Journal of Life Sciences Volume 5 Number (2)
J LS
Journal of Life Sciences
Volume 5, Number 2, February 2011 (Serial Number 34)
Contents
Research Papers
81 Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
Chiara Nobili, Massimo Reverberi, Alessandra Ricelli, Valeria Scala, Gabriella Aureli, Flavia Pinzari, Anna Adele Fabbri and Corrado Fanelli
91 Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. (mint) from Kenya Based on AFLP Markers
Lexa G. Matasyoh, Francis N. Wachira, Miriam G. Kinyua and Anne W. Thairu Muigai 100
Study on Flavonoids and Triterpenoids Content of some Euphorbiaceae Plants
Laila Abdel-Ghany Refahy 108
Agrobiodiversity for Livelihood Security: A Case Study of Agroforestry Technologies in Mexico
P. Krishna Krishnamurthy and Krishnamurthy Laksmi Reddiar
120 Production of High Quality Functional Labneh Cheese Using Exopolysaccharides Producing
Encapsulated Lactobacillus Bulgaricus
Kawther El-Shafei, Fayza M. Assem, Azzat B. Abd-El-Khalek and Osama M. Sharaf 129
Identification of Pathogenic Germs and Antibiotics Residues in the Raw Milk and Their Effects on Human Health
Tadjine Nacera, Tassist Amina, Bradea Maria-Stela, Tarzali Dalila and Guetarni Djamel
132 Impact of Selenium on Microtubules Polymarisation of Spermatozoa of Endosulfan Exposed
Swiss Albino Mice
Ranjit Kumar, Jitendra Kumar Singh, Mohammad Ali and Arun Kumar
136 Prevalence of Muscardine Disease in Different Silkworm Hybrid, Bombyx Mori L. under Agro-climatic Conditions in Aurangabad (M.S.), India
Bhalerao Rajendra S., Hiware Chandrashekhar J. and Avhad Sunil B.
Methods and Techniques
The Use of HPLC in Determination of Endogenous Hormones in Anthers of Bitter Melon
Yi Tang, Li Wang, Chao Ma, Ji Liu, Bin Liu and Huanxiu Li
143 Potential of the Integrated Control of Cucumber Root Rot Using Natural, Biological and Chemical Methods
Mohamed El Khaleely Barakat, Abdel Radi Taher Bakeer and Wallaa Fathy Mostafa 157
Ethanol Production by Enzymatic Hydrolysis of Elephant Grass
Isaías Barbosa Soares, Olga Martins Marques, Mohand Benachour and César Augusto Moraes de Abreu
Reviews
Termination of DNA Replication and the Role of Enzymes in Recombination
Naila Rozi and Nasir Uddinkhan
Journal of Life Sciences 5 (2011) 81-90
Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
1 2 3 4 4 Chiara Nobili 5 , Massimo Reverberi , Alessandra Ricelli , Valeria Scala , Gabriella Aureli , Flavia Pinzari , Anna
Adele Fabbri 2 and Corrado Fanelli 2
1. ENEA, Via Anguillarese 301, Roma 00123, Italy 2. Università degli Studi “Sapienza”, Largo Cristina di Svezia 24, Roma 00165, Italy 3. ICB-CNR, P.le Aldo Moro 5, Roma 00185, Italy 4. QCE-CRA, Via Cassia 176, Roma 00100, Italy 5. Istituto Centrale per il Restauro e la Conservazione del Patrimonio Librario, Via Milano 76, Roma 00184, Italy
Received: July 09, 2010 / Accepted: September 25, 2010 / Published: February 28, 2011.
Abstract: The aim of this work was to verify if the wheat susceptibility/tolerance phenotype to Fusarium head blight (FHB) into the field could be related to the ability of the re-activated seeds to hamper deoxynivalenol (DON) synthesis by Fusarium graminearum into non optimal (i.e. high humidity) storage conditions. On this purpose, two Fusarium graminearum strains, Fg126 and Fg8308, were separately inoculated on active but not germinating kernels of Triticum aestivum L. cv “Sagittario” (FHB-susceptible) and cv “Blasco” (FHB-tolerant). The growth of the fungal strains on wheat kernels was monitored from 0 to 15 days post inoculation through a quantitative SYBR green real-time PCR, as well as ergosterol content through HPLC for comparison. DON biosynthesis into the contaminated wheat seeds was quantified by HPLC method in the same time intervals as above. Significant differences in the ability to grow and synthesize DON, acetylated and glucosylated forms, emerged between the strains also in relation to the wheat variety on which the pathogen developed. The results obtained indicated that the wheat variety which from the epidemiological studies carried out into the field is FHB-tolerant, i.e. Blasco, resulted as the more efficient in hampering DON synthesis in both F. graminearum strains. Thus, this preliminary study could represent a contribution to find out less time consuming methods for screening the wheat varieties tolerant to DON accumulation in non optimal storage conditions.
