fig. 3c). To study the reduction of all ij) were tested: 0.05, 0.15 and 0.95. In patches 1, 2
the parameter β, the authors also compared three and 3, the authors maintained the same value of β as in
cases, similar to those performed for the parameter a: a
Simulation of Spread of Infectious Diseases and Population
Mobility in a Deterministic Epidemic Patch Model
0.15 a 1 =a
m id
a 1 =a 2 = 0.011 m
ij = 0.05
id
m ij = 0.15
em 0.12 indiv st
d indiv ste 0.09 m ij = 0.95
a 1 = 0.002, a 2 = 0.020
le sy fe ec ol h o 0.06
n h i 0.06
inf of ew h
n of
on t 0.03
io in the w 0.03
Time (au)
Time (au)
0.15 a 1 =a 2 = 0.020 al u
als u
a 1 =a 2 = 0.011 id
v id
m ij = 0.05
a 1 = 0.002, a 2 = 0.020 d3 0.12 indiv an
indi
m ij = 0.15
m ij = 0.95
of in
inf he 0.06 of tc
ti in pa 0.03
0.00 Time (au) P
Time (au)
0.15 a 1 =a 2 = 0.020 s
u al
a 1 = 0.002, a 2 = 0.020 u
id
al 0.10 m ij = 0.95
id a 1 =a 2 = 0.011 0.08
n di
m ij = 0.15
ed i
ct ch 0.09
d indiv 4 0.06
m ij = 0.05
n of i
inf 0.04 in pa
ti on 0.02
P rop
0 20 40 60 80 or
Time (au) rop
0.00 P
Fig. 3 Reduction of the flow of people from the external
population to a system of two patches: evolution of the Fig. 2 Influence of the mobility between patches on the
Time (au)
prevalence. Three different situations were compared: a evolution of the prevalence for a system of four patches.
control case (a 1 =a 2 = 0.020), a uniform reduction of 45%in Three different values of m ij were tested: 0.05, 0.15 and 0.95.
both patches (a 1 =a 2 = 0.011), and a localized reduction of
90% percent only in patch 1 (a 1 = 0.002, a 2 = 0.020). The conditions and the rest of the parameters are as in the
Parameter β: β 1 β = 2 = β 3 = 0.005, β 4 = 0.001. The initial
initial conditions and the rest of the parameters are as in benchmark case. A: Whole system.; B: Patches 1, 2 and 3;
the benchmark case. A: Whole system; B: Patch 1; C: Patch C: Patch 4. Time expressed in adimensional units (au).
2. Time expressed in adimensional units (au).
control case ( β 1 = β 2 = 0.005), a uniform reduction of reduction of β produced a greater decreased of peak
40% in both patches ( β 1 = β 2 = 0.003), and a localized
prevalence than the localized measure (Fig. 4a). In
patch 1, the localized reduction was the one that 0.005). In contrast to what was observed for the
reduction of 80%only in patch 1 ( β 1 = 0.001, β 2 =
caused the greatest decrease of prevalence (Fig. 4b), parameter a, in the whole system the uniform
whereas the opposite occurred in patch 2 (Fig. 4c).
Simulation of Spread of Infectious Diseases and Population
Mobility in a Deterministic Epidemic Patch Model
A patch system has been designed. In contrast to the
ls ua
0.15 stochastic model previously developed by the authors id
v ndi em 0.12
[13], the present design includes births, deaths and the st
di displacement of people between patches and an
te le sy 0.09 β 1 = 0.001, β 2 = 0.005
ec o external population. Through computer simulations, nf i 0.06 e wh
2 the behaviour of the system was illustrated and some n
of
h t io in 0.03
particular aspects of its dynamics were studied. o rt
The SIR scheme is appropriate for infectious rop
diseases that induces permanent immunity, such as the
Time (au)
caused by a particular strain of influenza virus [5]. The exponential growth of the prevalence to a peak
s 0.15
and its subsequent decline observed in the simulations u al
β 1 = β id
of the present model, is the typical behavior seen in iv d 0.12 n
real scenarios of influenza (for example, Ref. [23]). i ed 1 ct
0.09 However the present model is further characterized in n fe atch
i p 0.06 that the proportion of infected individuals reaches a
of in
plateau. Brauer and van den Driessche [14] and Li et on ti
0.03 or
al. [15] demonstrated analytically for SIR models that, 0.00
when there is immigration of infected individuals P rop
Time (au)
towards a spatially homogeneous population, which is not divided into patches, a disease-free equilibrium
C cannot be reached and exists a globally asymptotically
s 0.15
stable endemic equilibrium. That is, the disease u al id
iv persists at an endemic level in the recipient population.
d 0.12 β 1 = 0.001, β 2 n = 0.005 i
Numerical simulations of our model are compatible ed 2
ct 0.09 with these results and, in addition, they show how n fe i atch p
in 0.06 β
1 of = β 2 = 0.003
endemic levels are reached in all patches of the system.
Furthermore, our results suggest that, in on ti
0.03 or
metapopulation systems that receive immigrants from 0.00
P rop
the outside, the mobility of individuals between
Time (au)
patches, and the distribution among different patches
Fig. 4 Reduction of the infection rate in a system of two
of the total immigration flow and of control measures
patches: evolution of the prevalence. Three different
situations were compared: a control case ( β 1 = β 2 = 0.005), a
that reduce the infection rate, are all factors which
uniform reduction of 40%in both patches ( β 1 = β 2 = 0.003),
may influence the dynamics of epidemics in these
and a localized reduction of 80% only in patch 1 ( β 1 = 0.001,
systems and should be considered in health policy
β 2 = 0.005). The initial conditions and the rest of the
planning. In the previously mentioned work [13], the
parameters are as in the benchmark case: A: Whole system; B: Patch 1; C: Patch 2. Time expressed in adimensional
authors have already shown how the distribution of
units (au).
control measures among subpopulations could affect the efficacy of these strategies.
4. Discussion
Clearly, like its predecessors [14-17], the present In the present study, a deterministic SIR model in a
model is an abstraction of the reality and may be
Simulation of Spread of Infectious Diseases and Population
Mobility in a Deterministic Epidemic Patch Model
modified in various ways. For example, to obtain a future swine flu (H1N1), BMC Medicine 7 (30) (2009) 1-8.
more realistic representation of the spread of influenza, [6] B. Mielczarek, J. Uziałko-Mydlikowska, Application of the population could be divided into age groups, since
computer simulation modeling in the health care sector: the incidence and the mortality may vary considerably
A survey, Simulation: Transactions of the Society for with age during outbreaks of flu [24]. Thus, future
Modeling and Simulation International 88 (2) (2012) 197-216.
works could be directed to complicate this model in [7] S. Riley, Large-scale spatial-transmission models of
order to study other aspects of the spread of epidemics infectious disease, Science 316 (5829) (2007) 1298-1301. in a system with mobility of individuals between
[8] J. Arino, Diseases in metapopulations, Series in patches and migration from outside.
Contemporary Applied Mathematics 11 (2009) 65-123. [9] A. Lunelli, A. Pugliese, C. Rizzo, Epidemic patch models
5. Conclusion
applied to pandemic influenza: Contact matrix, stochasticity, robustness of predictions, Mathematical
In the present work, the authors have developed a Biosciences 220 (1) (2009) 24-33. deterministic model for the study of the spread of
[10] A. Sani, D.P. Kroese, P.K. Pollett, Stochastic models for the spread of HIV in a mobile heterosexual population, epidemics in populations connected by mobility of
Mathematical Biosciences 208 (1) (2007) 98-124. individuals between them, and also affected by the
[11] M. Jesse, P. Ezanno, S. Davis, J.A.P. Heesterbeek, A exchange of people with an external population. It is a
fully coupled, mechanistic model for infectious disease dynamics in a metapopulation: movement and epidemic
theoretical model that could be the starting point for duration, Journal of Theoretical Biology 254 (2) (2008)
generating hypotheses about these systems. In
331-338.
particular, the model explorations performed in the [12] Y.-A. Rho, L.S. Liebovitch, I.B. Schwartz, Dynamical present study suggest that the spatial structure and the response of multi-patch, flux-based models to the input of infected people: Epidemic response to initiated events, spatial dynamics of the population should be taken
Physics Letters A 372 (30) (2008) 5017-5025. into account in the study of the behaviour and control
[13] A.F. Gualtieri, J.P. Hecht, Dynamics and control of of epidemics.
infectious diseases in stochastic metapopulation models, Journal of Life Sciences 5 (7) (2011) 503-508.
Acknowledgments
[14] F. Brauer, P. van den Driessche, Models for transmission of disease with immigration of infectives, Mathematical
The authors gratefully acknowledge the University of Biosciences 171 (2) (2001) 143-154. Buenos Aires for providing financial support (grant
[15] J. Li, J. Zhang, Z. Ma, Global analysis of some epidemic models with general contact rate and constant
numbers UBACyT O405, UBACyT 20020090200258). immigration, Applied Mathematics and Mechanics 25 (4) (2004) 396-404.
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Mar. 2013, Vol. 7, No. 3, pp. 259-266 Journal of Life Sciences, ISSN 1934-7391, USA
Tests of Antibiotic Properties of Algerian Desert Truffle against Bacteria and Fungi
Samir Neggaz and Zohra Fortas Laboratory of Biology of Microorganisms and Biotechnology (LBMB), Department of Biotechnology, Faculty of Sciences, University of Es-Sénia, BP 1524 El M’naouer, Oran 31000, Algeria
Received: October 23, 2012 / Accepted: December 15, 2012 / Published: March 30, 2013.
Abstract: In the present paper, the ethyl acetate extract from the fruiting bodies of Tirmania pinoyi (Maire) was obtained by Soxhlet extraction. Six fractions were separated from this extract using two chromatographic methods. All these fractions were submitted to antimicrobial activity against four clinically important bacteria Staphylococcus aureus ATCC6538, Enterococcus faecalis ATCC6538, Pseudomonas aeruginosa ATCC14028, Escherichia coli ATCC25922 and one pathogenic fungus Candida albicans ATCC10231. The in vitro antimicrobial activity was performed by agar disc diffusion method. The fractions with the greatest antimicrobial activity were fractions 02 and 06 which inhibited growth of both Gram negative and Gram positive bacteria and had significant antifungal activity against Candida albicans. The present study validates the folk use of the boiled truffle water-extract and indicates that it could be effective potential candidates for the development of new strategies to treat bacterial or fungal infections.
Key words: Agar diffusion assay, Tirmania pinoyi, antimicrobial activity, chromatographic methods, Soxhlet extraction.
1. Introduction
have been found in the Kalahari Desert [3-5], in North America [6], Australia [7, 8] and China [9].
Tirmania and Terfezia, so-called desert truffles, are Several studies on their chemical composition have hypogeous ascomycota fungi (they form their shown that the dry matter (about 20% by weight)
fruitbodies below ground). Ecologically, they are consists of: 20-27% protein, some 85% of which is mycorrhizal, forming mutually beneficial associations digestible by humans; 3-7.5% fat, including with the roots of host plants, especially with unsaturated as well as saturated fatty acids; 7-13% Helianthemum species [1]. Taxonomically, Tirmania crude fiber; close to 60% carbohydrates; and and Terfezia were shown to belong to the Pezizaceae appreciable amounts (2-5%) of ascorbic acid [10-16]. rather than to the distinctly hypogeous Terfeziaceae Desert truffles have been used as traditional family, which was therefore abolished [2]. These medicine in Arabia for over two millennia without truffles are edible and their geographical distribution known toxic harmful effects to its users [17]. As a is limited to arid and semi-arid lands, mostly in fungal drug remedy, a boiled truffle water-extract is countries around the Mediterranean basin: (Turkey, highly recommended by Bedouins for the treatment of Italy, Spain, Portugal, France, and Hungary), North one of the most common eye diseases at that time, e.g., Africa (Tunisia, Algeria, and Egypt) and the Middle trachoma [18]. This practice developed the following East (Saudi Arabia, Kuwait, Iraq, Iran, Lebanon, Syria, recommendations of the Prophet Mohammad (Peace and Jordon). In addition, some desert truffle species
be upon him) whom was reported to have said: “Truffles are from man (as they grow naturally
Corresponding author: Samir Neggaz, Ph.D., research field: without man’s care) and their water is a cure for the biological activities of desert truffle. E-mail:
samir_neggaz@yahoo.fr.
eye”.
