Trichoderma sp. (Isolate Td 22 )
Trichoderma sp. (Isolate Td 22 )
Yan Ramona 1 and Martin A. Line 2
1. Integrated Laboratory for Biosciences and Biotechnology, Udayana University, Bali 80362, Indonesia 2. School of Agricultural Science, University of Tasmania, Tasmania 7001, Australia
Received: February 16, 2013 / Accepted: May 15, 2013 / Published: July 30, 2013.
Abstract: The main objective of this research was to investigate the ability of a Trichoderma sp. (Td 22 ), inhibitory to Sclerotinia minor Jagger, to grow and survive in mature wood fibre waste (WFW) compost of paper mill origin following nutrient amendment. The growth and survival of the fungus in the WFW compost was assessed by serial dilution plate count method followed by confirmation of the fungal identity using pectic enzyme analysis as described in Cruickshank and Pitt [1]. It was found in this study
that the population densities of Td 22 achieved under non-sterile conditions in the WFW compost following nutrient amendment was approximately in the range of 7.0 log 10 CFU/g dw – 8.5 log 10 CFU/g dw after 28 days, depending on pre-treatment. The efficacy of this WFW compost-grown Td 22 for protection of lettuce from attack by S. minor was also demonstrated in glasshouse trials. This study indicates that cellulosic paper mill waste compost could provide an abundant low-cost growth medium for the large-scale cultivation of fungal antagonists, improving prospects for cost-competitiveness with chemical treatments.
Key words: Compost, biological control, Trichoderma sp., Sclerotinia minor.
1. Introduction Problems related to the large-scale production of biological control agents in low cost materials include
High concentration of pesticide usage often leads to difficulties in handling, transport, and storage. These increased pathogen resistance, resulting in ever higher problems have been largely overcome by maintenance pesticide applications in order to kill the same of bacterial antagonists in carriers such as peat or pathogen [2-4]. Furthermore, pesticide residue can vermiculite for periods of five months or more [12-14]. have adverse effects on reproduction and Cost of the cultivation medium, its transport, and field developmental processes in a variety of animals dispersal are critical factors in any assessment of including humans [5-7]. Due to these detrimental biological control relative to chemical treatments, a effects, reduced pesticide usage in agricultural practice problem sometimes exacerbated by the perceived need has been championed [8, 9] and one of the best broad for proprietary media formulations. spectrum pesticide/fumigants (methyl bromide) is no Norske-Skog Paper Mill Limited produces longer used in developed countries. Alternatives have approximately 33,000 tonnes of wood fibre waste been proposed, but none appears to be as effective [10]. (WFW) per annum, to be dumped as landfill [15]. In anticipation of such future reduction, Painuly and This cellulosic waste was considered worthy of Dev [11] proposed some possible alternative control, examination in view of its attributes of excellent such as biological control, use of pathogen-resistant water-holding and/or aeration capacity, its freedom plant varieties, and integrated pest management. from toxic elements, its free availability, and its
Corresponding author: Yan Ramona, Ph.D., research fields: potential utilization as a source of energy and carbon by environmental microbiology, composting and biocontrol.
cellulose-utilizing fungi, including the Trichoderma sp. E-mail: yan_ramona@yahoo.com.
Use of Wood Fibre Compost for the Cultivation of Trichoderma sp. (Isolate Td 22 )
isolate Td 22 . then either briefly autoclaved for 5 minutes prior to Based on the above background, the potential use of
inoculation (Compost A), or inoculated directly with wood fibre waste compost as a possible substrate for
inoculum (Td 22 suspension described above) the low-cost cultivation of a fungal biological control
(Compost B) to give an initial density of 5.5 log 10
CFU/g compost. Incubation was at 25 °C for 4 weeks together with assessments of the efficacy of the
agent (Trichoderma sp. isolate Td 22 ) was investigated,
with periodic assay of CFU on Potato Dextrose Agar compost-grown Td 22 to protect lettuce seedlings from
(PDA) for Td 22 and on 0.1% Trypticase Soya Agar attack by S. minor in pot trials or glasshouse level
(TSA) for check for bacterial contaminants. The experiments.
identity of the Td 22 was confirmed by analysis of pectic enzymes using the method as described by
2. Materials and Methods
Cruickshank and Pitt [1].
2.1 Isolate Td 22
2.5 The Efficacy of WFW Compost-Grown Td 22
Trichoderma sp., isolate Td 22 was kindly provided
Lettuce seeds were sown in pots containing field soil by Dr. Dean Metcalf (DPIWE Tasmania). For long amended with suppressive compost (Compost A) at term storage, the fungus was maintained at 4 °C in levels of 2.5, 5.0, 10 and 20% v/v. sterilized and moist millet seeds. The spore density of the antagonist (Td 22 ) in the
2.2 Wood Fiber Waste compost was 9.21 log 10 spores/g wet weight of compost. Lettuce pathogen (S. minor) grown on millet
The WFW used in this study was the same as that seed was evenly inoculated approximately 20 mm used by Ramona and Line [15]. It is a mix of eucalypt below the soil surface at the rate of 2.0 g inoculum per and Pinus radiata, comprising holocellulose as its pot. Soil without compost amendment, amended with primary constituent with very low levels of metal pathogen only, or without pathogen, served as controls. contaminants. It was also deficient (from a recycling Five replicate pots per treatment, each containing five perspective) in N and P. In the present trial, seeds, were maintained for four weeks in a shade house. composting of this material was for three months Pots were irrigated as required and the germinated (followed the method specified by Jackson and seeds counted one week after sowing. Disease Line [16] and Jackson [17]) until its toxicity to radish incidence on the lettuce seedlings was recorded from seeds was eliminated.
two weeks after sowing.