Key words: Triticum aestivum, Fusarium graminearum, FHB-susceptible, FHB-tolerant, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), 3-glucosyldeoxynivalenol (3-GDON).
1. Introduction
In particular, Fusarium graminearum can cause the Fusarium head blight (FHB) of wheat [1]. This disease
Fungi can grow on many food commodities. Some leads to a severe reduction of grain yield and quality. fungal genera, such as Fusarium include several The FHB-affected wheat grains are small, light, species which can produce, under suitable conditions, shrivelled and sometimes covered with a blank or pink secondary metabolites, i.e. mycotoxins, which are down. It has been assessed that one of the main dangerous for the health of humans and animals. As aggressiveness factor involved in FHB disease is F. matter of fact, mycotoxigenic fungi are a real issue, graminearum toxigenic strains production of especially for cereal industry. deoxynivalenol (DON) [2, 3]. If this mycotoxin is
present in wheat grains, the edible part of plant, it could Corresponding author: Chiara Nobili, Ph.D., research field:
food diagnostic. E-mail: chiara.nobili@enea.it. have toxic effects also on human and animal health. In
82 Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
particular it is known that DON intoxication can affect favourable to the development of Fusarium strains as growth (anorexia and decreased nutritional efficiency),
well as to early detect the most aggressive F. can cause emetic effects and haemorrhagic diathesis,
graminearum strains for checking their development in bone narrow aplasia and a general affection of the
both field and storage conditions. In relation to this, immune function (enhancement or suppression), and of
there is a real need to develop in seed bio-assays able to reproduction (reduced litter size) [4, 5]. The toxicity
rapidly individuate wheat varieties tolerant to DON in and the high diffusion of this contaminant has induced
order to reduce the health risks related to toxin the European Union to establish a maximum tolerable
contamination [10, 11]. Moreover a reliable analytical level for its presence in different food commodities
method in order to early identify DON producing (Commission Regulation No. 1881/2006).
isolates would be useful since the identification of In wheat plants, DON delays the germination and
fungi solely based on morphological features might be growth of wheat plants and inhibits the growth of the
difficult, time-consuming and usually requires grain and the coleoptiles tissues. DON is probably able
taxonomic expertise.
to circulate in the phloem, with a concentration
F. graminearum become evident only when the following a descending gradient from the rachis,
grains are heavily-contaminated. Recent advances in through lemmas and grains to the peduncle [6]. Briefly,
DNA-based techniques confer to real-time PCR assays DON presence can inhibit the expression of some
an important role among molecular screening methods because of the accelerated diagnostic outcome, and of
proteins involved in plant defence mechanisms and it is the ability to perform an early detection of the
also phytotoxic, causing chlorosis, necrosis and considered fungi, so that, this method is providing new
apoptosis in planta [7]. FHB is influenced by different tools for fungal detection and quantification in kernel,
aspects, some are related to the environmental in other plant tissues and complex matrix [12-15].
conditions like temperature and humidity, to the attack The aims of this study were the set up of an in seed by pests and microorganisms and to the competition post harvest assay capable of partly reproducing the in dynamics between Fusaria and the other colonizers planta in field screening of wheat varieties, i.e. fungi [8]. individuating a DON-tolerant seeds profile in However intrinsic factors like plant variety and FHB-tolerant plants, an analytical method to quantify F. isolate characteristics are very important to predict the graminearum strains through a SYBR green Real-time probability of the onset of disease at its extent. For PCR assay [16-19], and the evaluation of the instance it is already known that some wheat varieties relationship between fungal DNA concentrations are more susceptible to FHB and that some F. growth and DON production by the mean of the graminearum isolates are particularly able to cause this Principal Components Analysis (PCA). disease. Moreover, the conidia of Fusarium can be
present also onto the kernels and transported into seeds
2. Materials and Methods
storage. In non optimal storage conditions, i.e. a water
2.1 Fungal Strains
activity of the seeds higher than 0.9, the conidia can germinate and secondary infections cycles occur. In
DON-producing Fusarium graminearum strains, these new infection cycles, the pathogen is able to
Gibberella zeae perfect stage, Fg8308 and Fg126, were produce other DON, heightening the toxin content into
supplied from the collection of CNR-ISPA Bari-Italy. the seeds [9]. It is then important to know which are the
2.2 Wheat Varieties
FHB- and DON-susceptible plant varieties in order to limit their diffusion into environments which are
Two soft wheat Triticum aestivum varieties, the
Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant
Varieties of Triticum Aestivum
FHB-tolerant (T) “Blasco” and the FHB-susceptible (S)
Table 1 Wheat samples.
“Sagittario” were supplied by CRA-QCE Roma-Italy.