Tests of Antibiotic Properties of Algerian Desert Truffle against Bacteria and Fungi
Rougieux (1963) was the first researcher to Recently, in another study, Al laith (2010) [24] investigate the biological activity of desert truffle
investigated the antioxidant capacities of Tirmania extracts on bacterial growth in vitro. He reported that
nivea from Bahrein, Iran, Morocco and Saudia Arabia. the extract of Terfezia boudieri Chatin had a slightly
These truffles showed varying degree of antioxidant inhibitory for Staphylococcus aureus [19].
antiradical activities based on four analytical methods: Al-Marzooky (1981) [20] has tested the biological
FRAP (ferric reducing ability), DPPH (2, activity of desert truffle extracts on bacterial growth in
2-Diphenyl-1-Picrylhydrazy), deoxyribose and nitric vitro . He reported that all aqueous, polar and
oxide. He found that the Iranian truffles possessed the non-polar extracts of Terfezia claveryi exhibited good
highest DPPH values (30.6% ± 13%), the strongest antibacterial activity against broad spectrum of the
nitric oxide radical scavenging activity (EC 50 = 102 tested bacterial species, in particular, the causal
mg/mL) as well as highest percent of inhibition of organism of trachoma Chlamydia trachomatis.
deoxyribose breakage (91%). Both Bahraini and Saudi Consequently, preliminary results of a pilot clinical
truffles possessed the highest FRAP values (18.62 study conducted by Al-Marzooky (1981) [20] on
mmol/100g and 18.06 mmol/100g, respectively) [24]. Terfezia claveryi aqueous sterilized extracts for the
Janakat and Nassar (2010) [25] evaluated the treatment of trachoma infected patients proved useful
hepatoprotective activity of the desert truffle (Terfezia but required longer time compared with standard eye
claveryi ) in the rat with different solvent extracts antibiotic treatments.
(water, methanolic and petroleum ether), using a Moreover, Chellal and Lukasova (1995) [21] have
potent hepatotoxin carbon tetrachloride (CCl 4 ) in reported that antibiotics extracted from the desert
comparison with the hepatoprotective activity of a truffles Terfezia and Tirmania spp. proved effective
reference plant Nigella sativa. The authors found that against bacteria. The most important bacteriostatic
the aqueous extract of T. claveryi demonstrated a very activity against Gram (+) bacteria including Bacillus
powerfull hepatoprotective activity against CCl 4 . subtilis , Streptococcus pyogenes and Staphylococcus
The present study is as an attempt to find an aureus was indicated by methanolic extract at 45 °C
alternative antimicrobial preparation from natural from Terfezia. Finally, they noticed that extracts of
desert truffles (Tirmania pinoyi). In our search we Tirmania were less bacteriostatic than those of
established antimicrobial activity of different fractions Terfezia .
extracted from fruiting bodies of T. pinoyi against Furthermore, Janakat et al. (2004) [22] have studied
bacterial and fungal reference strains. the antimicrobial activity of aqueous and methanolic
extracts from Terfezia claveryi against Staphylococcus
2. Material and Methods
aureus in vitro. They showed that 5% of aqueous
2.1 Fungal Material
extract inhibited the growth of S. aureus by 66.4%, however, the methanolic extract did not cause a
Fresh ascocarps of T. pinoyi were purchased from significant growth inhibition. In 2005, Janakat et
the local market of El Bayed (Algeria) in January al. [23] have investigated the antimicrobial activity of
2009 (Fig. 1). The specimens were authenticated by aqueous and methanolic extracts from Terfezia
Professor Fortas from the Laboratory of Biology of claveryi against Pseudomonas aeruginosa in vitro.
Microorganisms and Biotechnology, Faculty of Five percent of aqueous extract inhibited the growth
Sciences, University of Oran, Algeria. Asci and of P. aeruginosa by 40.9%, while methanolic extract
ascospores of T. pinoyi were studied under electronic was ineffective.
microscope.
Tests of A Antibiotic Pro operties of Al gerian Deser rt Truffle agai nst Bacteria and Fungi
Ent terococcus f faecalis AT TCC6538, P Pseudomonas s aer uginosa
Escheri ichia coli i ATC CC25922 and d Candida a albicans ATC CC10231. All l
ATCC14028, A ,
mic croorganisms were obtai ined from I IPA (Pasteur r Inst titute of Alge eria). Microor rganisms wer re maintained d at
4 °C on n nutrient aga ar slants (fo or bacteria), , Sab bouraud slants s (for yeast).
Fig. 1 Fresh h ascocarps of T. pinoyi .
2.5 Antimicrobia al Assay
2.2 Soxhlet E Extraction T The antimicro obial assay wa as performed d by agar disc c
diff fusion method d [28].
The fruiti ng bodies of T. pinoyi wer re cut into pi eces,
2 2.5.1 Antibact terial Assay
sun dried an nd powdered i in a grinder a and then 100 g of Each bacteri E al strain tes sted was st treaked onto o the powder red fruiting bodies were e extracted w with
nutr rient agar m medium to o obtain isolat ed colonies. . 600 mL of ethyl acetat te by Soxhle et extractor. The
Aft er incubation n at 37 °C ov vernight, we s selected four r solvent was heated to re eflux. The pr rocedure was s run
or five well-iso olated colon nies and tra ansferred the e for 1 h to facilitate at least three c cycles of Sox xhlet
gro wth to a tube e of sterile nu utrient broth (10 mL) and d extraction. The crude e extract was concentrated d in
then n incubated at 37 °C f for 24 h. Af fter that the e vacuum usin ng a rotary ev vaporator and d then weighe ed to
bac cterial suspen nsion was ag gitated on a v vortex mixer r calculate the e yield of the e extracts an nd stored at 4 4 °C
imm mediately th en compared d to the 0.5 5 McFarland d until requir red. Before extraction procedure, all
stan 8 ndard (10 C FU/mL) to a adjust the tur rbidity of the e glassware w was thoroughl y washed and d dried at 150 0 °C
inoc culum suspe ension. 15 m L of the mo olten Mueller r for 3 h to av void any organ nic contamin ation [26, 27] ]
Hin nton Agar wa as poured eve enly into Petr ri plate (9 cm m
2.3 Separati ion of the Com mpounds diam meter). Usin g a sterile p ipette, 1 mL L of the 24 h h
test 6 t bacterial br roth culture (1 × 10 CF FU/mL) was s We subjec cted the crud de ethyl aceta ate extract to o CC
spre ead over the surface of th he dried agar r plates using g (column ch hromatograph hy) and TL LC (thin l layer
a st terile glass sp preader, allow wed to absor rb in the agar r chromatogra aphy) to sepa arate different t compounds. .3g
10 min and t the plates dri ied, inverted, , at 37 °C for r of the crude e extract was chromatogra aphed over s ilica
for
app proximately 3 30 min until the bacterial l overlay had d gel 60GF 254 4 using petr roleum ether r and increa asing
drie ed on the s surface. Ster rile filter di iscs (6 mm m concentratio on of ethyl a acetate in eth her petroleum m as
diam meter) were impregnate ed with 20 µL of each h eluents, and then each fra action collect ted was analy yzed
frac ction of the crude extra ct of T. pin oyi , initially y by the thi in layer ch hromatograph hy using a 1:6
diss solved in et thyl acetate which was s previously y petroleum e ether: ethyl a acetate mixtu ure as the mo obile
test ted for anti imicrobial a activity agai inst all test t phase. S ix bands were
bac cteria and fungi and found to o have no o p -anisaldehy yde [27].
identified
by
anti imicrobial ac ctivity, air-dr ried and three e filter paper r disc cs were take en and place ed equidistan ntly onto the e
2.4 Assay M Microorganism ms surf face of one Petri plate. Negative co ontrols using g
20 µL of ethy yl acetate pr repared for each assay. . extracted fr from fruiting g bodies of f T. pinoyi was
In vitro antimicrobia al activity o of six fract tions
Inh ibition zone s around filt ter discs we ere measured d determined
24 h-48 h, a and then the e microorgani isms: Staphy lococcus aur reus ATCC6 538,
against
the fol llowing
panel p
afte er incubation n periods of
mea an values we ere calculate ed. All exper riments were e
Tests of A Antibiotic Pro operties of Al gerian Deser rt Truffle agai nst Bacteria and Fungi
carried out i in triplicate [ 29].
(a a)
(b b)
2.5.2 Anti ifungal Assay y In vitro antifungal activity was s tested aga ainst Candida alb bicans which was determi ined as descr ribed previously in Section 2
2.5.1 except t we have u used
Sabouraud a agar instead of nutrient a agar and Mu eller Hinton Aga ar. After the e agar solidi ified, 20 µL L of different fra actions of th he crude extr ract of T. pi inoyi dissolved in n ethyl acetat te were adde ed to filter p aper
Fig. . 2 (a): Ascus with ascospor res of T. pinoyi (GR 1500); (b): Spore of T. pin noyi (GR 5000 ).
disks (6 mm m diameter.) w which were p laced on the agar surface afte er solvent ev vaporation. N Negative cont trols
(C. albicans ). T The control treatment (e ethyl acetate) ) using 20 µL L of ethyl ac cetate were p prepared for e each
had d no inhibi itory effect on any o of the test t assay. After
48 h of incub bation at 25 ° °C, the inhibi ition mic croorganisms . The resul lts of this screen were e zones from the centre of f the disc to the inner ma argin
sum mmarised in T Table 1.
of the surro ounding fung gal growth w was measure d in T The results o of the agar diffusion as ssay showed d millimetres and recorde ed. All tests s were done e in
rem markable antim microbial ac tivity for the e fractions 2 2 triplicate [29 9].
and d 6; however the rest of f fractions yiel lded small or r no i inhibition zon nes. We foun nd that these f fractions (i.e. .
3. Results and Discus ssion
and 6) inhibi 2 a ited the grow wth of both bacteria and d In this stu udy, the identi ity of T. pinoy yi was confir rmed
fun gi.
using electro onic microsc ope. As show wn in Fig. 2, this The fraction 2 T 2 showed sign nificant inhib bitory activity y desert truffle e has spheric al spores, the e asci contain n 4-8
aga ainst Gram p positive bact teria (Fig. 3 3) especially y spores at ma aturity, and ha ave ellipsoid form.
Ent terococcus fa aecalis ATCC C6538 and Gr ram negative e The yield d of ethyl ace etate extract of the powd dered
4) espec ially Esche erichia coli i fruiting bod dies of T. p inoyi was 4 4%. In total, six
bac teria (Fig.
ATC CC25922.
fractions we ere separated from the cru ude ethyl ace etate T The fraction 6 showed also a goo od inhibitory y extract of T T. pinoyi by column chro omatography and
acti ivity against Gram positi ive bacteria (Fig. 5) and d thin layer ch hromatograph hy.
Gra am negative b bacteria (Fig. 6). These fra actions were e screened f for antimicro obial
A Among tested d fungi specie es, the growth h of Candida a activity agai inst two Gram m-positive ba acterial specie es (S.
albi icans was markedly inhibited (d diameter of f aureus and
E. feacalis ), two Gram-n negative bact erial inhi ibition zone was 20 mm) by these fra actions (i.e. 2 2 species (E. co oli and P. aer ruginosa ) and one yeast spe ecies
and d 6) (Figs. 7 a and 8).
Table 1 Stra ains used and d mean inhibit tion zones in d disc diffusion assays with th he six fraction ns separated fr rom the crude e extract of T. p pinoyi .
Inh hibition zone (m mm) Species F1 F2 F3 F4 F5 F6
Gram positive e bacteria Staphylococcu us aureus ATCC C6538
6 15 0 0 7 13 Enterococcus f faecalis ATCC C6538
0 18 6 0 0 6 Gram negativ e bacteria Escherichia c oli ATCC25922 2 0 16 0 0 0 8
Pseudomonas s aeruginosa AT TCC14028 0 13 0 7 0 14 Yeast-like fun ngi Candida albic cans ATCC1023 31 7 20 00 0 16
Tests of A Antibiotic Pro operties of Al gerian Deser rt Truffle agai nst Bacteria and Fungi
Fig. 3 Inhib bition growth o of Gram positiv ve bacteria in t the presence of f the fraction 2 2 and their neg gative controls .
a1: Enterococc cus faecalis AT TCC6538; a2: N Negative control l; b1: Staphyloc coccus aureus ATCC6538; b2: A Negative contr rol.
a1 a2
b1 b2
Fig. 4 Inhib bition growth o of Gram negati ive bacteria in the presence o of the fraction 2 and their ne gative controls s.
a1: Escherichi ia coli ATCC25 5922; a2: Negat tive control; b1 : Pseudomonas s aeruginosa AT TCC14028; b2 : Negative cont trol.
Tests of A Antibiotic Pro operties of Al gerian Deser rt Truffle agai nst Bacteria and Fungi
a1 a2
b1 b2
Fig. 5 Inhib bition growth o of Gram positiv ve bacteria in t the presence of f the fraction 6 6 and their neg gative controls .
a1: Enterococc cus faecalis AT TCC6538; a2: N Negative contro ol; b1: Staphyloc coccus aureus ATCC6538; b2 A 2: Negative con ntrol.
a1 a2
b1 b2
Fig. 6 Inhib bition growth o of Gram negati ive bacteria in the presence o of the fraction 6 and their ne gative controls s.
a1: Escherichi ia coli ATCC25 5922; a2: Negat tive control; b1 : Pseudomonas s aeruginosa AT TCC14028; b2 : Negative cont trol.