2.3 Inoculum Preparation
2.6 Data Analysis
The active starters used in this experiment were The data obtained from this experiment was prepared according to the methods as specified in statistically analyzed by using analysis of variance Ramona [18]. The potency of this inoculum, measured (ANOVA) using Minitab software for Windows. The in CFU, was determined by serial dilution plating (in significance of differences between means was further triplicates) before being used to inoculate the WFW. tested using the least significant different (LSD) test at
2.4 Assessment of Growth of the Td 22 P < 0.05 following ANOVA.
The WFW compost (at a self-generated temperature
3. Results
of 55-60 °C) was air-dried in a glasshouse, mixed with
3.1 The Growth and Survival of Td 22 in WFW Compost 2% (w/v) Phostrogen® solution, and placed into 750
mL flasks (100 g quantities each). These flasks were The growth and survival of Td 22 in compost over 28
Use of Wood Fibre Compost for the Cultivation of Trichoderma sp. (Isolate Td 22 )
days following different pre-treatments is presented in
Fig. 1. Significant increases in CFU of Td 22 in
steam-treated or untreated composts were observed in the first seven days, after which time the populations
Rf 1.0
remained relatively constant at approximately 8.5 log 10 CFU/g dw in compost A or approximately 7.0 log 10 CFU/g dw in compost B.
Rf 0.24
The identity of Td 22 cultivated in non-sterile
compost was confirmed by both morphological Rf 12
Rf 0.0
characteristics and isozyme profile (Fig. 2).
The initial bacterial populations (contaminants) in
Fig. 2 Contact print following polyacrylamide-gel
the mixes depended on the method of preparation.
electrophoresis to confirm survival of Td 22 in three-month
The population of the mesophilic bacteria in compost
old WFW compost. Dark bands indicate polygalacturonase
A was recorded at approximately one order of
and light bands indicate pectinesterase. Wells 1, and 9 – 14
magnitude lower than that in compost B after two
contained the control reference, Td 22 , while wells 2 – 8 contained re-isolated fungi from compost B on day 7 of
weeks incubation. However after four weeks of
sample collection.
incubation, the growth rate of the indigenous
mesophilic bacteria in compost A exceeded that of
compost B, to result in a population of 9.3 log 10 CFU/g in compost A, in comparison with 8.5 log 10
CFU/g in compost B.
3.2 The Efficacy of Td 22 -Grown Compost to Protect
Lettuce from S. minor Attack
The Td 22 -grown compost was found to be non toxic
as c between
88%-100% of the lettuce seeds were
t Fig. 3 Protection of lettuce seedlings from attack by S.
minor. Each value in the graph ± standard error bar is an pos
average of 5 replicates. Bars with the same letter are not
om 7
statistically significant at P < 0.05.
1. Nil pathogen and antagonist addition; 2. Pots amended with S. minor only;
gd 5
Compost A
fu/
3. Pots amended with 2.5% compost-grown Td 22 and S. minor;
Compost B
4 4. Pots amended with 5% compost-grown Td 22 and S. minor;
log c
0 5 10 15 20 25 30 5. Pots amended with 10% compost-grown Td 22 and S. minor;
Incubation time (days)
6. Pots amended with 20% compost-grown Td 22 and S. minor.
Fig. 1 Population density of Trichoderma sp. (isolate Td 22 )
observed to germinate in soil amended with
in three months old WFW compost, either briefly treated
for 5 min at 121 °C prior to inoculation (Compost A), or
5-20% (v/v) of this compost. No inhibition of seed
directly inoculated following re-wetting (Compost B). Both
germination was evident even higher rate of
compost types were brought to approximately field capacity
Td
22 -grown compost was applied 4 weeks after
with 2% (w/v) Phostrogen
solution (~350 mL/L). Each
sowing.
value in the graph is an average of three replicate
determinations ± standard error.
The effectiveness of the Td 22 -grown compost
Use of Wood Fibre Compost for the Cultivation of Trichoderma sp. (Isolate Td 22 )
amendment to protect lettuce seedlings from attack by and other enzymes are important in the degradation of S. minor is presented in Fig. 3. As indicated in Fig. 3,
complex carbon sources contained in WFW and the the rate of amendment of the compost/fungus was
ability to produce them is advantageous when in proportional to disease control, ranging from 32%
competition for available major energy sources. provided by 2.5% (v/v) amendment to 100%
Assay of isoenzyme profiles (e.g. pectolytic enzyme protection provided by 20% (v/v) amendment at four
profiles as undertaken in the present study) of fungal weeks after sowing. This was statistically significant
isolates provided a convenient tool to differentiate when compared to the control treatment (pots Td 22 from other isolates of Trichoderma sp. following inoculated with S. minor only) where 100% mortality
re-isolation from non-sterile samples. This assay has was observed at four weeks after sowing (Fig. 3).
been applied by many workers [24-26] to distinguish their fungal isolates from other related fungi.