Sample Description
Time of analysis 1 Non inoculated tolerant (T) variety /
2.3 Inoculation of Two Varieties of Soft Wheat with F. Non inoculated susceptible (S) 2 variety
/ Graminearum 3 T variety inoculated with Fg 126
Each wheat samples (20 g) was superficially 4 T variety inoculated with Fg 8308 Zero
5 sterilized (2% chlorex solution), rinsed threefold by S variety inoculated with Fg 126 6 S variety inoculated with Fg 8308
sterilized distilled water, moistened up to 0.94 a W and 7 T variety inoculated with Fg 126 then inoculated with 100 μl of F. graminearum
8 T variety inoculated with Fg 8308
1 (Fg126 and Fg8308) conidia suspension (10 /ml). The
9 S variety inoculated with Fg 126
10 moistening did not allow an early germination of the S variety inoculated with Fg 8308 11 T variety inoculated with Fg 126
seeds, even if moistened un-inoculated seeds are able 12 T variety inoculated with Fg 8308
to germinate after 15 days of incubation in the same 3 13 S variety inoculated with Fg 126
conditions as below. As it is shown in Table 1, the 14 S variety inoculated with Fg 8308
15 wheat inoculated with the pathogen was analysed after T variety inoculated with Fg 126 16 T variety inoculated with Fg 8308
0, 1, 3, 7, 10 and 15 d.p.i. at 25 ℃. 7 17 S variety inoculated with Fg 126
2.4 DNA Extraction from Wheat Grains 18 S variety inoculated with Fg 8308 19 T variety inoculated with Fg 126
DNA of contaminated wheat seeds has been 20 T variety inoculated with Fg 8308 10
21 extracted according to Färber method with slight S variety inoculated with Fg 126 22 S variety inoculated with Fg 8308
modifications [20]. An amount (100 mg) of fresh 23 T variety inoculated with Fg 126 wheat grains were grounded in liquid nitrogen and
24 T variety inoculated with Fg 8308 15 incubated with 500 µl of extraction buffer (Tris-HCl
25 S variety inoculated with Fg 126 200 mM pH8.0, NaCl 250 mM, EDTA 25 mM, SDS
26 S variety inoculated with Fg 8308
0.5%) for 60 min at 65 ℃ in an orbital shaker. After rpm for 30 min at 4 ℃ the pellet was washed with incubation, samples were put on ice for 10 min and
70% cold ethanol, centrifuged at 13,000 rpm for 10 centrifuged at 12,000 rpm for 15 min at 4 ℃. The
min, dried at 64 ℃ and resuspended in 30 µl of sterile supernatant was collected in a 2 ml tube and 3/10
water containing 20 mg/ml RNAse. volume of sodium acetate 4 M was added. This
2.5 Optimization of End-Point PCR Conditions solution was placed on ice for 30 min and centrifuged
at 12,000 rpm for 10 min at 4 ℃ and the supernatant Before starting analyses on real samples, F. was transferred, extracted with hand shaking graminearum strains were tested inoculating cultures
phenol-chloroform-isoamylic alcohol (25:24:1 v/v/v) of Potato Dextrose Broth in the dark at 25 ℃ without and centrifuged at 12,000 rpm for 10 min at 4 ℃. The
shaking. For each strain, the mycelium was harvested, supernatant was transferred again and added with
washed with sterile water, lyophilized, and stored at chloroform (1:1 v/v), was shaken for 5 min, centrifuge
-20 ℃. In order to ascertain if the F. graminearum for 15 min at 4 ℃ and precipitated by adding 0.5
strains provided, Fg126 and Fg8308 strains were high- volume of cold 2-propanol. One more precipitation
or low-DON producers, a method designed by Bakan
[21] was followed. Extracted DNA was analysed by CH 3 COONa (3 M pH5.2) and 440 µl of ethanol. After
step was done adding 200 µl of H 2 O, 20 µl of
end-point PCR using N1-2 (for_CTT GTT AGG CTA
20 min incubation at -20 ℃ and centrifuge at 12,000 AGC GTT TT; rev_AAC CCC TTT CCT ATG TGT
84 Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
TA) and 4056-3551 primers (for_ATC CCT CAA afterwards as a reference standard for the extrapolation AAA CTG CCG CT; rev_ACT TTC CCA CCG AGT
of quantitative information for DNA targets of ATT TC). Both primers were designed by Konietzny
unknown concentrations. The efficiency of the PCR and Greiner [22], in the trichothecene gene cluster
reaction (Fg126 = 94% and Fg8308 = 83%) was intergenic region Tri5-6. According to this method, the
obtained from calibration curves slope (E = 10 -1/slope − N1-2 primer pair designed into the tri5-tri6 sequences
1). Real-time PCR amplification reactions were carried yielded a 200-bp fragment for high-producing strains,
out in triplicate from two independent experiments and whereas no amplification should be observed for
the results were expressed as ng(DNA)/g(wheat grain). low-producing strains. Conversely, with the 4056-3551
2.7 Ergosterol Extraction and Determination primers pair, the PCR amplification yielded a 650-bp
fragment for low-producing strains, whereas no In order to extract the whole of ergosterol forms amplification should be observed for high-producing
present in wheat, samples were treated as described by strains. End-point PCR was prepared in 25 μl reaction
Gessner [23]. Ergosterol analysis was carried out, on mixture which contains buffer 1X (SIGMA), MgCl 2 3 the whole set of samples, by high-pressure liquid
mM, dNTPs 0.4 mM, Taq 1 U/ μl and 0.4 μM of each chromatography (HPLC Agilent 1200 series) coupled primer. PCR was performed with the following cycling
to a DAD analyser (Agilent) and equipped with a conditions: 94 ℃ for 2 min, followed by 35 cycles of
Symmetry C18 column (150 mm × 4.6 mm, 5 μm)
94 ℃ for 1 min, 55 ℃ for 30 s and 72 ℃ for 40 s. preceded by a pre-column of the same material Thermal cycle ended with an annealing step for 8 min
(Waters). Quantitative determination was performed at 72 ℃. PCR products were run on a 1% agarose gel
by analysing the signal emitted at 280 nm. Methanol and analyzed.