The resu ults of the a antibacterial activity aga ainst foun nd out that t ethyl aceta ate extract of Tirmania a Gram positi ive bacteria come in agr reement with h the
inhi ibited the g growth of G Gram positi ive bacteria. . findings of Chellal and d Lukasova ( (1995) [21] who
Mo oreover, the an ntibacterial a activity of aqu ueous extract t
Tests of A Antibiotic Pro operties of Al gerian Deser rt Truffle agai nst Bacteria and Fungi
a1 a2
Fig. 7 Inhib bition growth o of Candida albi icans ATCC102 231 in the pres sence of the fra action 2 and ne egative control l.
a1: Candida a albicans ATCC C10231; a2: Ne egative control. .
a1 a2
Fig. 8 Inhib bition growth o of Candida albi icans ATCC102 231 in the pres sence of the fra action 6 and ne egative control l.
a1: Candida a albicans ATCC C10231; a2: Ne egative control. .
anti imicrobial p properties t that can b be used as s (diameter o of inhibition n zone wa as 8 mm) and
of Terfezia claveryi ag ainst Staphy ylococcus au ureus
anti imicrobial ag gents in new drugs for th he therapy of f
infe ectious diseas ses caused by y pathogens. I In conclusion n, was:10 mm) ) was reporte ed by Jankat et al . (2004 and
Pseudomona as aeruginosa a (diameter o of inhibition z zone
it i s obvious th hat we are d dealing with a promising g 2005) [23, 24]. It is p ossible to co onclude that the
anti ibiotic, which h needs to be characterized d. fractions 2 and 6 separ rated from t the ethyl ace etate
Ac knowledgm ments
extract of T T. pinoyi had a broad spec ctrum of acti ivity against man ny bacterial species, as s well as th hese
Thanks the T editor-in-chi ief and the anonymous s fractions we ere more activ ve against ba acterial specie es as
revi iewers for th heir detailed comments an nd criticisms s compared to o fungal speci ies.
that t helped a lo ot to improve e the presen tation of the e pap per. This w work was supported by LBMB B
4. Conclus sion
(La aboratory of f Biology o of Microorg ganisms and d In the pr resent work, the antimicr robial activity y of
Bio otechnology).
different fra actions sepa arated from the crude e ethyl acetate extr ract of T. p pinoyi tested against var rious
Re ferences
microorgani isms allow us s to conclude that the fract tions [1] Z. Fortas, G . Chevalier, Ef ffect of culture e conditions on n
2 and 6 exhibited a antibacterial and antifu ungal the mycorrhi ization of Heli ianthemum gutt tatum by three e species of the e genus of Ter fez: Terfezia an nd Tirmania of activities tha f at support tra ditional use o of the extract of T. Algeria, Can n. J. Bot. 70 (1 1992) 2453-24
60. (in French h pinoyi in the e treatment o of some disea ases. We con nfirm
with English abstract)
that the fra ctions 2 and d 6 possess compounds w with [2] T. Laessoe, K K. Hansen, Truf ffle trouble: Wh hat happened to o
Tests of Antibiotic Properties of Algerian Desert Truffle against Bacteria and Fungi
the Tuberales?, Mycological Research 111 (2007) A1-Mahammad, Chemical composition and nutritive 1075-1099.
value of truffles of Saudi Arabia, J. Food Sci. 50 (1985) [3] Y. Ferdman, S. Aviram, N. Roth-Bejerano, J.M. Trappe,
450-453.
V. Kagan-Zur, Phylogenetic studies of Terfezia pfeilii and [17] A.N. Al-Rahmah, Truffle of Deserts and Jungles, King Choiromyces echinulatus (Pezizales) support new genera
Saud University Publications, Riyadh, Saudi Arabia, for southern African truffles: Kalaharituber and
2001, p. 272. (in Arabic)
Eremiomyces, Mycological Research 109 (2005) [18] H.A. Bokhary, Desert truffles ‘Al-Kamah’ of the 237-245.
Kingdom of Saudi Arabia, occurrence, identification and [4] W.F.O. Marasas, J.M. Trappe, Notes on southern African
distribution, Arab Gulf Journal of Scientific Research B 5 Tuberales, Bothalia 11 (1973) 139-141.
(1987) 245-255.
[5] J.M. Trappe, Use of truffles and false truffles around the [19] R. Rougieux, Ntibiotic and stimulating actions of the world, in: M. Bencivenga, B. Granetti (Eds.), Proceedings,
desert truffle (Terfezia Boudieri Chatin), Ann. Inst. Paris Atti del Secondo Congresso Internazionale sul Tartufo.
105 (1963) 315-318.
Comunita Montana dei Monti Martini e del Serano, [20] M.A. Al-Marzooky, Truffles in eye disease, in: Spoleto, Italy, 1990, pp. 19-30.
Proceedings of the International Conference on Islamic [6] J.M. Trappe, M.A. Castellano, Keys to the genera of
Medicine, Kuwait, 1981, pp. 353-357. truffles (Ascomycetes), McIlvainea 10 (1991) 47-65.
[21] A. Chellal, E. Lukasova, Evidence for antibiotics in the [7] T. Lebel, M.A. Castellano, Australasian truffle-like fungi.
two Algerian truffles Terfezia and Tirmania, Pharmazie IX. History and current trends in the study of the
50 (1995) 228-229.
taxonomy of sequestrate macro fungi from Australia and [22] S. Janakat, S. Al-Fakhiri, A.K. Sallal, A promising New Zealand, Australian Systematic Botany 12 (1999)
peptide antibiotic from Terfezia claveryi aqueous extract 803-817.
against Staphylococcus aureus in vitro, Phototherapy [8] J.M. Trappe, A.W. Claridge, D.L. Claridge, L. Liddle,
Research 18 (2004) 810-813.
Desert truffles of the Australian outback ecology, [23] S. Janakat, S. Al-Fakhiri , A.K. Sallal, Evaluation of Ethnomycology and Taxonomy, Economic Botany 62 (3)
antibacterial activity of aqueous and methanolic extracts (2009) 497-506.
of the truffle Terfezia claveryi against Pseudomonas [9] B.C. Zhang, Ascospore nuclear number and taxonomy of
aeruginosa , Saudi Medical Journal 26 (2005) 952-955. truffles, Micologiae Vegetazione Mediterranea 7 (1992)
[24] A.A.A. Al-Laith, Antioxidant components and 39-42.
antioxidant/antiradical activities of desert truffle (Tirmania [10] L.G.J. Ackerman, P.J. Vanwyk, L.M. Du Plassis, Some
nivea ) from various Middle Eastern origins, Journal of aspects of the composition of the Kalhari truffle of Nabba,
Food Composition and Analysis 23 (2010) 15-22. South Afr. Food Rev 2 (1975) 145-147.
[25] S. Janakat, M. Nassar, Hepatoprotective activity of desert [11] K.S. Al-Delaimy, Protein and amino acid composition of
truffle (Terfezia claveryi) in comparison with the effect of truffle, Journal of the Canadian Institute of Food Science
Nigella sativa in the rat, Pakistan Journal of Nutrition 9 and Technology 10 (1977) 221-222.
(2010) 52-56.
[12] M.M.A. A1-Shabibi, S.J. Toma, B.A. Haddad, Studies on [26] I. Karabegovic, M. Nikolova, D. Veli kovic, S. Stojievic, Iraqi truffles, proximate analysis and characterization of
V. Veljkovic, M. Lazic, Comparison of antioxidant and lipids, Journal of the Canadian Institute of Food Science
antimicrobial activities of methanolic extracts of the and Technology15 (3) (1982) 200-202.
Artemisia sp. recovered by different extraction techniques, [13] H.A. Bokhary, A.A. Suleiman, M.O. Basalah, The fatty
Chinese Journal of Chemical Engineering 19 (3) (2011) acid components of the desert truffle “AlKamah” of
504-511.
Saudi Arabia, Journal of Food Protection 52 (1989) [27] G. Tan, W. Lei, S. Juan, H. Cheng-lin, F. Li, Antioxidant 668-669.
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Cooke & Massee, Food Chemistry 127 (2011) 1634-1640. Vera, M. Honrubia, P. Parras, Antioxidant activity of
[28] A.W. Bauer, W.M.M. Kirby, J.C. Sherris, M. Turck, edible fungi (truffles and mushrooms) losses during
Antibiotic susceptibility testing by a standardized single industrial processing, Journal of Food Protection 65
disk method, Am. J. Clin. Pathol. 45 (1966) 493-496. (2002) 1614-1622.
[29] J.M. Wilkinson, M. Hipwell, T. Ryan, M.A. Cavanagh, [15] F.A. Sakri, Chemical composition and mineral content of
Bioactivity of Backhousia citriodora, antibacterial and Iraqi truffles, Iraqi. J. Sci. 30 (1989) 421-424.
antifungal activity, Journal of Agriculture and Food [16] W.N. Sawaya, A. A1-Shahat, A. A1-Sogair, M.
Chemistry 51 (1) (2003) 76-81.
Mar. 2013, Vol. 7, No. 3, pp. 267-275 Journal of Life Sciences, ISSN 1934-7391, USA
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic Antigen (Chromomycin) in Healthy People in an Endemic Area
Conceição de Maria Pedrozo e Silva de Azevedo 1 , Antônio Augusto Moura da Silva 1 , Sirlei Garcia Marques 1 , Oscar Bruña-Romero 2 , Gilnara Fontinelle Silva 1 , Cecília Silva de Lima 1 , Flávia Raquel Fernandes do
1 Nascimento 2 and Maria Aparecida de Resende Stoianoff 1. Department of Medicine, Federal University of Maranhão, São Luís City 65604010, Maranhão, Brazil
2. Department of Microbiology, Federal University of Minas Gerais, Belo Horizonte 31270901, Minas Gerais, Brazil
Received: October 16, 2012 / Accepted: January 04, 2013 / Published: March 30, 2013.
Abstract: The CBM (chromoblastomycosis) is a disease caused by dematiaceous fungi, which has the species Fonsecaea pedrosoi as main agent. This fungus is found in warm and moist climates, characteristically found in the amazonic lands of the region, where its environmental isolation was once described. This research aimed to identify the healthy population exposed to agent F. pedrosoi in four villages located in the Legal Amazon. In order to clarify the risk factors for allergic immune exposure, a survey was conducted by the technique of delayed skin reaction (IDR) with the metabolic antigen (chromomycin) in 449 healthy individuals. The results showed that 14.9% (67 subjects) were IDR positive, with induration ≥ 5 mm. Multivariate analysis with logistic regression for risk factors: living in the village of Zé Pedro (municipality of Bacabeira in the state of Maranhão, Brazil), (OR = 190.8, P < 0.01, CI =
12.1 to 2,997.4), age over 40 years (OR = 19.5, P < 0.05, CI = 1.65 to 230.8), density home > 5 people (OR = 12.1, P < 0.01, CI = 2.051 to 71.4) and work in breaking the babassu coconut (< 4 h/day: OR = 2147.7, P < 0.01, CI = 67.2-68601.9; > 4 h/day: OR = 3,483.4, P < 0.01, CI = 17.2 to 7,412.7). The dosage of anti- F. pedrosoi performed in 47 healthy individuals with IDR + showed positive in 97.5%, with average titer of 2.109 (S.D. + 3.676) and the average: 1.174 (S.D. + 0.456), showing positive correlation with
area induration of the IDR (mm 2 ).
Key words: Fonsecaea pedrosoi, chromomycin, risk factor.
1. Introduction Chromoblastomycosis most commonly affects male rural workers between the age of 30 and 50 years.
Chromoblastomycosis is endemic in various regions Agriculture is the main activity related to this mycosis of Brazil and is particularly prevalent in the states of [1, 6, 9]. Among agricultural activities, breaking of the Rio Grande do Sul [1] and Paraná [2], in the Amazon babassu coconut has been indicated as a probable risk region [3-5], and in the state of Maranhão [6]. The factor for the development of chromoblastomycosis disease affects the skin and subcutaneous tissue and
caused by F. pedrosoi.
infection results from the traumatic implantation of Silva et al. [10] reported two cases of propagules of different dematiaceous fungi into the chromoblastomycosis in the gluteal region of coconut skin. Fonsecaea pedrosoi and Cladophialophora breakers, a body area that comes in direct contact with carrionii are the most common etiological agents
soil and coconut shells.
[7, 8]. Marques et al. [11] cultured 68 different substrates
Corresponding author: Conceição de Maria Pedrozo e collected in the village of Fortaleza, Municipality of
Silva de Azevedo, Ph.D., research fields: medical mycology, Pinheiro, Maranhão, Brazil, and detected the growth focused in black yeast. E-mail: conceicaopedrozo@gmail.com.
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic Antigen (Chromomycin) in Healthy People in an Endemic Area
of F. pedrosoi in decomposing babassu coconut shells. humid and dry areas. The annual mean temperature of In that study, the coconut tree was found to be an
the state ranges from 25.4 ºC to 27.4 ºC [18]. important plant source of dematiaceous fungi, with the
The municipalities were chosen by convenience. isolation of these fungi from 66.6% of the All villages of these municipalities are located in the environmental samples analyzed [11].