4. Discussion and Conclusion
The effectiveness of Trichoderma sp. (Td 22 ) grown Air drying compost in a glasshouse for three weeks
in wood fibre waste compost to protect against S. minor or brief steam-treatment (rather than sterilizing) with a
in glasshouse trials has been demonstrated in the view to minimizing the indigenous microbiota, prior
present study. Application of compost-grown Td 22 at
rates up to 20% (v/v) was non-toxic (relative to The CFU of this fungus cultured under non-sterile
to inoculation with Td 22 , gave encouraging results.
controls) to lettuce seed in all treatments (Fig. 3). conditions increased by two to three orders of
The utilization of composted WFW as the authors magnitude, although they were always lower than
described could be achieved in large scale at moderate those obtained under sterile conditions (Fig. 1). The
cost while avoiding the problems of liquid cultures as CFU were found to be higher in the briefly autoclaved
outlined by Hadar et al. [27]. Application of compost (compost A) than those in the directly
compost-grown Td 22 after allowing growth for two inoculated compost (compost B) following re-wetting.
weeks or more appeared to be advantageous, because it Application of heat treatment to the WFW compost
was mostly in the form of spores (unpublished data). (by briefly autoclaving at 121 °C) may have changed
This reduces concerns relating to viability in the field, its composition. However the most probable reason
because fungal spores will be relatively more resistant
than mycelia to environmental stress [28]. near-elimination of competition by mesophilic
for the better growth of Td 22 in this medium was the
bacterial survivors in compost A relative to compost B.
Acknowledgment
Similar results were also demonstrated by Nakasaki et The authors would like to acknowledge Dr. Dean al. [19] who found a reduced growth rate of a strain of
Metcalf, Norske Skog Paper Mill Limited, and Bacillus subtilis following inoculation of this AusAid for the provision of Trichoderma sp. isolate
antagonist into non-sterile (as compared with sterile), Td 22 , wood fiber waste (WFW), and financial support, but freshly-cut grass clipping compost. This reinforces
respectively.
the notion that competition between the indigenous microbiota and the inoculated antagonists is important
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July 2013, Vol. 7, No. 7, pp. 700-704
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Evaluation of the Insecticidal Activity of the Aerial Part of Pseudocytisus integrifolius (Salisb) Rehder on Grain Borer, Rhyzopertha dominica Fab. (Bostrychidae) and Wheat Weevil, Sitophilus granarius Linn. (Curculionidae)
Kassemi Naima 1 , Khelil Mohamed Anouar 1 and Bendimerad Nassima 2 1. Research Laboratory Valuation of Human Actions for Environmental Protection and Enforcement in Public Health, Department of
Biology and Environment, Faculty of Science, University Abou Bakr Belkaïd, Tlemcen 13000, Algeria 2. Natural Products Research Laboratory, Faculty of Science, University Abou Bakr Belkaïd, Tlemcen 13000, Algeria
Received: December 21, 2012 / Accepted: March 21, 2013 / Published: July 30, 2013.
Abstract: This study aims to evaluate the potentialities of Pseudocytisus integrifolius (Salisb) Rehder (Brassicaceae) as a botanical insecticide for the protection of stored wheat against two pests of stored grain. The essential oil of Pseudocytisus integrifolius was tested at different doses on adults of Rhyzopertha dominica Fab. (Bostrychidae) and Sitophilus granarius Linn. (Curculionidae), at a temperature of 28 °C and a relative humidity of 75%. This oil has insecticidal activity and gives, for the studied adults, a very
significant reduction. The essential oil extracted from Pseudocytisus integrifolius is toxic on Rhyzopertha dominica with LD 50 = 2.61 μL/30g grain and LD 50 = 3.99 μL/30g grain for Sitophilus granarius, the chemical composition analysis shows the richness of the
essential oil compounds known for their insecticidal properties as dimethyl disulfide (DMDS) and dimethyl trisulfide explains the results.
Key words : Plant protection, insecticidal activity, Rhyzopertha dominica, Sitophilus granarius, Pseudocytisus integrifolius, DMDS.
1. Introduction
gelechiid moths and coleopteran beetles are the major insect pests of stored cereal and legume grains with
Globally, cereal stocks losses are estimated at over Sitotroga cerealella Olivier, Sitophilus spp., 100 million tons of which 13 million are due to insects. Prostephanus truncatus Horn, Rhyzopertha dominica In Africa, these losses reach often 30%, and, in F., Tribolium castaneum Herbst, Acanthoscelides developed countries, they are around 3% [1]. obtectus Say and Callosobruchus spp. being the most Insect damage in stored food grains pose the
destructive species [3, 4].
greatest challenge to the provision of quality food Currently, recommended pest control measures in with 10-60% losses in countries where modern storage durable stored food products rely heavily on use of technologies are yet to be fully adopted [2, 3]. synthetic insecticides which pose possible health The magnitude of loss is dependent upon the insect hazards to warm-blooded animals, risk of species involved, storage duration and pest control environmental pollution, development of resistance by methods among other factors. In the tropics, the
insects and pest resurgence [2].