100% at 0.6 ml·min -1 was used for isocratic elution and a volume of 20 μl sample was injected. Stock
2.6 Real-Time PCR Operative Conditions (Optimized solutions were prepared by dissolving the solid for SYBR Green) commercial molecule (SIGMA) in methanol 100% at
In order to monitor fungal growth onto the wheat concentration of 2.5 ng· -1 μl . This solution was used to seeds, a specific SYBR green qPCR method was set by
made a standard curve injecting 25, 50, 75, 100, 125 using N1-2 primers, mentioned above. Real-time PCR
ng of ergosterol. The results were expressed as was prepared in 20 μl reaction mixture which contains
nanograms of ergosterol per gram of dry matter. SYBR green JumpStart Taq Ready Mix 1X (SIGMA),
2.8 DON Extraction and Analysis MgCl 2 3mM and 0.5 μM N1-2 primes. Real-time PCR was performed in a LineGene K PCR detection systems
An amount (10 g) of fresh wheat grains were (Bioer, Japan) with the following cycling conditions:
grounded in liquid nitrogen and extracted with 30 ml of
H 2 O: acetonitrile (84: 16) mixture by shaking for 1 min. s, 55 ℃ for 30 s and 72 ℃ for 1 min. Standard
95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15
After 7 min the extract was agitated by vortexing for 1 calibration was performed plotting the real-time PCR
min twice and then filtered through filter paper signals obtained for F. graminearum genomic DNA
(Whatman 4). One more extraction step was performed extracted from both fungal strains in the concentration
washing samples with 10 ml of extracting mixture and range 5 ng - 1 pg (data not shown). The equation
samples were evaporated to dryness under a stream of describing the increase of DNA concentration were
air. Prior to further analyses clean up was made with calculated (Fg126: y =
“MycoSep® 227” columns (Biopure). A HPLC Fg8308: y =
−3.47x + 29.5, R 2 = 0.9972;
−3.79x + 33.7, R 2 = 0.9991) and used instrument (Agilent 1,200 series) coupled to a DAD
Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant
Varieties of Triticum Aestivum
analyzer (Agilent) was employed for the detection. performed using XLSTAT Addinsoft software [25]. Two different methods were used to detect DON, its
3. Results and Discussion
glucosylated (3-GDON) and acetylated (3- and 15-ADON) chemotypes. For DON and 3-GDON a
3.1 F. Graminearum Identification volume of 25 µl sample was separated on an Symmetry
As shown in Fig. 1, a discrete band is obtained only C18 column (150mm × 4.6mm, 5 μm) preceded by a with the amplification with N1-2 primers whereas the pre-column of the same material (Waters). Acetonitrile: amplification with 4056-3551 primers results in an water (10:90) mixture was used for isocratic elution.
aspecific pattern, indicating that both F. graminearum The 0.6 ml·min mobile phase flow rate and 25 ℃ strains (Fg126 and Fg8308) are high-DON producers. column temperature were found as optimal. For the
acetylated forms a volume of 25 µl sample was
3.2 Detection of F. Graminearum in Contaminated separated on a Zorbax-C18, 150 × 4.6 mm, 5 µm
Wheat: A Double Approach through Biochemical and column. Acetonitrile in water (10:90) (mobile phase A)
Molecular Markers
was used for isocratic elution with 14% of acetonitrile The wheat varieties were chosen from an in-field (mobile phase B). The 0.6 ml·min -1 mobile phase flow pre-screening. The epidemiological results indicated rate and 25 ℃ column temperature were found as
that “Sagittario” is a FHB-susceptibile whereas optimal. For all the injected molecules, 10 ng· μl stock “Blasco” is a FHB-tolerant variety [26, 27]. Thus, in solutions were prepared by diluting each standard this study the authors intend to propose a preliminary (BIOPURE) in mobile phase (A). These solutions was approach for screening the seeds of wheat varieties used to made a standard curves. The results were which are resistant, i.e. able to actively hamper, to expressed as ng of DON (or derivatives) per gram of
DON accumulation.