Amazon region of Maranhão: Icatu (village of Clinically, chromoblastomycosis is characterized by
Salgado), Alcântara (village of Peroba de Cima), slow-growing pleomorphic lesions that begin as a
Rosário (village of Zé Pedro), and Pinheiro (village of papule or nodule and can reach large body areas
Fortaleza) (Fig. 1). At least one subject with a before the patient has access to medical care [12, 13].
diagnosis of chromoblastomycosis lived in each The existence of reservoir areas of these fungi has
village of these municipalities. The prevalence of the been demonstrated in several studies, in which the
mycosis (per 1,000 inhabitants) among patients agents were isolated from endemic foci such as
monitored by the mycosis outpatient clinic of UFMA “Baixada Maranhense”, located in the Amazon region
(Universidade Federal do Maranhão) was 0.23 in Icatu, of Maranhão [11], state of Pará [5], and Japan [14].
0.45 in Alcântara, 0.09 in Pinheiro, and 0.34 in The frequency of contact of asymptomatic individuals
Bacabeira.
with causative agents of chromoblastomycosis was evaluated in areas of occurrence of the disease by skin
2.2 Study Population
testing methods using F. pedrosoi methylic antigen. An immunological survey using chromomycin The results showed that certain population groups
(crude F. pedrosoi antigen) was conducted. All were infected with the fungus [15, 16].
residents of the villages older than 10 years (615) The objectives of the present study were to describe
were invited and 449 subjects agreed to participate in the results of an immunological survey of the
the study. All subjects were submitted to delayed population living in reservoir areas of the
hypersensitivity skin testing with F. pedrosoi microorganism in the Amazon region of Maranhão
metabolic antigen (chromomycin) according to the using F. pedrosoi metabolic antigen (chromomycin),
following distribution: 152 subjects in the village of to identify risk factors for infection with this fungus,
Fortaleza (Pinheiro), 30 in the village of Zé Pedro and to analyze the presence of anti-F. pedrosoi
(Bacabeira), 123 in the village of Salgado (Icatu), and antibodies in subjects with a positive skin reaction to 144 in the village of Peroba de Cima (Alcântara). chromomycin. There was no loss of any of the subjects tested and the
2. Methods
skin test could be read in all cases. For immunological study, IgG antibodies were measured in 47 subjects
2.1 Study Place with a positive skin reaction to chromomycin.
The state of Maranhão is located in the northeastern The members of the community that agreed to region of Brazil, occupying the most western part of
participate in the study signed a statement of consent. the country between 1º1 ′ and 10º21′7″ latitudes and
They were interviewed with a questionnaire for the 41º48 ′30″ and 48º50′51″ longitudes [17]. The state
purpose of gathering demographic data. After presents a transition climate between the humid
interpretation of the skin tests, 67 subjects with a climate of the Amazon region and the semi-arid
positive test were invited, but 47 agreed to participate climate of northeastern Brazil. However, most of the
in the second phase of the study, which consisted of territory is characterized by a sub-humid climate
the collection of 20 mL blood for the measurement of which defines the transition between effectively
IgG antibodies.
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic
Antigen (Chromomycin) in Healthy People in an Endemic Area
Fig. 1 Map of the state of Maranhão (Brazil) showing the northeastern mesoregion with the municipalities of Pinheiro, Alcântara, Bacabeira, and Icatu.
2.3 Preparation of Metabolic Antigen
2.4 Skin Test
Metabolic antigen was prepared in the Laboratory For the skin test, 0.1 mL of the antigen (57 µg/mL of Mycology, Department of Microbiology, protein) was injected intradermally into the anterior Universidade Federal de Minas Gerais, according to
surface of the right forearm of each subject. Smith the technique of Oliveira [19]. An F. pedrosoi
medium (0.1 mL) injected into the anterior surface of reference strain deposited in the American Type
the left forearm was used as control. The test was read Culture Collection (ATCC 46428) was used. The
48 h after inoculation of the antigen by measuring the fungus was cultured in Sabouraud agar in an inclined
diameter of the induration in two directions. An glass bottle for 30 days. Next, 1.0 g of the fungus
induration ≥ 5 mm was defined as a positive test [22]. triturated in a blender was inoculated into 100 mL
2.5 Fungal Somatic Antigen
Smith medium. After 15 days of rest, the culture was filtered through a sterile Seitz filter. The filtrate was
Fonsecaea pedrosoi (ATCC 46428) was cultured in stored in a sterile flask wrapped with aluminum foil in
Sabouraud broth under mechanical shaking for 15
a refrigerator. A sample of the filtrate was incubated days at 28 ºC. The culture was filtered and the in brain-heart infusion broth, blood agar and supernatant was used as somatic antigen. The antigen Sabouraud agar (Difco Laboratories Detroit, MI, USA)
was stored at -20 ºC until the time of use. Protein to confirm the sterility of the material.
concentration was determined by the method of Partial chemical analysis of the antigen previously
Bradford [23] and was 97 µg/mL. performed by Barros and Resende [20] showed the
2.6 Enzyme Immunoassay (ELISA) following composition: 0.23 mg/mL lipids, 0.57 mg/mL
protein, and 10.73 mg/mL carbohydrates. The ELISA was used for the measurement of IgG chromomycin used has been validated by Marques et
antibodies in serum samples of subjects with a al. [21], who showed a sensitivity of the antigen of
positive reaction to chromomycin. For this purpose, 90% and specificity of 98.8%. TM 96-well plates (Maxisorp , Nunc) were sensitized
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic Antigen (Chromomycin) in Healthy People in an Endemic Area
with fungal somatic antigen at a concentration of 3 adopting a level of significance of P < 0.05. µg/mL (50 µL/well) diluted in 0.06 M sodium
3. Results
carbonate, pH 9.6, and incubated overnight at 4 ºC. Next, the plates were washed three times with PBS-T
Analysis of the distribution of gender and [0.3% (v/v) Tween 20 in phosphate buffer, pH 7.4]
educational level in the population studied showed a and nonspecific binding sites were blocked by
male-to-female ratio of 1.1:1 and 56.1% of the incubation of the plates with 3% (v/v) casein in PBS-T
subjects had incomplete elementary school. The for 1 h at 37 ºC. The plates were again washed three
dwellings had mud walls in 79.7% of the sample, a times with PBS-T and different human sera were
dirt floor in 67.7%, and a tile roof in 51.2%. Water added at dilutions ranging from 1:100 to 1:25,600,
was obtained from a common well by 56.1% of the followed by incubation of the plates for 1 h at 37 ºC.
participants. Waste disposal was inadequate in 80.6% After three washes with PBS-T, the of the dwellings. peroxidase-conjugated anti-human IgG secondary
The average household income was US$ 150. antibody (ZyMax TM , Invitrogen), diluted 1:1000, was
Analysis of living habits showed that 94.7% (n = 425) added to each well. The plates were incubated for 1 h
of the subjects worked with soil and plants, with at 37 ºC. Next, the plates were washed five times with
76.5% (n = 325) being involved in agricultural PBS-T and two times with pure PBS and the reaction
activities and 29.8% (n = 130) in the activity of was developed by the addition of TMB as chromogen.
coconut breaking. The occurrence of traumas during The color intensity was measured in a activities in the field was reported by 73.3% (n = 329) spectrophotometer at 490 nm. All sera were tested in
of the subjects, with treatment of the lesions in duplicate and the mean optical density was calculated.
78.7% (n = 259).
The cut-off value was determined as the mean optical The skin test performed on 449 subjects was density of normal control sera plus 3 standard
positive in 67 (14.9%), with an induration ≥ 5 mm. deviations (x ± 3 SD).
Univariate analysis showed that residence in Zé Pedro, male gender, age > 40 years, living with more
2.7 Statistical Analysis than five persons in the dwelling, a household income
Skin tests were performed as described above to < US$ 250 coconut breaking, trauma, and lack of determine the level of exposure to F. pedrosoi of the
treatment of the lesion after trauma were associated inhabitants selected in the different municipalities.
with a positive skin test (Table 1). Data obtained with a semi-structured questionnaire
Multivariate analysis showed that residence in Zé were compared between exposed and unexposed
Pedro, age > 40 years, living in a dwelling with more subjects from the reservoir areas selected. The data
than five persons, coconut breaking and untreated were processed using the Epi-Info 6.0 program.
trauma involving soil and plants were strongly
associated with infection with F. pedrosoi. The larger Yates correction was applied. All variables that were
Univariate analysis using the chi-square ( 2 ) test with
the number of hours spent per day on coconut found to be significant upon univariate analysis were
breaking, the greater the association with this activity submitted to multivariate logistic regression using the
(Table 2).
STATA 8 program. The level of significance was set Anti- F. pedrosoi IgG antibodies were detected by at 5% (P ≤ 0.05). The correlation between antibody
ELISA in serum samples of 45 (97.5%) of the 47 levels and induration area in healthy subjects with a
healthy subjects, with a mean (± standard deviation) positive skin test was evaluated using Spearman’s test,
titer of 2,109 (± 3,676) and a mean optical density of
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic
Antigen (Chromomycin) in Healthy People in an Endemic Area
Table 1 Univariate analysis of risk factors for infection with Fonsecaea pedrosoi in four villages of the Amazon region of Maranhão, Brazil (2009).
Positive skin test
Negative skin test
Variable Statistical analysis
Village Fortaleza 27 40.3 125 32.7 Zé Pedro
20 29.9 10 2.6 χ² = 76.23, P < 0.05 Salgado 11 16.4 112 29.3 Peroba de Cima
Gender Male
55.0 χ² = 2.67, P < 0.05 Age < 20 years
31.4 χ² = 11.95, P < 0.05 ≥ 40 years
20-39 years
No. of persons in the dwelling
48.2 χ² = 5.22, P < 0.05 Household income
> R$ 500.00 11 16.4 46 10.2 χ² = 2.52, P < 0.05 Breaking of babassu coconut No
< 4 h/day 19 28.3 29 7.6 χ² = 151.3, P < 0.05 ≥ 4 h/day
Trauma Yes
30.6 χ² = 18.59, P < 0.05 Treatment of trauma Yes
No 54 84.3 16 6.1 χ² = 252.8, P < 0.05 The skin test was performed using F. pedrosoi metabolic antigen (chromomycin).
Table 2 Multivariate analysis of risk factors for infection with Fonsecaea pedrosoi in the villages of Salgado (Icatu), Fortaleza (Pinheiro), Peroba de Cima (Alcântara), and Zé Pedro (Bacabeira), Maranhão, Brazil (2009).
Variable Odds ratio P value (probability associated with F. pedrosoi infection) 95% confidence interval Village
Peroba de Cima
0.87-83.70 Fortaleza
Zé Pedro
12.14-2997.4 Salgado
0.37-31.10 Age
0.31-43.79 > 40 years
< 20 years
20-40 years
1.65-230.88 No. of persons in the dwelling
2.05-71.40 Breaking of babassu coconut
67.23-68601.9 > 4 h/day
No
< 4 h/day
105.42-115100 Trauma
0.011-0.91 17.23-7412.7
Untreated
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic Antigen (Chromomycin) in Healthy People in an Endemic Area
gender. With respect to educational level, 73.8% of the subjects had more than 4 years of schooling, but 26.2% of the population was semi-illiterate or illiterate. The subjects lived under inadequate conditions of housing and sanitation, with more than 40% of the dwellings consisting of mud walls, a dirt floor, and a thatched roof. Basic infrastructure services were also inadequate, with most households not having access to home health units. These findings agree with Ribeiro and Barbosa [27], who reported precarious housing
2 Fig. 2 Correlation between induration area (mm conditions in the hinterland of northeastern Brazil. In ) in the skin
addition, 2008/2009 data from the IBGE [28] showed
test and anti-F. pedrosoi IgG antibody levels (P < 0.05).
that the poorest housing conditions, corresponding to 1.174 (± 0.456). A positive correlation was observed
22% of the households studied, are found in between anti- F. pedrosoi IgG antibody levels and
northeastern Brazil.
induration area (mm 2 ), Spearman’s test, (P < 0.05) The skin test to F. pedrosoi metabolic antigen (Fig. 2).
(chromomycin) was positive in 14.9% (n = 67) of cases, a finding demonstrating that an important
4. Discussion
percentage of the population had contact with the In the present study, rural workers between the age
fungus. An immunological survey of of 30 and 60 years were most commonly affected by
chromoblastomycosis was conducted for the first chromoblastomycosis in the productive stage of life,
time by Baquero in 1959 [29], who applied skin in agreement with the studies of Londero et al. (1976)
testing methods using F. pedrosoi antigen to the rural [24] conducted in Rio Grande do Sul and Silva et al.
population of Cuba. The tests were positive in 25% (1999) in Amazonas [4].
of the sample. In the study of Albornoz [16], 4.5% of In Brazil, the state of Maranhão occupies third
180 subjects aged 5 to 70 years from the state of position in terms of the number of cases of
Falcón, Venezuela, presented positive reactions to F. chromoblastomycosis after Amazonas and Rio Grande
pedrosoi. Celis et al. [15] conducted a similar study do Sul. The existence of an endemic area has been
in two rural municipalities in Colombia, in which established in the state. This area is located in the
116 subjects (88 from Sahagun and 28 from northern mesoregion, which belongs to the Amazon
Medellin) were tested. The skin tests were positive in region of Maranhão [1, 4, 25].
54.5% and 78.6% of cases, respectively. The study The socioeconomic characteristics of the population
involved subjects older than 10 years since younger investigated were similar to those reported in studies
children are considered to be less exposed to risk conducted by the Brazilian Institute of Geography and
factors.