The extent of damage caused by the beetles in grain Corresponding author: Kassemi Naima., M.Sc., assistant
is in connection with their feeding behavior. Indeed, professor, research field: economic entomology. E-mail:
proenv2007@yahoo.fr. pests, the best suited to mechanical disturbances that
Evaluation of the Insecticidal Activity of the Aerial Part of Pseudocytisus integrifolius
(Salisb) Rehder on Grain Borer and Wheat Weevil
occur during storage, live inside the grains or simply in the laboratory at room temperature (26-28 °C) for 8 need grains necessarily to survive. This is the wheat
days.
weevils (Sitophilus granarius) and grain borer
2.2 Breeding of the Insect
(Rhyzopertha dominica etc.). Grain weevils are generally among the most
The mass rearing for obtaining adult R. dominica damaging insects during storage or conservation [5].
and S. granarius for bioassays were conducted in Unlike many storage insects, the damage of
glass containers (15.5 cm and 8 cm in diameter) Rhyzopertha caused by the larvae as well as by the
containing grains of durum wheat (Triticum durum), very voracious adults, sparing almost no grain [6].
which were covered by a fine mesh cloth for Stored products are generally protected by the
ventilation. The cultures were maintained in an oven application of insecticides or fumigators. But the
set at a temperature of 28 °C and a relative humidity presence, in food, of toxic residues and the appearance
of 75%. Strains of insects used were those of the grain of strains of insects resistant to these insecticides are
and vegetable cooperative (Abu Tachfine cooperative, becoming more frequent.
Tlemcen, Algeria).
In the search for alternative control methods, the plant kingdom offers many possibilities. Many studies 2.3 Witness Breeding
are currently being developed to isolate or identify We mixed 1 mL acetone with 30 g of durum wheat secondary substances extracted from plants that have
grains in petri box. After evaporation of solvent, we insecticidal, repellent or anti-appetent vis-à-vis the
introduced 20 adult insects not sexed and aged less insects. Some of these compounds have been isolated
than 2 days with four repetitions. from products used in food [7].
2.4 Test Essential Oil P. integrifolius oils, were the subject of many research in order to
The compounds allelochimic, in particular essential
For bioassays, durum wheat grains, which have not reduce the losses caused by the devastating insects of
been chemically treated, come from a warehouse in the grains stored by there insecticidal effects [8, 9].
the region of Tlemcen. These were carried out in an The sulfur substances characteristic of crucifers
oven at temperature and relative humidity maintained (Glucosinolates, Isothiocyanates) play an essential
at 28 °C and 75%, respectively.
role in plant defense against external aggression [10]. For each test, 1 mL of acetone solution, containing We observed that the essential oil of the aerial part
each of essential oils at 1, 2, 3, 4, 5, 6, 7 and 8 µL/mL of P. integrifolius contains insecticidal compounds.
of acetone, was added to 30 g of grains contained in The present study therefore consisted of extraction
Petri dishes, and then all was well mixed. The tests and chemical characterization of P. integrifolius
were repeated 4 times for each dose. All boxes were essential oils then evaluation of their insecticidal
infested with 20 adult insects not sexed and aged less effects in relation to the grain borer (R. dominica) and
than 2 days. Counts of dead insects are made every 24 wheat weevil (S. granarius).
hours for a period of six days, the observed mortality
2. Materials and Methods
is expressed after correction by Abbott’s formula [11].
Po – Pt
2.1 Plant Material Pc = × 100 100 – Pt
The plant, P. integrifolius, was collected in western where: Pc: corrected mortality in %; Pt: mortality Algeria (South of Tlemcen). The aerial part was dried
observed in the control; Po: mortality observed in
Evaluation of the Insecticidal Activity of the Aerial Part of Pseudocytisus integrifolius
(Salisb) Rehder on Grain Borer and Wheat Weevil
the test. increased significantly by using essential oil P. LD 50 values were calculated for the toxicity of
integrifolius tested against a control. essential oil in the beans. Thus, the mortality
Depending on the dose factor in essential oil, there percentages were transformed into probit units and the
is significant variation between the mortality of values obtained were correlated with the logarithm of
weevils with F = 23.61 for P = 5.6548 -11 10 . the doses to obtain the lethal dose for 50% of the
Depending on the duration of exposure factor, population of insects, for each plant material to test [12].
statistical analysis showed a difference between the mortality rate of bruchids with F = 158.51 for
2.5 Data Statistical Analysis
P = 8.28 10 -28 .
The results are to be tested for the variance to two The transformation of mortality percentage after classification criteria, useful for studying the action of
two days of exposure in probit and the regression of two factors [13].
these data against the logarithm of the of essential oils This type of analysis was used to test the effect of
dose, yielded the following equations: dose and exposure duration of essential oils on the rate
Rhyzopertha dominica
of the insect mortality. 2 Y = 3.76967 + 2.94583x (R = 57.1%) Statistical analysis was performed with Microsoft
LD 50 values determined from these equations were Office Excel 2007.
2.58 μL/30g of grain
Sitophilus granarius
3. Results and Discussion
Y = 3.34364 + 2.75769 x (R 2 = 47%). The mortality rate of adult (R. dominica and S.
LD 50 values determined from these equations were granarius) exposed to essential oils P. integrifolius, is
3.99 μL/30g of grain.
increasing as a function of dose and duration of
3.3 Discussion
exposure. Our results show that there is a variation on the
3.1 Effect of Essential Oils on the Mortality of R. lesser grain borers and weevils mortality rate which dominica depends on the dose and exposure duration of P.
Depending on the dose factor in essential oil,
integrifolius essential oils.
statistical analysis showed that there is a highly Chemical analysis of the essential oil of the aerial significant variation between the mortality rates of the
part shows a high content of sulfur compounds Grain borers with F = 197.42 for P = 3.43 -26 10 .