dry matter or as parts per billion. The two fungal strains showed a similar trend of
2.9 Statistics growth on “Blasco” and “Sagittario”, i.e., exponential increase in ergosterol and DNA amount during the
All the experiments were carried out in three time course. In particular, Fg126 achieved the highest replicates. The values presented in the following amount of ergosterol concentration in both wheat figures are the mean ± S.E. of 6 different results. The varieties already after 7 days of incubation (Fig. 2). mean values were compared by using the
Mann-Whitney test; P values above 0.05 were considered not significant. Principal Components Analysis (PCA) [24] was performed on the whole set of data (DON and DNA concentrations of the two fungal strains over different incubation times). PCA is a projection method for the extraction of systematic variations in a dataset. Analysis of variance (ANOVA) was applied in the comparison of the treatments, and significance of differences were tested at 95% confidence by Fisher’s LSD test which is a least
significant difference (LSD) method consisting in a Fig. 1 Agarose 1% gel electrophoresis. M: 1 kb marker; strain Fg126: amplified respectively with
two-step testing procedure for pair wise com- N1-2 and tri 5-6 primer pairs; strain Fg8308: amplified parisons of several treatment groups. Calculations were
respectively with N1-2 and tri 5-6 primer pairs.
86 Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
In Fig. 3, DNA quantification of Fg126 and Fg8308 order to avoid an auto-intoxication by the DON itself partially overlap with the results obtained with
[33]. In relation to this, Fg126 strain only seems able to ergosterol quantification, even if the DNA assay lacks
activate this auto-defence mechanism. in sensitiveness (i.e. no significant increase between
Plants either can detoxify DON by converting a part
10 and 15 d.p.i.) in the the late times of growth. The of the toxin produced by Fusarium in the less toxic use of primers N1-2 could be fundamental for the set
3-GDON [34]. It was found that also the re-activated up of a rapid screening test into the field. In fact, these
stored wheats seeds are able to convert part of the DON primers are quantitative and qualitative molecular
into 3-GDON. The concentration of this less toxic markers able to discriminate the presence of minimal
masked mycotoxin corresponds to 5 up to 10 percent of amount of fungal propagula of high-DON producer
the total DON produced by F. graminearum onto strains of F. graminearum directly onto viable kernels.
wheat seeds (Fig. 6).
Fungal growth of the Fusarium strains on the different
Triticum varieties was compared and analysed by
n rai
ANOVA for evaluating the significance of the
-1 (g
differences. The test confirmed that Fg126 grown
)g g
more than Fg8308 on both the wheat varieties (Fisher
(er ng
test, P < 0.05). The results also showed an almost precise overlap, at least up to 7 d.p.i., with the quantification of fungal growth obtained by ergosterol quantification.
Fig. 2 Ergosterol quantification through HPLC on “blasco” (T) variety and “sagittario” (S) variety.
3.3 Occurrence of DON and Its Derivatives in
Inoculated Wheat
It has been demonstrated that, within the same
rain) -1 (g
species and in the same culture conditions, toxin
)g
production by Fusarium strain may vary sharply; some strains produce large amounts of trichothecenes,
(DNA g n
whereas others produce small or undetectable amounts [28-32]. Most important differences in DON biosynthesis are evident after 10 days of incubation
onto wheat seeds. An higher DON amount is produced Fig. 3 DNA quantification (qPCR of N1-2 fragment) on
by Fg126 (~ 60 μg·g ) in comparison with Fg8308 strain (~ 20 -1 μg·g ) after 15 days of incubation onto the
“blasco” (T) variety and “sagittario” (S) variety.
susceptible variety (Fig. 4).
Indeed, both F. graminearum strains synthesise an
ng
higher amount of DON onto “Sagittario” than in
“Blasco” (~ 5 -1 μg·g for Fg8308, ~ 8 μg·g for Fg126). Moreover, concerning the DON acetylated forms, only
Fg8308 strain produces 15-ADON (Fig. 5a), whereas 3-ADON is produced only by Fg126 (Fig. 5b).
3-ADON is usually produced by Fusarium strains in
Fig. 4 DON quantification through HPLC method.
Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant
Varieties of Triticum Aestivum
first two principal components F1 and F2. In the plot -1 showing the variables (Fig. 8) a weak correlation
g between the variables DON and DNA at time 15 is ng
shown, whereas DON values at 0 to 7 d.p.i. are correlated with DNA values at time 0. These results are relevant to the whole set of samples. A higher degree of correlation between DON and DNA data
a could be obtained considering separately the different
fungal strains with single wheat variety (data not shown).
g The PCA plot obtained with the components F1 and ng
F2 accounted for the 61.81% of the variability and the observations were grouped according to both the fungal strains and the different wheat varieties (Fig. 9). On the whole, the PCA analysis showed a different growth and different degree of DON production of
b fungal strains on respect to the wheat variety infected.