Statistics (IBGE) [17] and by Lemos [26]. Agriculture Comparison of the percentage of positive skin tests plays a key role in the region and the techniques used
between localities showed a higher percentage in the are still rudimentary, a fact exposing the population to
village of Zé Pedro, municipality of Bacabeira (66.6%) direct contact with different pathogenic fungi present
(P < 0.05). This result is surprising since the sample in soil and plants [17, 26].
tested in this community corresponded to less than The sample was equally distributed in terms of
10% of the whole sample. The percentage of a
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic
Antigen (Chromomycin) in Healthy People in an Endemic Area
positive skin test was 56.7% among males, with P < fungus during contact with the infection source [2, 4,
0.05 upon univariate analysis. However, no 31]. The association between trauma and a positive association with a positive skin test was observed
skin test was higher (OR = 360) when the lesions were upon multivariate analysis. In a similar study, Celis et
not treated. It is possible that treatment after injury, al. [15] observed positive skin tests among 63.6% and
which includes cleaning of the wound, mechanically 89.3% of men from two different localities in
removes the fungal agent from the port of entry into Colombia. However, despite the larger number of
the human organism.
positive skin tests among men the authors found no ELISA performed on healthy subjects with a significant association with gender.
positive reaction to chromomycin was positive in 45 Multivariate analysis showed that age > 40 years
samples (95.7%), demonstrating that individuals was significantly associated with a positive skin test,
living in endemic areas who are exposed to the fungus with 59.7% of the subjects older than 40 years testing
may develop a specific humoral response but not the positive (P < 0.05) (OR = 19.5). These findings differ
disease as suggested by Esterre et al. [32]. These from those reported by Celis et al. [15], who observed
authors conducted a seroepidemiological survey of
a larger number of positive skin tests among subjects chromoblastomycosis among residents of eastern aged 10 to 19 years from Sahagun and among subjects
Madagascar. IgG specific for F. pedrosoi was aged 20 to 29 years from Medellin. Albornoz [16]
measured by ELISA in 374 serum samples and observed a higher percentage of positive skin tests in
subclinical infection was detected in 6% of the the group of subjects aged 24 to 35 years.
samples. L. Oberto-Perdigon et al. [33] used somatic With respect to the number of residents per
antigen from Cladophialophora carrionii for the dwelling, exposure to F. pedrosoi was found to be
measurement of specific antibodies by ELISA in 84 higher, the larger the number of individuals living in
healthy subjects, 45 related to and 39 not related to the same house (P < 0.05) (OR = 12.1).
patients with chromoblastomycosis caused by C. The reservoir areas of chromoblastomycosis in the
carrionii. In that study, all samples tested negative. state of Maranhão are located in the Amazon region,
On the basis of the theory of existence of where parts of the coconut plantations are found. The
asymptomatic infection, the authors concluded that a main coconut palm grown in this area is the babassu
study including a larger number of healthy subjects palm (Orbignya phalerata Mart). This palm plays an
from the endemic area of Falcón and the use of skin exceptional role in the transition zones from the
tests is needed to establish a determinant criterion and semi-arid northeast to the savannah where it to have better knowledge of the disease. predominates the grazing landscapes as the main plant
Previous studies have shown the limitations of and in terms of its socioeconomic importance [30]. As
investigating humoral immunity in
a consequence, a strong association was observed chromoblastomycosis. In this respect, Iwatsu et al. [34] between breaking of babassu coconut and a positive
reported that the cellular response is more effective skin test, demonstrating the importance of this activity
than the humoral response. According to Kurita [35], as a risk factor for infection with F. pedrosoi (OR =
serum antibody levels are very low in patients with 2,147.7 and 3,483.4).
chromoblastomycosis, a fact impairing their detection Other variables associated with F. pedrosoi by laboratory methods. In the present study, a positive infection were related to environmental factors and the
correlation was observed between the induration area main route of disease transmission since skin trauma
in the skin test and anti-F. pedrosoi IgG antibody or microtrauma is necessary for penetration of the
levels. This finding shows that laboratory detection of
Detection of Delayed Hypersensitivity to Fonsecaea pedrosoi Metabolic Antigen (Chromomycin) in Healthy People in an Endemic Area
cellular and humoral immunity in asymptomatic on Amazonic region (Brazil), Mycopathology 143 (3) (1998) 171-175.
subjects is possible and that these responses are
C.G. Salgado, W.G. da Silva, S.S. Yamano, U.I. Salgado, interrelated. Further studies are needed to establish the
J.A. Diniz, J.P da Silva, Cutaneous localized annular role of cellular and humoral immunity in these
chromoblastomycosis, Journal of Cutaneous Pathology individuals.
36 (2009) 257-261. [6] C.M.P. Silva, Clinical and epidemiological
5. Conclusion
chromoblatomycosis in the state of Maranhão, São Luís, Master’s Thesis, Federal University of Maranhão, São
The finding of positive skin reaction to Luís, Brazil, 1998, p. 105. (in Portuguese) chromomycin and the detection of anti-F. pedrosoi
F. Queiroz-Telles, M.R. MCGinnis, I. Salkin, J.R. Graybill, Subcutaneous mycoses, Infectious Disease
IgG antibodies (by ELISA) in healthy subjects led us Clinics of North American 17 (2003) 59-85.
to conclude that contact with these fungi is followed [8] G.S.D.E Hoog, D. Attili-Aagelis, V.A. Vicente, H.A.D. by asymptomatic infection in endemic areas of
Gerrits Van Den Ende, F. Queiroz-Telles, Molecular Maranhão. Also, it is possible to use skin reaction and ecology and pathogenic potential of Fonsecaea species, Medical Mycology 42 (2004) 405-416. ELISA serology to identify these individuals, thus
[9] M.R. Mcginnis, Chromoblastomycosis and permitting long-term monitoring of any clinical sign
phaeohyphomycosis: New concepts, diagnosis and of active disease. In addition, knowledge about the
mycology, Journal of the American Academy of Dermatology 8 (1) (1983) 1-16.
risk factors for exposure to infection with F. pedrosoi [10] C.M.P. Silva, R.M. Rocha, J.S. Moreno, M.R.F.C. contributes to the establishment of measures to
Branco, R.R. Silva, S.G. Marques, et al., The babassu prevent chromoblastomycosis, as protection during
coconut (Orbignya phalerata Martins) as a probable risk break of babassu coconut and treatment of the injuries factor of human infection of chromoblastomycosis in the state of Maranhão, Brazil, Journal of the Brazilian
that occur after trauma in the work of farming. Society of Tropical Medicine 28 (1) (1995) 49-52. (in Portuguese)
Acknowledgments
[11] S.G. Marques, C.M. Pedrozo e Silva, P.C. Saldanha, M.A. Resende, V.A. Vicente, F. Queiroz-Telles, et al., Isolation
The authors gratefully acknowledge the Foundation of Fonsecaea pedrosoi from the shell of the babassu
for Research Support of Maranhão (FAPEMA) and coconut (Orbignya phalerata Martins) in the Amazonic National Counsel of Technological and Scientific
region of Maranhão, Brazil, Japanese Journal of Medical Development (CNPq).
Mycology 47 (2006) 305-311. [12] C.P. Milan, N.A. Fenske, Chromoblastomycosis,
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Mar. 2013, Vol. 7, No. 3, pp. 276-281 Journal of Life Sciences, ISSN 1934-7391, USA
Inhibitory Effect of the Essential Oil from Hyptis suaveolens (L.) Poit on the Growth and Aflatoxins Synthesis of Aspergillus flavus
Ana Carolina Pessoa Moreira 1 , Egberto Santos Carmo 2 , Paulo Alves Wanderley 3 , Evandro Leite de Souza 4 and
Edeltrudes de Oliveira Lima 1
1. Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa 58051-900, Brazil 2. Academic Unit of Health, Federal University of Campina Grande, Cuité 58175-000, Brazil 3. Department of Agriculture, Federal University of Paraíba, Bananeiras 58220-000, Brazil 4. Laboratory of Food Microbiology, Department of Nutrition, Federal University of Paraíba, João Pessoa 58051-900, Brazil
Received: September 19, 2012 / Accepted: December 03, 2012 / Published: March 30, 2013.
Abstract: H. suaveolens (L.) Poit has been used in folk medicine revealing a wide pharmacological potential. This study aimed to evaluate the efficacy of the essential oil from H. suaveolens leaves in inhibiting the growth of A. flavus LM257 and synthesis of
aflatoxins B 1 and B 2 . The essential oil at 20, 40 and 80 µL/mL strongly reduced the biomass and spore germination of A. flavus. A 100% inhibition was found at 40 µL/mL and 80 µL/mL, while at 20 µL/mL the growth depressed over 50% for both tested parameters. The essential oil at all assayed concentrations totally inhibited the production of aflatoxin B 1 and B 2 . From these results, the essential oil from H. suaveolens leaves may be considered as an alternative and effective inhibitor of A. flavus, in addition to offer some protective effect to the production of aflatoxins.
Key words: Hyptis suaveolens, essential oil, Aspergillus flavus, mycotoxins, inhibitory effect.
1. Introduction especially, A. flavus and A. parasiticus. A. flavus, the
most common causal fungus, produces aflatoxins B 1 Aspergillus genus, which presents species inserted and B 2 . The occurrence of aflatoxins in food and feed in the group of infesting living plants (e.g. A. flavus) commodities has been a potential threat to consumer and infesting stored food products fungi (e.g. A. safety and to the world market due to the extremely parasiticus , A. ochraceus, A. fumigatus, A. chevalieri low tolerance levels [5-7]. The special risk posed by and A. clavatus), is responsible for many causes of aflatoxins provokes studies on development of novel food contamination all over the world [1-3]. The technologies for inhibiting the growth of aflatoxigenic growth of Aspergillus in foodstuffs is toxicologically moulds and/or the synthesis of aflatoxins in foods.
significant since some species are known to
Thus, the presence and growth of this fungus in food produce mycotoxins when exposed to suitable and feed threatens human and animal health. conditions [4, 5]. The use of chemical or synthetic agents with Aflatoxins are toxic and hepatocarcinogenic antifungal activity (as inhibitors, growth reducers or polyketides produced by some Aspergillus species, even inactivators) is one of the oldest techniques for
controlling fungal growth and mycotoxins production Corresponding author: Ana Carolina Pessoa Moreira, Ph.D., professor, research field: mycology. E-mail: in foods. The application of preservatives to foods is carolpessoa25@gmail.com.
Inhibitory Effect of the Essential Oil from Hyptis suaveolens (L.) Poit
on the Growth and Aflatoxins Synthesis of Aspergillus flavus
fundamental if their safety is to be maintained [5, 7]. Brazil), where a voucher specimen was kept under a The essential oils of plants and their constituents with
number 11367. H. suaveolens essential oil was obtained antifungal properties could feature as interesting
by hydrodistillation using a Clevenger apparatus. The alternative for this purpose. Very few publications
essential oil was assayed at 20, 40 and 80 µL/mL, and have documented the antimicrobial activity of the solutions were prepared according to Souza et essential oil from Hyptis suaveolens (L.) Poit. against
al. [15].
fungal species [8]. In earlier study, the authors
2.2 Test Microorganism
observed that the essential oil from H. suaveolens leaves at low concentrations provided interesting
A. flavus LM-257 was obtained from the inhibition of the radial mycelial growth of different
microorganism collection, Laboratory of Mycology, Aspergillus species and abnormalities in their Health Sciences Center, Federal University of Paraíba morphology [8]. These findings thus, indicate the
(João Pessoa, Brazil). Stock cultures were kept on possibility of exploiting H. suaveolens essential oil as
sterile Sabouraud agar slants under 7 °C (± 1 °C). For an effective inhibitor of this biodegrading and
anti-mould assays were used 7 days-old cultures storage-contaminating fungi. The genus Hyptis Jacq.
grown on sterile Sabouraud agar at 25-28 °C. Mould consists of over 300 species that occur in tropical
spores were taken by adding sterile NaCl (0.85 g/100 America [9]. Hyptis suaveolens (L.) Poit. is a
mL) on the media growth followed for gentle shaking completely aromatic aggressive annual weedy species
for 30 s. Each suspension was filtered through sterile distributed in tropic and subtropic regions, and
triple layer cheesecloth to remove mycelial fragments. normally restricted to places where soils have been
Mould spores number was counted using profoundly disturbed [10, 11]. The essential oil
hemocytometer, and the spores suspension was obtained from this specie has showed a high degree of
adjusted using sterile NaCl (0.85 g/100 mL) to contain variability in quantity and types of components
approximately 10 6 spores/mL [16]. differing according to the geographic origin [12-14].
2.3 Determination of Mycelial Dry Weight anti-Aspergillus property of the essential oil obtained
Here we report the further investigation of the
Inhibition caused by the essential oil on the from H. suaveolens leaves, particularly, its inhibitory
mycelial dry weight/biomass was determined using effect on the mycelial mass, spore germination and
the poisoned substrate technique (dilution in liquid aflatoxins synthesis of A. flavus. To our knowledge,
medium). For this, 10 6 spores/mL of the assayed fungi this is the first report about the inhibition of
was inoculated in Erlenmeyer flasks containing sterile mycotoxins synthesis by the essential oil from H.