Depending on the factor duration of exposure, The P. integrifolius essential oil contains statistical analysis showed a difference between the
components known for their insecticidal properties; mortality rates of the lesser grain borers with this is the case of dimethyl disulfide (DMDS),
F = 22.13 for P = 6.02 10 -10 . Pent-4-enenitrile and dimethyl trisulfide (DMTS). So, there is a variation on the grain borers mortality
Many studies have reported the insecticidal rate, which depends on the dose used and of exposure
potential of sulfur compounds from the alliaceae. duration of P. integrifolius essential oils.
These molecules are mainly disulfides: dimethyl disulfide (DMDS), dipropyl disulfide (DPDS) and
3.2 Effect of Essential Oils on the Mortality of S.
diallyl disulfide (DADS).
granarius Toxicity tests conducted on different insect species
The mortality rate among S. granarius adults have shown that sulfur volatiles emitted by these
Evaluation of the Insecticidal Activity of the Aerial Part of Pseudocytisus integrifolius
(Salisb) Rehder on Grain Borer and Wheat Weevil
Fig. 1 Evolution of adult mortality of R. dominica function of time and doses of Pseudocytisus integrifolius essential oil.
in relation to insects not thrall to plants producing sulfur compounds [14]. The toxicity of DMDS is even roughly equal to that of methyl bromide, the fumigant, the largest commercially used, for the protection of stored products. The LC50 of Sitophilus oryzae exposed to DMDS and methyl bromide under the same experimental conditions were 1.23 µL/L and
1.05 µL/L, respectively [17]. Finally, the DMDS is synthesized industrially in
Fig. 2 Evolution of adult mortality of S. granarius
large quantities at low cost and could be very
function of time and doses of essential oil of Pseudocytisus
interesting for potential commercialization if the high
integrifolius.
toxicity of this substance is confirmed [14]. plants, thiosulfinates and their degradation products,
4. Conclusion
including disulfides and in particular dimethyl disulfide (DMDS), are toxic in relation to all the
By observing the results obtained, we deduce that species tested [14].
these essential oils are effective against the attacks of In Plutella xylostella, Lepidoptera thrall to brassicas,
the R. dominica grain borers and the S. granarius larvae susceptible to toxicity of isothiocyanates [15]
weevils. Indeed, it influences directly affects the prevent the formation of the latter from the precursor
mortality rates. The statical analyses showed that the glucosinolate [16].
factor proportions out of oils essential with a highly There are some scattered indications in the
significant incidence on the mortality of the two literature on the specific activity of some sulfur
ravagers. This repulsive capacity of essential oils substances against various pathogenic organisms: the
extracted from Pseudocytisus integrifolius is due case for example with disulfides acting on insects, like
primarily to their chemical compositions. It is beetles or moths, present in stored food stuffs [17]
significant to avoid the disadvantages of the chemical and diallyl disulfides having a fungicidal action on
fight, the use of the essential oils extracted from the sclerotia of Sclerotium cepivorum [18].
plants, having an insecticidal activity can constitute an In general, the toxicity of DMDS is very important
at the same time effective and economic solution.
Evaluation of the Insecticidal Activity of the Aerial Part of Pseudocytisus integrifolius
(Salisb) Rehder on Grain Borer and Wheat Weevil
Acknowledgments
Effectiveness of organic powders and essential oils of the leaves of Chenopodium ambrosioides et Eucalyptus
The author is grateful to Professor Bouazza saligna against pulse beetle, Callosobruchus maculatus Mohammad, Laboratory of Ecology and Management Fab. (Coleoptera, Bruchidae), Cahiers Agricultures 12 (6)
(2003) 401-407.
of Natural Ecosystems, Abou Bekr Belka University,
A. Kellouche, Study of the weevil chickpea, Tlemcen (Algeria) for his vital encouragement and
Callosobruchus maculatus (Coleoptera: Bruchidae): support.
Biology, physiology, reproduction and control, Ph.D. Thesis, Univ. Tizi-Ouzou, Algérie, 2005, p. 154.
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Plant oils as fumigants and contact insecticides for the [12] P. Dagnelie, Theories and statistical methods, The control of stored-product insects, J. Stored Prod. Res. 33 Agronomic Press of Gembloux, A.S.B.L. 2 (1975) (1997) 7-15.
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[3] J.O. Ogendo, A.L. Deng, E.O. Omolo, J.C. Matasyoh, I.M. Tabu, Grain pests management practices in
[13] S. Dugravot, The secondary sulfur compounds of Allium: Role in the defense systems of leek and actions on the
subsistence agriculture: A case of farmers in Suba District, Nyanza Province, Kenya, Farm Survey biology of insects, Ph.D. Thesis, Univ de Tours, 2004. Report, 2003. (AICAD Grant No. [14] Q. Li, S.D. Eigenbrode, G.R. Stringham, M.R. AICAD/RD-07/SED/02-001).
Thiagarajah, Feeding and growth of Plutella xylostella [4] J. Gressel, A. Hanafi, G. Head, W. Marasas, A.B. Obilana,
and Spodoptera eridania on Brassica juncea with varying J. Ochanda, et al., Major heretofore intractable biotic
glucosinolates concentrations and myrosinase activities, J. constraints to African food security that may be amenable
Chem. Ecol. 26 (2000) 2401-2419. to novel biotechnological solutions, Crop Protection 23
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J. Kroymann, Disarming the mustard oil bomb, Proc. Natl. [5]
H. Yadi, Qualitative criteria and technology associated Acad. Sci. USA 99 (2002) 11223-11228. with the proliferation of rice weevil, Sitophilus oryzae (L.)