Fig. 5 Quantification through HPLC method of 15-ADON (a)
In fact, a clear cut separation was observed on the basis
and 3-ADON (b).
3.4 F. Graminearum Growth Related to Deoxynivalenol (DON) Levels
To evaluate if a positive correlation between fungal
growth and DON synthesis may exist, the concentration of Fusarium DNA (fungal growth) was compared to the concentration of DON in the wheat samples at different incubation times. In PCA analysis, quite low degrees of correlation between DNA and
Fig. 6 Quantification through HPLC method of 3-GDON.
DON concentrations were observed. This is due to the significant differences emerged in the ability to grow and synthesize DON between the F. graminearum
strains in relation with the different varieties of wheat
e (%)
on which the pathogens developed. A constant lativ
ility increase of fungal DNA concentration in seeds with mu
g envalue
Cu ariab incubation time, did correspond for an equivalent
Ei
v increase of DON synthesis in all the combinations of
fungal strain/wheat variety The Scree plot of the PCA (Fig. 7) shows that the first four eigenvalues (F1-4) correspond to the whole
percentage of the variance in the dataset.
Fig. 7 Scree plot of the PCA. The first three Eigenvalues
Plots of the observations and variables in the factor
(F1-3) correspond to a high % of the variance, and will be
space were reported in Figs. 8 and 9 according to the
used to build the biplots.
88 Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant Varieties of Triticum Aestivum
XLSTAT2010.2.03 – Principal Component Analysis (PCA) Biplot (axes F1 and F2: 75.76%)
Fig. 8 Plot of the observations and variables in the factor space according to the first two Principal components F1 and F2.
XLSTAT2010.2.03 – Principal Component Analysis (PCA) Biplot (axes F1 and F3: 60.73%)
Fig. 9 Plot of the observations and variables in the factor space according to the first and third Principal components F1 and F3.
of the fungal strains, which were sharply separated in Thus, differences in DON concentration seem the PCA plots (Fg126 all on the left, Fg8308 all on the
more related to fungal strain and wheat seeds variety right). A separation was observed in the plots also
typical features than to the pathogen development between the wheat varieties (S and T).
per se .
Evaluation of Fusarium Graminearum Growth and Deoxynivalenol Content in Susceptible and Tolerant
Varieties of Triticum Aestivum
4. Conclusions
are grateful to Dr. M. Pasquini of CRA-QCE for having provided the wheat kernels of the two varieties,
Since fungal diseases which lead to mycotoxin “Blasco” and “Sagittario” and CNR-ISPA for having accumulation into feed- and foodstuff are widely
provided the fungal strains.
diffused it is of paramount importance to establish preventive strategies aimed to restrain their diffusion.
References
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Journal of Life Sciences 5 (2011) 91-99
Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. (mint) from Kenya Based on AFLP Markers
1 2 3 Lexa G. Matasyoh 1 , Francis N. Wachira , Miriam G. Kinyua and Anne W. Thairu Muigai 1. Department of Botany, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya
2. Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536, Egerton, Kenya 3. Moi University, Biotechnology Department, P.O. Box 1125, Eldoret, Kenya
Received: April 12, 2010 / Accepted: June 09, 2010 / Published: February 28, 2011.
Abstract: Ocimum gratissimum L. native to Africa is a shrubby essential oil containing plant with medicinal, antimicrobial and antihelminthic properties. It is an important herbal medicine not only among Kenyan communities but also in the sub-Saharan Africa. No systematic assessment of genetic variability in O. gratissimum of Kenya has been carried out. In this study, amplified fragment length polymorphism (AFLP) analysis was used to estimate genetic diversity and genetic differentiation in 139 samples from all the different provinces of Kenya. Seven primer pairs, the Eco+ACT plus Mse+CAA, Eco+ACT plus Mse+CTG, Eco+ACA plus Mse+CAA, Eco+ACA plus Mse+CTG, Eco+AGC plus Mse+CAA, Eco+ACC plus Mse+CAA, and Eco+ACC plus Mse+CAC were the best combinations and generated polymorphic 655 bands with fragment ranging in size from 50 - 473 bp in size. Populations from central Kenya had the highest levels gene diversity. Most of the variability was partitioned into within populations 71%; P < 0.001 implying that collection strategies for conservation should focus on a few populations with many individuals across the ecological amplitude of the population. Genetic differentiation was GST = 0.286, an indication of genetic variation among the populations.
Key words: Ocimum gratissimum L., AFLP, genetic diversity.