Sabouraud broth added of the essential oil at suaveolens .
concentrations of 20, 40 and 80 µL/mL. After 15 days
2. Materials and Methods
of exposure at 28 °C, the dry weight of mycelium was determined. For this, flasks containing mycelia were
2.1 Plant autoclaved (121 °C for 30 s) in order to inactivate the
Leaves of H. suaveolens L. (Poit) were collected in spores, and the content of the flasks were filtered by January 2007 from the experimental plant collection of
Whatman No. 1 and washed twice with sterile distilled the Department of Agriculture, Center of Technologists
water. Mycelia were allowed to dry at 60 °C for 6 h Formation, Federal University of Paraíba (Bananeiras,
and then 40 °C overnight. Filter paper containing dry Brazil). The plant was authenticated by the National
mycelia was weighed. Control flasks without essential Herbarium Prof. Jaime Coelho de Moraes (Areia,
oil were tested similarly. Percent of mycelia growth
Inhibitory Effect of the Essential Oil from Hyptis suaveolens (L.) Poit
on the Growth and Aflatoxins Synthesis of Aspergillus flavus
inhibition on the basis of dry weight was calculated in at 20 µL/mL the growth depressed over 50% for both comparison with the control assay [17].
tested parameters. Slight sporulation was observed in spores exposed to the essential oil (data not showed).
2.4 Spore Germination Assay Although at 20 µL/mL of essential oil some spores
Aliquots of the essential oil at concentrations of 20, were able to germinate, it was noted that these spores
40 and 80 µL/mL were mixed (1:1 v/v) with the produced smaller germ tubes (early growing hyphae) mould spores suspension (approximately 10 6 as compared with the control assay (data not shown).
spores/mL). The mixture was placed on separated In accordance with earlier researches [18, 20] the glass slides and incubated in a moist chamber at
inhibition of spore germination caused by H. 25-28 °C for 24 h. At the end of the incubation period,
suaveolens essential oil was in a dosage response each slide was fixed with lacto-phenol-cotton blue
manner. The exact mechanism by which antimicrobial stain and observed under the light microscope for
compounds inhibit the conidia germination has not spore germination. Control flasks without essential oil
been well understood, although some researchers have were tested similarly. About 200 spores were counted
purposed a reduction of the permeability to nutrients and the percent of spore germination was calculated in
in conidia cell wall, thereby affecting the germination comparison with the control assay [18].
[21, 22]. The high inhibition rates of spore
2.5 Aflatoxins Production germination caused when the tested fungi was exposed to the H. suaveolens essential oil is an
Three amount of essential oil (20, 40 and 80 µL/mL) interesting result because the spore is known to be an were added to flasks containing 100 mL of sterile
important structure for the survival and spread of Sabouraud broth, inoculated with 10 mL of the spore
fungi; therefore, the application of substances that suspension (approximately 10 6 spores/mL) and strongly inhibit spore germination could arise as
incubated for 10 days at 25 ºC. After the incubation promising tool for the biocontrol of spore populations period, flasks were sampled by aseptically removing
on various substrates or environments. duplicate portions of 0.1 mL of broth for
Moreira et al. [8] found that the essential oil from H.
suaveolens at 40 µL/mL and 80 µL/mL caused a Filtenborg et al. [19]. Control flasks without adding
determination of aflatoxin B 1 and B 2 according to
decrease of the radial growth of A. flavus LM-257 in the essential oil were tested similarly.
solid media over 90% along 14 days. These results are
3. Results and Discussion
in agreement to our findings and reveal that the essential oil from H. suaveolens is equally effective in
3.1 Mycelial Dry Weight and Spore Germination
liquid or solid medium.
A strong antifungal activity for the essential oil suaveolens essential oil at 20, 40 and 80 µL/mL was
Inhibition of the growth of A. flavus LM-257 by H.
from H. suaveolens leaves at 500 µL/mL and 1,000 estimated by the dry weight of the mycelial mass
µg/mL against Saccharomyces cerevisiae, Mucor sp. (biomass) and spore germination of A. flavus
and Fusarium moniliforme was found by Malele et al. LM-257 by Hyptis suaveolens leaves essential oil are
[14]. Asekun et al. [13] reported that the essential oil shown in Table 1. As can be seen, the biomass and
from H. suaveolens leaves (5 mg/L) displayed spore germination of A. flavus was strongly reduced
significant inhibitory activity against gram-positive in response to the assayed concentrations of H.
(Staphylococcus aureus and Bacillus cereus) and suaveolens essential oil. A 100% inhibition was
gram-negative (Escherichia coli and Pseudomonas found at 40 and 80 µL/mL of the essential oil, while
aeruginosa ) bacteria and yeast (Candida albicans).
Inhibitory Effect of the Essential Oil from Hyptis suaveolens (L.) Poit
on the Growth and Aflatoxins Synthesis of Aspergillus flavus
Table 1 Inhibition of the mycelial dry weight and spore germination of Aspergillus flavus LM-257 by Hyptis suaveolens leaves essential oil.
Essential oil (µL/mL)
Inhibition of spore germination (%) Control 0 20 52
Inhibition of mycelial dry weight (%)
Table 2 Effect of the essential oil from Hyptis suaveolens
B 2 . In fact, at positive control assay (without the
leaves on aflatoxins production by A. flavus LM-257.
essential oil) the aflatoxins B 1 and B 2 were found at
Aflatoxin (ng/mL)
Essential oil (µL/mL)
B 1 B 2 24.22 and 19.37 ng/mL, respectively.
20 ND ND Previous researchers have purposed that the
40 ND ND inhibition of aflatoxins production by A. flavus caused
80 ND ND by essential oils cannot be completely attributed to
24.22 19.37 insufficient mycelium growth [27, 28]. Since ND not detected.
Control
aflatoxins are synthesized extramitochondrially from Although relatively few samples of essential oils
acetylcoenzyme A during a period of rapid glucose from H. suaveolens have been analyzed, it appears
utilization, it has been suggested that the restriction of from the reported data that the essential oils obtained
carbohydrates catabolism in A. flavus caused due to from the leaves of this plant exhibits different
the interference of essential oils compounds on some compositions characterized for the occurrence of
key enzymes may result in decrease of its ability to β-caryophyllene (Malaysia and Nigeria), 1,8-cineole
synthesize aflatoxins [28]. This hypothesis is and sabinene (USA, India and Aruba), 1,8-cineole and
reinforced for the findings of other studies where plant β-pinene [23, 24] as the majority compounds.
compounds were able to penetrate inside the fungal Moreira et al. [8] evaluated the composition of the
cell and react with active sites of key enzymes or act essential oil from H. suaveolens assayed in this study
as H + carrier, depleting adenosine triphosphate pool and found the terpene alcohol 1,8 cineol/eucaliptol
and ultimately resulting in disturbance of the fungal (47.64%) as the majority compound, followed for the
metabolism [29].
terpene hydrocarbons gama-ellemene (8.15%), Moreira et al. [8] found that the essential oil from H. beta-pynene (6.55%), (+)-3-carene (5.16%), suaveolens caused morphological changes in A. flavus
trans-beta-cariophyllene (4.69%) and germacrene including lack of sporulation, loss of cytoplasm (4.86%). Compounds frequently found in the essential
content, loss of pigmentation and distorted oil, such as germacrene D and germacrene B [25, 26],
development of hyphae. Regarding these findings the were not found suggesting that the assayed essential
authors purposed that the anti-Aspergillus activity of oil could correspond to a new chemotype.
the assayed essential oil probably includes attack on the cell wall and retraction of the cytoplasm in the
3.2 Aflatoxins Production hyphae resulting in death of mycelium. The results of the inhibitory effect of H. suaveolens
4. Conclusions
essential oil on the aflatoxin production by A. flavus LM-257 are shown in Table 2. Correspondingly to the
According to the present results, the essential oil results of biomass and spore germination, the essential
from H. suaveolens leaves revealed a fast and steady oil at all assayed concentrations (20, 40 and 80 µL/mL)
anti-A. flavus property with a strong inhibition of the
strongly inhibited the production of aflatoxin B 1 and
mycelial growth, fungi spore germination and
Inhibitory Effect of the Essential Oil from Hyptis suaveolens (L.) Poit
on the Growth and Aflatoxins Synthesis of Aspergillus flavus
aflatoxins production. Based on this study, it can be [8] A.C.P. Moreira, E.O. Lima, P.W. Souza, E.S. Carmo, E.L. Souza, Chemical composition and antifungal activity of
concluded that the essential oil from H. suaveolens Hyptis suaveolens (L.) Poit leaves essential oil against
leaves, if applied in sufficient amounts, possesses Aspergillus species, Brazilian Journal of Microbiology 40 fungitoxic activities, inhibiting the growth of A. flavus,
(2007) 1036-1041.
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inhibitor of the survival of this fungi in foodstuffs, in
(1988) 87-95.
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of seeds in Hyptis suaveolens, Ecology 54 (1973) 646-649.
Acknowledgments
[11] M.M. Iwu, C.O. Ezeugwu, C.O. Okunji, D.R. Sanson, M.S. Tempesta, Antimicrobial activity and terpenoids of
The authors thank the National Council for the essential oil of Hyptis suaveolens, International Scientific and Technological Development
Journal of Crude Drug Research 28 (1990) 73-76. (CNPq Brazil) for the financial support.
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Mar. 2013, Vol. 7, No. 3, pp. 282-288 Journal of Life Sciences, ISSN 1934-7391, USA
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the Number of Maiden Fruit Trees Produced in an Organic Nursery
Zygmunt Stanis ław Grzyb, Wojciech Piotrowski, Paweł Bielicki and Lidia Sas Paszt Department of Pomology, Research Institute of Horticulture, Pomologiczna 18, Skierniewice 96-100, Poland
Received: October 16, 2012 / Accepted: December 21, 2012 / Published: March 30, 2013.
Abstract: In an organic nursery, various bioproducts were used to stimulate plant growth: Fertigo (granulated bovine manure), Micosat, Humus UP (humic acids microbiologically enriched), Humus Active + Aktywit PM (beneficial microorganisms), BioFeed Amin, BioFeed Quality, Tytanit, and Vinassa. One of the aims of the study was to determine the effect of these products on the ability of grafted buds of apple and sour cherry cultivars to survive the winter. Losses resulting from the freezing of buds during winter dormancy were calculated as the difference between the number of buds deemed to have taken in autumn and the number of live buds that began to develop properly in the spring of the following year. In autumn, the number of maiden trees obtained in each fertilization combination was also determined in the same nursery. The greatest tendency to die in winter was shown by apple and sour cherry buds in those combinations in which the rootstocks were supplied with a mineral fertilizer with NPK components, and the smallest where humic preparations (so-called vermiculites) were used. The type of fertilizer had an indirect influence not only on the number of grafted buds that died in winter as a result of freezing, but also on the final number of maiden trees obtained from a given area of the nursery.
Key words: Bio-products, organic nursery, grafted buds mortality, maiden trees.
1. Introduction resistance of grafted buds to frost, and which of them increase it, and at the same time will reveal how those
In the available scientific literature on the products affect the final number of the trees propagation of fruit trees, one can come across reports
produced [1, 8-10].
on the impact of the type of rootstock on the taking of bud grafts [1-5], but there is no data on the effect of
2. Materials and Methods
the type of fertilizer (supplied to rootstocks) on this The study was conducted in 2009-2011 in an process. It is well known that excessive doses of experimental nursery located in Mokra Lewa near nitrogen fertilizers prolong the period of vegetative Skierniewice, on a podzolic soil (pH 5.5-6.0). In the growth in plants and thus do not allow them enough year preceding the experiment, a phacelia and vetch time to prepare for winter dormancy [6, 7]. Buds mixture of cover plants was grown on the plot and grafted on such rootstocks often freeze. In an organic incorporated into the soil at blooming to serve as a nursery, the use of bioproducts containing different means of organic fertilization and to reduce the proportions of minerals, including nitrogen, will make occurrence of soil-borne pathogens. The experiment it possible to determine which of them reduce the was set up in a randomized block design with four
replications, each consisting of 10 plants. Corresponding author: Zygmunt Stanis ław Grzyb, Ph.D.,
Prof., research fields: ecology, nursery, gene resources, stone In the first year of running the nursery, fertilization fruits. E-mail: Zygmunt.Grzyb@inhort.pl.