[16] J. Auger, F. Cadoux, E. Thibout, Allium spp. (Coleoptera: Curculionidae) in a stock of paddy rice in its
thiosulfinates as substitute fumigants for methyl bromide, storage in sealed enclosures under modified atmospheres
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[17] A.R. Entwistle, P.R. Merriman, H.L. Munasinghe, P. Bordeaux, 1987, p. 29.
Mitchell, Di-allyl Di-sulfide to reduce the numbers of [6]
G.H. Schmidt, E.M. Risha, A.K.M. Nahal, Reduction of Sclerotium cepivorum in soil, Soil Biology and progeny of some stored-products Coleoptera by vapours
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of Acorus calamus oil, J. Stored Prod. Res. 27 (1991) [18] N. Bendimered, Study of essential oils Pseudocytisus 121-127.
integrifolius (Salisb) Rehder and Sinapis arvensis L., [7] E.P. Lichtenstein, Insecticides occuring naturally in crps,
Crucifer of the western region of Algeria, Demonstration Adv. Chem. Ser. 53 (1966) 34-38.
of compounds and nutritional consequences, Ph.D. Thesis, [8] L.A. Tapondjou, C. Adler, H. Bouda, D.A. Fontem,
State Univ. Abou Bekr Belkaid, 2006, p. 152.
July 2013, Vol. 7, No. 7, pp. 705-711
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Structure - Antimicrobial Activity Relationship Investigation of Some Butadiene and Chalcone Derivatives
Hanoy AL-Amood 1 , Hadeel T. AL-Hadithi 2 and Ghazwan F. Fadhil 1, 3
1. Dept. of Chemistry, College of Science, University of Basrah, Basrah 61001, Iraq 2. Dept. of Biology, College of Science, University of Basrah, Basrah 61001, Iraq 3. Dept. of Chemistry, Faculty of Science, University of Duhok, Duhok 1048 BD, Iraq
Received: February 04, 2013 / Accepted: April 09, 2013 / Published: July 30, 2013.
Abstract: The biological activity against Staphylococcus aureus, and Eschericia coli were investigated implementing three series, the first series was 1-phenyl-2-(4'-X-phenyl)-4-(2,4-dichlorophenyl)-1,3-butadiene, where X = H, CH 3 , OCH 3 , NH 2 , Cl, F, NO 2 and COOEt; the second was 3,4-dichlorochalcone series namely 3-(3,4-dichlorophenyl)-1-(4'-X-phenyl)-2-propen-1-one, where X = H, CH 3 , OCH 3 , NH 2 , Cl, F, NO 2 and CN; and the third one was 2,4-dichlorochalcone series namely 3-(2,4-dichlorophenyl)-1- (4'-X-phenyl)-2-propen-1-one, where X = H, CH 3 , OCH 3 , NH 2 , Cl, F, NO 2 and COOEt. MIC, MBC and the percentage of inhibition (activity) at 20 µg/mL, 15 µg/mL and 10 µg/mL against Eschericia coli, and at 7.5 µg/mL, 5 µg/mL and 2.5 µg/mL against Staphylococcus aureus , were determined for each compound in the three series.
Highest MIC activity against E. coli and S. aureus were given by 2,4-dichlorochalcone series. Butadiene series was similar in behavior to 2,4-dichlorochalcone series in MIC activity against S. aureus. Results of MBC revealed that compounds in the three series exerted high activity against both types of bacteria. Compounds substituted with nitro or nitril exhibited higher activity than other compounds in the three series. Percentage of inhibition of halogenated compounds (4'-Cl and 4'-F) was almost equal in every series. Compounds with substituents (4'-H and 4'-CH 3 ) showed fluctuation in activity according to the nature of each series.
Key words: Structure activity relationship, chalcone, butadiene, Eschericia coli, Staphylococcus aureus.
1. Introduction shown significant antimicrobial activity [2]. Also, anti-candida albicans activity was found for
Compounds exhibiting antimicrobial activity vary chalcones [3]. A series of new greatly in their effect against different types of 1-(2',5'-dimethyl-3'-furyl)-3-(substituted aryl)-2- bacteria and these effects may also vary against propen-1-ones (chalcones (Fig. 1)) were prepared and different strains of the same species. Chalcones or demonstrated antimicrobial and anti-inflammatory 1,3-diaryl-2-propen-1-ones are natural or synthetic activities, some of them were found to possess compounds belonging to the flavonoid family. significant activity, when compared to standard 3-hydroxy Bezofuran chalcones have shown
drugs [4].
antibacterial activity against Staphylococcus [1]. A Minimum inhibitory concentration (MIC) of series of nine chlorine containing chalcones have 2',4',2-trihydroxychalcone; 4',3-trihydroxychalcone
and 2',4',4-trihydroxychalcone were examined and Corresponding author: Hadeel T. AL-Hadithi, Ph.D., Prof.,
compared to that of 2',4'-dihydroxychalcone [5]; research fields: microbiology, bacteriology. E-mail:
hadeelalhadithi@yahoo.com. which reported the following sequence:
Structure - Antimicrobial Activity Relationship Investigation of Some
Butadiene and Chalcone Derivatives
2. Materials and Methods
2.1 Compounds Syntheses
A B The synthesis of 2,4-dichlochalcone series was reported by Perjessy, et al. [7]. The synthesis of the
butadiene series and 3,4-dichlorochalcone series were Y reported in Ref. [6].