1. Introduction forest cover of 1.64 million hectares, only 1.22 million hectares is left [2]. As most rural people in Kenya turn
Access to conventional health services in most of to usage of traditional medicinal plants that include Africa is beyond the reach of most of the rural important weeds, it is important to consider their population. Traditional medicine provides an
genetic conservation.
alternative health care system in most of Africa. Ocimum gratissimum L. is among the seven weed Domestication of important medicinal plants is species identified as important medicinal herbs that increasingly serving as an avenue for increasing merit conservation in central Kenya [3]. The species household incomes as well as enhancing the belongs to the Lamiaceae family, which has close to availability of the curative plant products to healers and 252 genera and 6,700 species [4], most of which are other resource users [1]. used as medicine [5]. The essential oils found in leaves, In Kenya, 2.9 million people live within 5 km of seeds, flowers and roots of Ocimum species are used as forest areas and thereby exert high pressure on forests
medicine.
such that out of the original closed canopy indigenous Under in vitro conditions, the oils have been
Corresponding author: Lexa G. Matasyoh, Ph.D., research demonstrated to exhibit antibacterial activity [6]. The fields: botany, plant molecular biology. E-mail: lexa111@
oils also exhibit activity against such pathogenic fungus hotmail.com.
92 Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. (mint) from Kenya Based on AFLP Markers
as Candida albicans [6, 7]. distance of about five meters. The collected leaf Because of the biodiversity of its essential oils, O.
samples were wiped dry with cotton wool and then gratissimum has a high potential as a traditional
stored in the freezer. This method of preservation medicine [6, 7], and its incorporation into agricultural
allowed several months of storage without affecting the systems would not only make the species accessible to
yield and quality of the extracted DNA [15]. the majority of the rural population that uses it, but also
2.2 Genomic DNA Isolation
contribute to its genetic conservation. There is information based on volatile oils, flavanoids and
Genomic DNA was isolated using the modified SDS RAPD markers of O. gratissimum [8].
method with the reducing agent dithiothreitol as Biochemical and molecular techniques also provide
described [16].
an alternative approach for evaluating genetic diversity Amplified fragment length polymorphism analysis in crop plants. The application of DNA technology in
(AFLP-PCR) method was carried out following the agricultural research has progressed rapidly over the
standard procedure as described [17] and adapted in the last twenty years, especially in the area of cultivar
AFLP ® Plant Mapping protocol of the Applied identification and characterization [9] as well as
Biosystems (ABI), USA.
determination of population diversity in many plant
2.2.1 Template Preparation and Adaptor Ligation species [10-14].
Before carrying out the AFLP analysis, the In this paper, the level and structure of genetic
suitability of the restriction enzymes chosen to cut the diversity in wild populations of O. gratissimum was
O. gratissimum genomic DNA was firstly tested. This evaluated using AFLP markers.
was carried out by digesting the genomic DNA with MseI (frequent-4-base cutter-TAA) and EcoRI
2. Materials and Methods
(rare-6-base cutter-AATTC) restriction enzymes
2.1 Leaf Sample Collection separately and then in combination. 20 µl of the genomic DNA was incubated for 2 hrs at 37 ℃ with 2
Fresh leaf samples of O. gratissimum L. were µl of EcoRI/MseI (1.25 U/µl each in 10 mM Tris-HCl collected from 13 populations distributed throughout (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, Kenya. Sampling was carried out in such a way as to
0.1 mg/ml bovine serum albumen (BSA), 50% glycerol sample different silvicultural zones across the (v/v), 0.1% Triton ® X-100), and 5 µl of 5× reaction ecological amplitude of the species. 139 samples were buffer (50 mM Tris-HCl (pH 7.5), 50 mM Mg-acetate, collected from the 13 populations (Table 3) in Western 250 mM K-acetate). 5 µl of each of the digested Province, Rift Valley Province, Central Province, products sample in 5 µl of 1× blue dye was run on 1.5% Nyanza Province and Coast Province. agarose gel in 1× TBE to check for complete digestion Collection sites were largely chosen in the natural with DNA size markers (100 bp ladder). habitats of the species though a few collections were
A restriction-ligation enzyme master mix was also made from cultivated fields. Collection was done prepared by combining 1 µl of T 4 DNA ligase (1 U/µl only on plants that had not flowered. For each in 10 mM Tris-HCl (pH 7.5), 1 mM DTT, 50 mM KCl, population, a voucher specimen (805LG) was 50% (v/v) glycerol) with 24 µl adapter/ligation solution deposited at the Department of Botany Herbarium, (EcoRI/MseI adapters, 0.4 mM ATP, 10 mM Tris-HCl Egerton University, Kenya. A population comprised of (pH 7.5), 10 Mm Mg-acetate, 50 mM K-acetate). The clustered plants that were delimited from another adapter pairs were first annealed to make them double cluster. Within each population sampling was carried stranded by heating the tubes in a water bath at 95 ℃ out from individuals that were separated by an average
Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. (mint) from Kenya Based on AFLP
Markers
for five minutes. The tubes were then cooled to room Seven of the best primers (*) showing high temperature over a 10-minute period. This reaction
polymorphism were then used in full analysis of the mixture was then incubated at room temperature
test germplasm. The selective PCR was performed in overnight. The restriction-ligation products were then
20 µl volumes consisting of 3 µl of the diluted diluted by adding 18.9 µl of 1× TE to 10 µl of the
pre-selective amplification products, 1 µl reaction mixture in a 1.5 ml micro-centrifuge tube to
florescent-labeled EcoRI primer, and 1 µl of MseI give the appropriate concentration for subsequent PCR.