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the
Number of Maiden Fruit Trees Produced in an Organic Nursery
treatments were applied to M 26 apple rootstocks and 0.03% N, 1,050 mg·kg -1 P and 4,119 mg·kg -1 K. to cherry rootstocks (Prunus mahaleb L.), and in the
Aktywit PM is a soil improver containing 20.5% C, second year, after grafting, to maiden trees of two
0.92% N, 81.2 mg·kg -1 P and 42,990 mg·kg -1 K. apple cultivars (“Topaz” and “Ariwa”) and maiden
Humus Active was applied to the soil as a 2% solution trees of two sour cherry cultivars (“Debreceni
(2 mL·m -2 ) (20 L·ha -1 ) and Aktywit PM was applied to Bötermö” and “Sabina”).
the soil as a 1% solution—1 mL·m -2 (10 L·ha -1 ); The following types of fertilization treatment were
(7) BioFeed Amin (Agrobio Products B.V., the applied:
Netherlands)—an extract reinforced with amino acids (1) No treatment (control);
—an extract of vegetal amino acids containing 1.12% (2) Chemical NPK fertilization: at a dose of 17.64
C, 0.14% N, 347 mg·kg -1 P. The product was applied g·m -2 NH
4 NO
to the soil as a 0.5% solution (0.5 mL·m 3 -2 , 6.52 g·m ) (5 L·ha );
triple super phosphate, and
2 SO 4 , equivalent to 60 kg·ha N, 30 (8) BioFeed Quality (Agrobio Products B.V., the kg·ha -1 P, and 80 kg·ha -1 K (NPK);
16.0 g·m -1 K
Netherlands)—an extract from several seaweed
(3) Fertigo (Ferm-O-Feed, the species reinforced with humic and fulvic acids Netherlands)—granulated bovine manure contains
containing 0.6% C, 0.07% N, 32.6 mg·kg -1 P (applied 55% C, 1% N, 0.3% P and 1% K; besides these, also
to the soil as a 0.5% solution (0.5 mL·m -2 ) (5 L·ha -1 )); microelements and soil micro-organisms. The product
(9) Tytanit (Intermag, Poland) – titanium (Ti) was applied at a dose of 150 g·m -2 (1,500 kg·ha -1 ),
applied to the leaves as a 0.05% solution (0.05 mL·m -2 )
equivalent to 45 kg·ha -1 N, 13 kg·ha P and 17 kg·ha (0.5 L·ha );
K; (10) Vinassa (Józefów Sp. z o.o., (4) Micosat (CCS Aosta Srl, Italy)—microbial
Poland)—molasses residue from yeast production inoculum consisting of mycorrhizal fungi (Glomus -1 containing 12.0% C, 1.86% N, 949 mg·kg P, 17,615
mosseae and G. intraradices), and plant growth mg·kg -1 K. The product was applied to the soil as a
promoting bacteria (Pseudomonas fluorescence and -1 0.5% solution (0.5 mL·m ) (5 L·ha ). Bacillus subtilis strains). The product contains 40% C,
The plants treated with Micosat, BF Quality, BF 0.15% N, 431 mg·kg -1 P and 9,558 mg·kg -1 K, and a
Amin, Tytanit and Vinassa were planted in the soil granular formulation Micosat F12 WP was applied at
which had been fertilized with half the dose of planting to the soil at a dose of 10 g·m -2 (100 kg·ha -1 ),
granulated bovine manure (75 g·m -2 ). and a second application was carried out in mid-June
All the preparations except NPK and bovine in liquid form (Micosat F MS 200) at a dose of 1 g·m -2
manure (Fertigo) were applied twice: the first (10 kg·ha -1 ); time—in the rootstock nursery in mid-May, and in the
(5) Humus UP (Ekodarpol, Poland)—an extract nursery of maiden trees in late April/early May, and from vermicomposts containing 0.65% C, 0.03% N,
the second time: to rootstocks—in mid-May, and to
30.8 mg·kg -1 P and 4,535 mg·kg K. The product was maiden trees again in mid-June. After each application applied to the soil as a 2% solution (2 mL·m -2 ) (20
of the preparations, the soil around the plants was L·ha -1 );
thoroughly mixed by hand using hoes. (6) Humus Active + Aktywit PM (Ekodarpol,
In late July, for each fertilization combination, 10 Poland)—an extract from vermicomposts based on a
rootstocks were bud grafted in four replications. The product derived from molasses. Humus Active is a
number of buds that had taken was checked in late soil improver with active humus and population of
autumn after leaf fall, and the number of live buds beneficial microorganisms containing 0.78% C, beginning to grow was assessed in the second half of
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the
Number of Maiden Fruit Trees Produced in an Organic Nursery
May of the following year. The difference between the organic nursery on the survival of grafted buds of number of buds identified as “having taken” in the
apple cultivar “Topaz” was insignificant both in the autumn of the previous year and the number of buds
winter of 2010 and 2011 (Table 1). Depending on the starting to grow in the spring of the following year
year and type of fertilizer used in the rootstock was the basis for calculating the mortality rate (due to
nursery, the mortality rate ranged from 2.5% to 5.8% freezing) in winter. The number of maiden trees
in the winter of 2010, when there was a sufficiently obtained in each fertilization combination on the
thick snow cover, while in 2011, after a snowless experimental plots was determined immediately winter, the mortality rate was higher and ranged from before digging up the trees.
3.1% to 18.9%. The largest number of dead buds after The effects of the fertilization combinations on
the winter in the second year of running the nursery different plant growth parameters were assessed using
was in the combination without fertilization. Mineral one-way analysis of variance. Multiple comparisons
NPK fertilizers, and also BF Quality, were not of means for the combinations were performed using
conducive to their survival, either. Tukey’s test at a significance level of α = 0.05. In
In 2010, the number of maiden apple trees cv. each column, the data that are not significantly
“Topaz” varied from 64.7% to 91.3% depending on different are marked with the same letters.
the type of the product used as fertilizer. It was the highest in the combination where Humus UP was used,
3. Results
and the lowest for BF Amin. In the second year of the The influence of the type of fertilizer used in the
study, the variability in the results was higher than in
Table 1 Number of buds frozen in winter and the number of obtained maidens of “Topaz” and “Ariwa” apple trees budded on rootstock M 26 (Mokra Lewa, 2009-2011).
Number of maiden trees obtained in relation to Treatment
Winter losses in the number of buds [%]
the number of budded rootstocks (%)
2011 Cultivar “Topaz” Control 3.0 a 18.9 a 91.3 ab 62.5 a NPK 5.8 a 11.3 a 88.9 ab 67.5 a
2009-2010 2010-2011 2010
Fertigo Manure 2.5 a 6.1 a 64.7 a 75.0 a Micosat 5.0 a 8.4 a 70.6 ab 74.8 a
Humus UP 2.5 a 10.6 a 92.5 b 80.0 a Humus Active + Aktywit PM
2.8 a 3.5 a 86.0 ab 84.2 a BF Quality
2.8 a 18.0 a 86.7 ab 79.2 a BF Amin
3.0 a 3.1 a 71.4 ab 85.0 a Tytanit 3.8 a 4.5 a 80.6 ab 79.4 a Vinassa 2.8 a 7.6 a 83.0 ab 73.9 a
Cultivar “Ariwa” Control 3.8 ab 15.0 a 94.4 b 76.7 a NPK 14.3 b 20.0 a 66.1 a 78.3 a
Fertigo Manure 0.3 a 12.9 a 92.5 ab 80.8 a Micosat 6.3 ab 12.4 a 90.0 ab 85.7 a
Humus UP 0.5 a 3.7 a 92.5 ab 80.0 a Humus Active + Aktywit PM
0.5 a 9.0 a 95.0 b 76.7 a BF Quality
0.3 a 15.1 a 97.5 b 80.0 a BF Amin
0.5 a 11.3 a 91.7 ab 75.0 a Tytanit 2.5 a 7.5 a 90.0 ab 76.9 a Vinassa 0.3 a 21.0 a 84.2 ab 73.3 a
Values that do not differ from each other are designated in each column by the same letters.
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the
Number of Maiden Fruit Trees Produced in an Organic Nursery
the first year, and thus there was no significant tendency to die was shown by the buds on the difference between the treatment combinations in
rootstocks treated with humic preparations, including terms of the number of maidens produced. In the case
products such as Humus UP and Humus Active + of NPK fertilization, we can only talk about a
Aktywit PM, and Tytanit. In 2011, the number of tendency to reduce the production of maiden trees,
obtained maiden trees was lower than in 2010. The and about an increase in that production when using
type of fertilization did not have any major effect on such preparations as Humus UP, Humus Active +
this result.
Aktywit PM, BF Amin, BF Quality and Tytanit. Losses in the number of grafted buds of the sour In the case of the apple cultivar “Ariwa”, the winter
cherry cultivar “Debreceni Bötermö” in the winter of mortality rate of the buds grafted on rootstock M 26 in
2010 were small and ranged from 2.8% to 22.2% the first year of the study (2010) was significantly
(Table 2). Compared to the other treatments, a higher in those treatment combinations where mineral
significantly higher mortality rate was observed NPK fertilization was applied. In the second year of
following the use of NPK to fertilize the rootstocks. A the study (2011), the differences in bud mortality
tendency to reduce the dying of buds in winter was between the combinations were not significant. A
shown by the rootstocks fertilized with such more pronounced tendency to increased winter preparations as BF Amin and BF Quality. In the mortality was shown only by the buds on the
following year, however, the variability in the number rootstocks treated with NPK and the preparation
of frozen buds among the treatments was not Vinassa. In contrast to these preparations, a lower
significant and varied within a small range.
Table 2 Number of buds frozen in winter and the number of obtained maidens of sour cherry cultivars “Debreceni Bötermö” and “Sabina” budded on Mahaleb cherry seedlings (Mokra Lewa, 2009-2011).
Number of maiden trees obtained in relation to Treatment
Winter losses in the number of buds (%)
the number of budded rootstocks (%)
2011 Cultivar “Debreceni Bötermö” Control 5.0 ab 10.3 a 97.5 b 77.5 ab NPK 22.2 b 7.4 a 75.0 a 60.8 a Fertigo manure
2009-2010 2010-2011 2010
5.6 ab 4.7 a 89.7 ab 88.5 ab Micosat 10.3 ab 13.7 a 96.9 b 73.6 ab
Humus UP 10.7 ab 15.0 a 85.0 ab 76.3 ab Humus active + aktywit PM
7.8 ab 12.2 a 77.5 ab 81.7 ab BF quality
2.8 a 2.8 a 76.7 ab 91.7 b BF amin
2.8 a 7.8 a 75.0 a 85.0 ab Tytanit 5.0 ab 3.1 a 72.5 a 90.0 ab Vinassa 5.0 ab 10.3 a 72.5 a 80.0 ab
Cultivar “Sabina” Control 2.5 a 19.8 a 75.0 b 72.8 a NPK 41.7 b 5.0 a 57.5 ab 70.8 a
Fertigo manure 19.5 ab 9.2 a 55.0 a 71.5 a Micosat 8.4 a 8.5 a 55.0 a 70.5 a
Humus UP 13.6 a 7.7 a 60.0 ab 82.3 a Humus active + aktywit PM
17.5 ab 13.8 a 58.9 ab 70.0 a BF quality
5.0 a 11.3 a 65.0 ab 78.8 a BF amin
12.5 a 6.3 a 52.5 a 66.4 a Tytanit 2.5 a 18.1 a 68.6 ab 67.5 a Vinassa 18.9 ab 11.4 a 67.5 ab 69.2 a
Values that do not differ from each other are designated in each column by the same letters.
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the Number of Maiden Fruit Trees Produced in an Organic Nursery
In the combination containing mycorrhizal fungi their thickness and the activity of the cambium during and beneficial soil bacteria (Aktywit PM), and in the
bud-grafting, and the general state of their health. Too control combination without fertilization, we obtained
heavy nitrogen fertilization extends the growing
a significantly greater number of maiden trees of the period of the rootstocks and thus does not allow them sour cherry cultivar “Debreceni Bötermö” compared
time to prepare for winter dormancy [11, 12]. Our with the other fertilization combinations. In 2011, the
research shows that mineral NPK fertilization in apple lowest number of trees was obtained as a result of
causes a significantly higher mortality of grafted buds fertilizing sour cherry maidens with the NPK mineral
during winter and a lower crop of maidens than in fertilizer, and the largest number in those those combinations where no fertilizer is used, or in combinations where BF Quality was used. The action
those combinations where humic formulations of Tytanit was also beneficial for increasing the
(vermiculites) or Tytanit are used, which do not have number of the maiden trees produced.
high levels of nitrogen in their composition and thus In the case of the sour cherry cultivar “Sabina”,
do not extend the growing period of rootstocks and do fertilization of rootstocks with the mineral NPK
not intensify their growth compared with the other fertilizer in 2010 resulted in a very high mortality
preparations [1, 5, 10, 12, 13,].
among the grafted buds during winter, reaching It is worth mentioning that in some states of the almost 40%. In the combination without fertilization,
United States of America, where winters are more and where Tytanit was used as a fertilizer, their
severe, there is a common practice of sowing oats in mortality rate did not exceed 2.5%. In the next year,
rootstock nurseries immediately after bud-grafting. 2011, the extent of the dying of buds in winter was
The oat plants take up nitrogen from the soil and thus small and ranged from 5% to 18.1%, depending on the
shorten the period of vigorous growth of rootstocks. type of fertilizer. The differences between the
Moreover, being a spring plant, the oat freezes in combinations were not significant.
winter and forms a protective layer that protects the The number of maiden trees in 2010 was
grafted buds from freezing [4, 7, 12, 14,]. significantly higher in the control combination
As demonstrated by our research, excessive NPK without fertilization than in the other combinations.