Fig. 1 Chalcone.
2.2 Strains 2',4',3-(OH) 3 > 2',4'-(OH) 2 > 2',4',4-(OH) 3 > > MIC
Two standard bacterial species: Escherichia coli 2',4',2-(OH) 3 . These results showed that introduction
of an electron donating group (-OH) in the aromatic (Gram negative bacteria ATCC25922) and B-ring causes an increase in bioactivity, and the
Staphylococcus aureus (Gram positive bacteria intensity of action depends on the position of the OH
ATCC25923) were chosen to investigate antibacterial substitute. activity of the three series.
Two new series were prepared by Hanoy [6]:
2.3 Experiments
1-phenyl-2-(4'-X-phenyl)-4-(2,4- dichlorophenyl) -1,3
Antibacterial activities of three series were OCH 3 , NH 2 , Cl, F, NO 2 and COOEt and investigated by agar plate technique [8]. Stock 3-(3,4-dichlorophenyl)-1-(4'-X-phenyl)-2-propen-1-one
-butadiene (butadiene series), whereas X = H, CH 3 ,
solutions (1000 µg/mL) of the compounds were
prepared in isopropanol solvent and stored at 4 °C to OCH 3 , NH 2 , Cl, F, NO and CN series.
(3,4-dichlorochalcone series), whereas X = H, CH 3 ,
8 °C until used. 100 µg/mL of each compound was Previous antimicrobial investigations [1-5] have
prepared by the addition of 13.5 mL of melted Mueller revealed that chalcones compounds serve as good
Hinton agar to 1.5 mL of each compound in a test tube candidates for antimicrobial compounds. Hence the 2 and sterilized in the autoclave (121 °C at 15 lb/in )
aim of this study is to investigate the variation of then poured in small sterile Petri dishes (4.5 cm). A structure and substituent groups of the newly 6 suspension of 10 CFU/mL of log phase growth
synthesized chalcones and butadienes [6] compounds prepared by inoculating one colony from fresh agar against two types of bacteria: Staphylococcus aureus
culture into 5 mL of sterile nutrient broth, incubated at (Gram positive) and E. coli (Gram negative). The
37 °C for 6 hrs (The turbidity of suspensions was investigated three structurally related compounds
adjusted to reach an optical density equal to that of 0.5 series are shown in Fig. 2, namely: McFarland standard). A loopful from each culture was 3-(2,4-dichlorophenyl)-1-(4'-X-phenyl)-2-propen-1-one,
streaked onto the agar surface. The plates were abbreviated (2,4-dichlochalconel), whereas X = H,
incubated at 37 °C for 24 hrs. Plates were examined
for the presence (+ve) or absence (-ve) of the bacterial series was 1-phenyl-2- (4'-X-phenyl)-4- (2,4- growth [9-11]. dichlorophenyl) -1,3-butadiene, abbreviated
CH 3 , OCH 3 , NH 2 , Cl, F, NO 2 and COOEt, the second
2.3.1 Determination of Minimum Inhibitory
(butadiene series), whereas X = H, CH 3 , OCH 3 , NH 2 ,
Concentration (MIC)
A series of dilutions of each compound were 3-(3,4-dichlorophenyl) -1-(4'-X-phenyl)-2- propen-1-
Cl, F, NO 2 and COOEt, and the third series was
prepared in test tubes using nutrient broth to obtain the one, abbreviated (3,4- dichlorochalcone series), concentrations of: 75, 50, 25, 20, 15, 10, 7.5, 5 and 2.5
whereas X = H, CH 3 , OCH 3 , NH 2 , Cl, F, NO 2 and CN.
µg/mL. One colony of each bacterial strain was left to
Structure - Antimicrobial Activity Relationship Investigation of Some
Butadiene and Chalcone Derivatives
grow in 5 mL of sterile nutrient broth for 6 hrs at
following equation:
37 °C. One mL of growing bacteria was suspended in Conc. of organism in the tubes containing compounds
9 mL of sterile nutrient broth. 0.5 mL of each bacterial % Growth = 100 suspension was added to the tubes containing 4.5 mL
Conc. of organism in the control tube dilutions of the prepared compounds. Controls were % Inhibition = 100 - % Growth
prepared by addition of 0.5 mL of each bacterial
3. Results and Discussion
suspension to 4.5 mL sterile nutrient broth free from compounds. The final inoculums size was 10 4 Minimum inhibitory concentration values clarifies that 2,4-dichlorochalcone series has higher activity
CFU/mL as counted by Petroff-Hauser Chamber. than 3,4-dichlorochalcone series and the latter is more
After incubation for 24 hr at 37 °C, the minimal active than Butadiene series, against E. coli (Table 1).