primer and 15 µl of AFLP core mix. The selective PCR The diluted products were stored at 4 ℃ for use within
amplification was programmed for an initial two one month or – 20 ℃ for longer period.
minutes at 94 ℃ followed by one cycle of 94 ℃ for 20
2.2.2 Pre-selective Amplification seconds, 66 ℃ for 30 seconds and 72 ℃ for two PCR amplification of the adapter-ligation restriction
minutes. This cycle was repeated 12 times with a fragments was performed for subsequent selective
lowering of the annealing temperature of 1 ℃ per cycle. amplification. The PCR reactions were performed in a
This was followed by 20 cycles of 94 ℃ for 20 seconds,
56 ℃ for 30 seconds and 72 ℃ for two minutes and a ligation DNA products, 1.0 µl AFLP pre-selective
20 µl volume consisting of 4.0 µl diluted restriction-
further hold time of 30 minutes at 60 ℃. primer pairs and 15 µl core mix. Pre-amplification was
2.3 Selective Amplification Product Resolution carried out at initial hold time of two minutes at 72 ℃
followed by 20 cycles of 20 seconds at 94 ℃, 30 The ABI capillary system from Applied seconds, at 56 ℃, and 72 ℃ and a further hold time of
Biosystems-ABI (Forster City, CA, USA) located at
30 minutes at 60 ℃. the International Livestock Research Institute (ILRI),
2.2.3 Verification of Successful Amplification in Nairobi, Kenya, was used to resolve the selective To check the success of the pre-selective amplification products in this study. amplification reaction, 10 µl of pre-selective
Samples were prepared for analysis on ABI 3130×l amplification products was mixed with 2 µl of 10×
genetic analyzer from the selective amplification loading dye and run on 1.5% agarose gel in 1× TBE
products. Each run consisted of 96 samples. Samples buffer at 4 V/cm for 3.30 hrs. The gel was stained with
were loaded through the use of an autoloader, which
1 µg/ml ethidium bromide and viewed on a UV transfered a small aliquot of purified sample from a 96 transilluminator. The presence of a smear of products
well plate. A loading buffer mix was prepared by from 100-1,500 bp meant that the pre-amplification
adding 12 µl of Gene Scan 500 LIZ internal size was successful. For each sample, 10 µl of the
standard (ABI) to 1 ml (1,000 µl) deionised formamide pre-selective amplification product was then diluted
HiDi. 9 µL of the size standard mix was added to 1 µl with 19.0 µl 1× TE buffer, and stored at 4 ℃.
of the selective amplification products in a MicroAmp
2.2.4 Selective Amplification Reaction
Table 1 AFLP selective amplification primers screened in
Selective amplification was conducted using various
Ocimum gratissimum L..
combinations of two AFLP primers specific for EcoRI Mse I primers and MseI primer adapters on a test panel of -CAA -CAC -CTG -CAG
√* √ representative samples. These primers have three
-ACT √*
√* √ additional 3’ nucleotides. The EcoRI primers are
EcoR I
-ACA √*
√ √ florescent-labelled (Table 1). Initially, 16 primer
primers -ACC √*
√ √ combinations were used for screening in a √ implies primer combination during screening; * implies primer combination that detected high polymorphism for
-AGC √*
representative sample of four accessions (Table 1).
further analysis.
94 Genetic Diversity of the Medicinal Plant Ocimum Gratissimum L. (mint) from Kenya Based on AFLP
Markers
PCR Plate. From the ABI 3130xl genetic analyzer, the Cluster analysis based on genetic distances was sample data was directed to the GeneMapper Software
undertaken according to equations of Nei [21] using version 3.0 which analysed and displayed the sizing
unweighted pair-group method with arithmetic results as electrograms and tabular data.
averaging (UPGMA) [22] to generate a dendrogram The Southern algorithm automatically calculated the
showing relationships among populations. The degree fragment sizes using the GeneMapper Software version
of polymorphism was also quantified using Shannon’s
3.0. The software was used to score the alleles.
index of phenotypic diversity [23].
Category bins were created in GeneMapper to be able The ARLEQUIN software version 2.000 [24] and to group peaks based on the sizes of the allele. The
GenA1Ex 6 were used to partition genetic variation category was defined by size in base pairs and an
into within and between populations components automatic standard deviation which was a maximum
according to an analysis of molecular variance shift that a fragment could show across the individual
(AMOVA) [25] based on Euclidean distance. capillaries and still be scored as the same allele.