fertilization in a sour cherry nursery results in a very The type of fertilizer in that year had no effect on the
high mortality of grafted buds during winter, much number of maidens obtained. A similar situation
higher than when fertilizing with other organic occurred in the year 2011, in which the differences
bioproducts or soil improvers. This has a strong between the combinations varied only within the
adverse effect on the number of maiden trees range 66.4%-82.3%. The smallest number of maidens
produced. The effect of fertilization on the quality of was found in the treatment with BF Amin, and the
maiden trees has been described in a separate largest, despite the difference not being significant,
publication [9, 10]. When it comes to the number of where Humus UP was used as fertilizer.
maidens produced from one hectare and the tendency of grafted buds to die during winter, it is better, as the
4. Discussion
results of our research show, to give up mineral In a nursery, the ability of apple and sour cherry
fertilization of rootstocks altogether than to exceed the buds to take and the number of maiden trees obtained
optimum doses of fertilizers, which, instead of from a given area are affected by many factors [6].
improving the results, clearly make them worse. The Important among them are such factors as the quality
number of grafted buds frozen in winter and the of the rootstocks planted in the nursery, especially
number of obtained “Topaz” and “Ariwa” maiden
Effect of Some Bioproducts on Winter Mortality of Grafted Buds and the
Number of Maiden Fruit Trees Produced in an Organic Nursery
apple trees grafted on rootstock M 26 are shown in sour cherry than in apple, and thus reduces the number Table 1.
of maiden trees produced;
Depending on the year, the cultivar, and the type of (2) Factors limiting the growth intensity of fertilization treatment, the results obtained differ quite
rootstocks in the second half of the growing season significantly from one another. Apart from (cessation of irrigation or fertilization) reduce the fertilization, the course of weather conditions during
susceptibility of grafted buds to freezing during the vegetation period also had an effect, as did other
winter;
factors. The number of young apple trees of the (3) Humic preparations (so-called vermiculites) that cultivar “Topaz” ranged from 62.5% to 91.3%, and of
do not intensify the vegetative growth of plants and the cultivar “Ariwa” from 66.1% to 94.4%, depending
are used as fertilizers in the nursery reduce the winter on the year and type of fertilization. For the sour
mortality of grafted buds to a greater extent in sour cherry cultivar “Debreceni Bötermö”, the cherry than in apple; corresponding figures ranged from 60.8% to 97.5%,
(4) The number of frozen buds, apart from affecting and for the cultivar “Sabina”— from 52.5% to 82.3%.
the yield per hectare, does not affect the quality of the The data quoted above show that an increase or loss
maidens produced;
of 10% to 30% in the number of apple maiden trees (5) The type of the biopreparations and soil produced depends to a large extent on our knowledge
improvers used in an organic nursery affects the and skills in dealing with the plants in the nursery, and
quality of maiden trees, including their thickness, in many cases also on chance factors (such as drought,
height and extent of branching. In the combinations severe snowless winter, etc.). In sour cherry, in which
with organic or mineral fertilization, the results the mortality of grafted buds in winter is generally
obtained are always better than in those without higher than in apple, the number of maidens produced
fertilization.
may be lower by a further 10%-20%, depending on
Acknowledgments
the year, compared with apple trees. By skillfully selecting products for use in fertilizing the organic
The work has been supported by a grant from the nursery, first of rootstocks, then of maiden trees, the
EU Regional Development Fund through the Polish winter mortality of grafted buds and the number of
Innovation Economy Operational Program, contract resulting maidens can be reduced for the former and
N0. UDA-POIG.01.03.01-10-109/08-00. significantly increased for the latter. An important
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Mar. 2013, Vol. 7, No. 3, pp. 289-292 Journal of Life Sciences, ISSN 1934-7391, USA
Comparative Evaluation of NPK Fertilizer and Tithonia diversifolia Biomass in Sweet Pepper (Capsicum annum) Production in Ado Ekiti, Nigeria
Ademiluyi Benson Oluwafemi Department of Plant Science, Ekiti State University, Ado Ekiti 36000, Nigeria
Received: October 23, 2012 / Accepted: December 15, 2012 / Published: March 30, 2013.
Abstract: The efficacy of the flower, leaf, stem and root of Tithonia diversifolia as organic fertilizer was comparatively studied with NPK fertilizer in improving the performance of sweet pepper (Capsicum annum) in the green house of the Department of Plant Science of Ekiti State University, Ado Ekiti, Nigeria. The different parts of Tithonia diversifolia were harvested, separately air dried and pulverized. 100 g each of the Tithonia parts and 10 g of NPK were respectively buried into each pot containing the pepper seedlings at two weeks after transplanting using the side dressing method. The study results showed that the leaf biomass of T. diversifolia was not significantly different from NPK fertilizer in improving the growth and yield of Capsicum annum. The stem and root biomass gave comparative growth and yield figures but higher than those recorded from the flower biomass. The control pots resulted to the lowest performance of capsicum annum. It is indicative in this study that Tithonia diversifolia plants ploughed into the soil at the tender stage before flowering will be useful in soil improvement for pepper production. It also revealed that the leave biomass contains nutrients in the required quantity that compared favourably with NPK for pepper production in the study area.
Key words: Capsicum annum, Tithonia diversifolia, NPK fertilizer.
1. Introduction pepper [5]. Also, the use of Tithonia diversifolia as organic fertilizer in soil improvement for various
Tithonia diversifolia is an annual weed growing crops yield has been reported [6, 7], but the exact parts aggressively along road paths and abandoned of the plant where the nutrients are concentrated for farmland and hedges all over Nigeria. It has been optimum plant yield has not been assessed in the study successfully used to improve soil fertility and crop area. The present study was therefore carried out to yield [1, 2]. The plant has attracted substantial comparatively evaluate the leave, flower, stem and research attentions because of the relatively high roots of Tithonia diversifolia and NPK fertilizer on nutrient concentration found in its biomass and its
pepper production.
ability to extract high amount of nutrients from the soil [3]. Tithonia diversifolia has been reported to
2. Materials and Methods
contain about 3.5% nitrogen, 0.82% phosphorus and The study was carried out at the experimental site 3.92% potassium with low contents of lignin 6.5% of the Department of Plant Science, Ekiti State and polyphenols 1.6% [2, 4]. University, Ado Ekiti, Nigeria (7°40 ′ N, 5°15′ E) Applications of organic and inorganic fertilizers between March 2009 and April 2010. Ado Ekiti has a have been reported to improve the growth of bimodal rainfall pattern with an annual mean of 1,400
Corresponding author: Ademiluyi Benson Oluwafemi, mm and a daily temperature of 27 °C Soil was Ph.D., research fields: plant physiology, weed science and
collected into horticultural pots with 15 L capacity and biological insect pests control. E-mail: femnikben@yahoo.com.
Comparative Evaluation of NPK Fertilizer and Tithonia diversifolia Biomass in Sweet Pepper (Capsicum annum) Production in Ado Ekiti, Nigeria
a depth of 28 cm. Green biomass of flower, leaf, stem those recorded in leaf and stem biomass of T. and root of Tithonia diversifolia were blended and
diversifolia in 2010. The shortest plants were incorporated into the soil at the rate of 200 g per pot
observed in the control experiment in both seasons. At (5 t/ha equivalent). These were applied a week before
8 WAT, the heights recorded in NPK and T. transplanting pepper seedlings. NPK fertilizer was
diversifolia leaf biomass were highest while those of added into another pot at the rate of 10 g per pot
control, flower buds and root biomass were lowest. (100 kg/ha equivalent) while the last pot was the
The root and stem biomass produced identical heights. control. There were six treatments in all which were
The highest number of green leaves of pepper replicated five times. Data determined were yield and
observed in the NPK fertilizer applied pots and yield attributing factors which include: fruit weight
Tithonia leave biomass applied pots were identical per plant, number of fruits per plant, plant height, and
and highest in both seasons of the trial. The least stem girth, number of green leaves at 8 WAT (weeks
number of green leaves was obtained in the control after transplanting) and number of days to flower.
experiment but not significantly different from those of Tithonia flower and root biomass. The Tithonia
3. Results
stem biomass produced significantly higher number of Table 1 shows the effects of NPK fertilizer and
green leaves than the flower and root biomass in both Tithonia diversifolia biomass on the height of pepper
seasons.
at 4 and 8 WAT and number of green leaves. The The effects of NPK fertilizer and T. diversifolia height recorded in the NPK applied soils and T.
biomass on the stem girth and days to flower are diversifolia leaf biomass applied soils were not
presented in Table 2. The stem girths recorded in the significantly different. The tallest pepper plants
NPK fertilizer and Tithonia leave biomass recorded in the NPK applied pots were identical to
were highest in both seasons at both 4 and 8 WAT. The
Table 1 Effect of NPK fertilizer and Tithonia diversifolia biomass on pepper height at 4 and 8 WAT and number of green leaves at 8 WAT.
Number of green leaves at 8 WAT Treatments 2009 2010 2009 2010 2009 2010
Plant height at 4 WAT (cm)
Plant height at 8 WAT (cm)
Control 15.0 c 15.4 c 19.2 c 19.1 d 14.5 c 13.9 c T. NPK
20.8 a 19.9 a 26.3 a 26.8 a 20.0 a 23.4 a T. Flower
16.0b c 15.8 c 20.5 bc 20.2 cd 14.4 c 14.2 c T. Leaf
18.9 ab 19.0 a 25.9 a 26.5 a 20.0 a 21.6 a T. Stem
17.0 b 18.4 a 22.2 b 23.3 b 18.5 ab 17.1 b T. Root
16.5 bc 17.9 b 20.6 bc 22.3 bc 15.8 bc 14.3 c Means with the same letter within column are not significantly different (P = 0.05) according to the Duncan multiple range test.
Table 2 Effect of NPK fertilizer and Tithonia diversifolia biomass on stem girth at 4 and 8 WAT and days to flowering.
Days to flower Treatments 2009 2010 2009 2010 2009 2010
Stem girth at 4 WAT (mm)
Stem girth at 8 WAT (mm)
Control 9 d 10 c 12 c 14 c 102 a 100 a T. NPK
19 a 18 a 31 a 29 a 85 c 83 c T. Flower
10 d 12 c 15 c 15 c 94 b 95 b T. Leaf
21 a 17 a 29 a 29 a 82 c 80 c T. Stem
16 b 14 b 21 b 22 b 83 c 84 c T. Root
13 c 12 c 14 c 14 bc 99 ab 101 a Means with the same letter within column are not significantly different (P = 0.05) according to the Duncan multiple range test.
Comparative Evaluation of NPK Fertilizer and Tithonia diversifolia Biomass in
Sweet Pepper (Capsicum annum) Production in Ado Ekiti, Nigeria Table 3 Effect of NPK fertilizer and Tithonia diversifolia biomass on number and weight (g) of fruits per plant.
Fruit weight per plant (g) Treatments 2009 2010 2009 2010
Number of fruits per plant
Control 5.1 c 4.7 c 193.4 d 204.5 c T. NPK
10.1 a 11.6 a 450.8 a 506.4 a T. Flower
5.4 c 4.6 c 199.1 cd 217.8 c T. Leaf
10.3 a 11.2 a 456.3 a 517.5 a T. Stem
8.4 b 9.0 b 365.2 b 410.6 b T. Root
5.8 c 5.1 c 208.5 c 214.6 c Means with the same letter within column are not significantly different (P = 0.05) according to the Duncan multiple range test.
control, flower and the root biomass produced the of NPK fertilizer application. The fact that neither the lowest stem girth of pepper. The leaf biomass, stem
root nor the flower biomass contributed significantly biomass and NPK fertilizer applied pots produced
to pepper performance when compared with the pepper plants that flowered first while the control and
control suggests that the nutrients needed for such root biomass flowered last (Table 3).
growth increase were not present in adequate quantity The highest number of fruits per pepper plant was
in either the flowers or the roots. It is point clear in the recorded in the Tithonia leaf biomass and the NPK
present study that nutrient composition of Tithonia fertilizer applied pots while the least was obtained in
diversifolia was highly concentrated in the leaves and the control, Tithonia flower and root biomass. The
tender stems of the plant. This is evident in the stem biomass was significantly higher than either the
comparatively higher yields obtained from their flower or the root biomass in terms of number of fruits
biomass. It had been observed that the N per plant. The highest fruit weight per plant was
concentration in Tithonia diversifolia leaves is higher recorded in pepper plants receiving Tithonia leaf
than the critical level of 2.0% to 2.5% below which no biomass but this was not significantly different from
immobilization of N would be expected [10]. the NPK fertilizer applied pots. The stem biomass
5. Conclusion
however produced higher fruit weight than either the Tithonia flower or root biomass. The control
The present study therefore suggests that nutrient experiment has the lowest fruit weight which was not
compositions were concentrated in the leaves and significantly different from those of root and flower
young stems of Tithonia diversifolia. It is suggested biomass in 2010 (Table 3).
that Tithonia diversifolia can be ploughed into the soil during the vegetative growth stage before flower
4. Discussion
production for optimum crop performance. The results from this study revealed that leave