inhibitory concentration (MIC, µg/mL) was determined by measuring turbidity using Bausch and
Activities of the two series 2,4-dichlorochalcone and Lomb Spectronic 20 at 686 nm [12]. butadiene are almost similar against S. aureus and it is
2.3.2 Determination of Minimum Bactericidal higher than that of 3,4-dichlorochalcone series (Table 2). Concentration (MBC)
It is obvious that all compounds in the three series
A loopful was removed from each test tube have bactericidal activity (MBC) at higher showing no visible growth and streaked onto surface
concentrations (Tables 1 and 2).
of sterile nutrient agar plates free from compounds. The bactericidal activity exhibited by the three After incubation for 24 hr at 37 °C, the growth of
series was higher against gram-positive bacteria (S. bacteria was examined. The tubes that failed to yield
aureus ) as compared with gram-negative bacteria (E. growth on the agar surface were recorded as the MBC
coli ). It is generally accepted that bacterial outer of compounds for the test organism [12].
envelope is responsible for the different microbial
2.3.3 Determination of Percentages of Growth responses to biocides challenges. Cytoplasmic (Inhibition) of Organisms
membrane in Gram negative bacteria is composed Percentages of growth (inhibition) of organisms
essentially of a phospholipid bilayer with embedded were determined by measuring the concentration of
proteins; it is semi permeable and regulates the the organism in test tubes using spectrophotometer at
transfer of solutes and metabolites in and out of the 686 nm. At concentrations 20, 15 and 10 µg/mL for E.
cell cytoplasm. It is also associated with several coli strain, and at concentrations 7.5, 5 and 2.5 µg/mL
important enzymes involved in various cell metabolic for S. aureus strain. Results were turned to the
functions [9]. Cytoplasmic membrane is often percentages of growth (inhibition) by using the
considered as the major target site for biocides.
Table 1 Values of MIC and MBC of three series (3,4-dichlorochalcone, 2,4-dichlorochalcone, butadiene) against E. coli.
3,4-Dichlorochalcone 2,4-Dichlorochalcone Butadiene X MIC MBC MIC MBC MIC MBC
* * * Not tested.
Structure - Antimicrobial Activity Relationship Investigation of Some
Butadiene and Chalcone Derivatives
Table 2 Values of MIC and MBC of three series (3,4-dichlorochalcone, 2,4-dichlorochalcone, butadiene) against of S. aureus.
3,4-Dichlorochalcone 2,4-Dichlorochalcone Butadiene X MIC MBC MIC MBC MIC MBC
H5 15 2.5 20 2.5 10 CH 3 -
10 2.5 20 2.5 10 OCH 3 5 10 2.5 10 15 10** NH 2 7.5 10 2.5 10 2.5 15 Cl 5
* * * Not tested; ** Opposite behavior.
Damage to the membrane can take several forms: (i) of comparisons were made: Firstly, a comparison physical disruption of the membrane; (ii) dissipation
between the percentage of inhibitions of the two series: of the proton motive force (PMF) and (iii) inhibition
2,4-dichlorochalcone and butadiene where the of membrane-associated enzyme activity [13]. carbonyl group was converted to double bond
In Gram-negative bacteria, passage across the outer substituted with phenyl group i.e. to increase the membrane (OM) depends upon the chemical nature of
conjugated of the compound (Fig. 2a). Secondly, a the inhibitor, hydrophilic compounds utilizing the porin
comparison between percentage of inhibitions of the channels (hydrophilic route) and hydrophobic compounds of the two series 2, 4-dichlorochalconeand compounds enter via the hydrophobic route. and 3, 4-dichlorochalcone to investigate the effect on Nevertheless, passive diffusion was the rout by which
the percentage of inhibitions on changing the position biocides pass through the staphylococcal cell wall
of one of the chlorine atoms from position 2 to 3 (in which might explain the higher susceptibility of S.
ring 2) in 2,4-dichlorochalcone series and aureus to the series applied in the present study [9, 14].
3,4-dichlorochalcone series (Fig. 2b). Th e -unsaturated ketone compounds and
3.2 Comparison between 2,4-Dichlorochalcone Series Butadiene compounds were classified to be an
and Butadiene Series
electrophiles /proelectrophiles. The bacteriostatic activity may be attributed to covalent reaction with
Percentage inhibition of all compounds was
decreased remarkably against E. coli, when carbonyl via a nucleophilic Michael-type addition reaction [15,
soft macromolecular nucleophiles (e.g., -SH, -NH 2 )
group was converted to a double bond, except for 16]. Nitro-substituted compounds exhibited much
4'-COOEt and 4'-CH 3 compounds. The percentage higher activity than the other compounds in three
inhibition due to 4'-COOEt compound has become series. Aromatic-nitro-containing compounds were
doubled. However, the Percentage inhibition owing to reported to be uncouplers of oxidative 4'-CH 3 compound was almost similar in both series phosphorylation, yielding the corresponding (Table 3). potentially highly active C-nitroso compounds [16].
These two series exerted variable percentage of inhibitions against S. aureus. A little decrease in
3.1 The Effect of Modifications on Percentage of percentage inhibitions due to 4'-H, 4'-Cl, 4'-F and Inhibitions of Compounds 4'-COOEt compounds was observed. Nevertheless, a
Effect of modification on percentage of inhibition considerable decrease in percentage of inhibitions of compounds under study was performed. Two types
owing to 4'-CH 3 , 4'-OCH 3 and 4'-NO 2 compounds was
Structure - Antimicrobial Activity Relationship Investigation of Some
Butadiene and Chalcone Derivatives
Cl
X 4'
6'
5 Cl
5'