Journal of Life Sciences Volume 7 Number (5)
J LS
Journal of Life Sciences
Volume 7, Number 5, May 2013 (Serial Number 61)
Contents
Molecular Biology and Medical Sciences
443 Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
Tirtha Bdr. Katwal, Dorji Wangchuk, Lhap Dorji, Namgay Wangdi and Rinzin Choney
453 Old Drug for New Use: Searching for Mitogen-Activated Protein Kinase Kinase 1 (MEK1) Inhibitor by the Computer Aided Drug Design
Po-Yuan Chen, Hong-Jye Hong, Mien-De Jhuo, Tzu-Ching Shih, Yu-Chi Wu, Chia-Hsing Cheng, Yen-Yu Huang and Tzu-Hurng Cheng
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
Md. Aminul Haque Khan, Mst. Rokshana Rabeya, Muhammad Saiedullah, Rukhsana Parvin, Sohel Ahmed and Md. Rezwanur Rahman
464 Development of in-vitro Susceptibility Testing for Pathogenic Bacteria
Fouad Houssein Kamel, Chiman Hameed Saeed, Ashti M. Amin and Saleem Saaed Qader 468
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic Fungi Causing External Ear and Skin Infections
Maha Akram Al-Rejaboo and Omar Mu’ayad Al-Obaidy 475
External Gastric Balloon in Obesity Treatment
Mesut Basak, H. Erdem Gozden, Gulay Turan, Hayrettin Mutlu and Emine Pakir
Biotechnology and Biological Engineering
483 Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
Hussein S. Taha, Hamida A. Osman, Mahmoud M.M.A. Youssef, Abdel Monem Y. El-Gindi, Hoda H. Ameen and Asmahan M.S. Lashein
Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi
Badireddy Madhusudana Rao, Floyd L. Inman III and Leonard D. Holmes
Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent under Field -Sunlight Conditions, in Egypt
Alexandra El-Helaly, Magda Khattab, Said El-Salamouny, Mohammed El-Sheikh and Salah Elnagar
Botany and Zoology
Study of Behavior Germination and Essays the Removing Tegumentary Inhibition of Seeds of Chamaerops humilis L. var. argentea André (Arecaceae)
Nadjat Médjati, Okkacha Hasnaoui, Nouria Hachemi, Brahim Babali and Mohammed Bouazza
507 Evaluation of Regeneration Stock Alternatives for Optimization of Growth and Survival of Field-Grown Forest Trees
Titus Fondo Ambebe, Lum Ayeoffe Fontem, Balgah Roland Azibo and Njoya Moses Tita Mogho
Microalgal Epibiontic Communities on Some Brachyuran Crabs in Suez Canal, Egypt
Nesreen K. Ibrahim and Abeer S. Amin
527 Comparative Study of Two Methods of Induction of Estrus and Fertility Following Artificial Insemination in Azawak Zebu in Niger
Issa Moumouni, Marichatou Hamani, Semita Carlo, Nervo Tiziana, Yénikoye Alhassane, Cristofori Francesco and Trucchi Gabriella
The Channel Catfish in Georgian Aquaculture
Rezo K. Goradze, Akaki Komakhidze and Irakli Goradze 539
Advocacy for Camel Research and Development in Kenya
Kisa Juma Ngeiywa and James Chomba Njanja
Nutritional Sciences
Lipids Data Composition of Edible Ant Eggs Liometopum apiculatum M. Escamoles
Melo Ruiz Virginia, Sánchez Herrera Karina, Sandoval Trujillo Horacio, Quirino Barreda Tomás and Calvo Carrillo Concepción
Development of Blast Chilling Method for Cooked Meat Dishes
Martins Rucins, Viesturs Rozenbergs and Imants Skrupskis
May 2013, Vol. 7, No. 5, pp. 443-452
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
Tirtha Bdr. Katwal 1 , Dorji Wangchuk 1 , Lhap Dorji 1 , Namgay Wangdi 1 and Rinzin Choney 2 1. Renewable Natural Resources Research and Development Centre, Wengkhar, Mongar, Bhutan 2. Department of Agriculture, Ministry of Agriculture and Forests, Thimphu, Bhutan
Received: March 26, 2013 / Accepted: May 07, 2013 / Published: May 30, 2013.
Abstract: Bhutan is a small landlocked country located in the eastern Himalayas. Over 69% of the population is engaged in agriculture. Rice, maize, wheat, barley, buckwheat and millets are the major cereal crops cultivated. Rice is the most preferred food crop of the Bhutanese. Maize is a primary food crop after rice and it ranks first among food crops in production. The cultivation ranges from less than 300 m asl (metres above sea level) nearly up to 2,800 m asl. In 2007, a new, extremely serious problem of GLS (gray leaf spot) in maize that was previously never reported in Bhutan was confirmed. This disease spread rapidly in the highland maize growing areas causing production losses of over 50% to 70%. All the maize varieties cultivated in the country were found to
be highly susceptible to the disease. In order to contain this devastating disease, the national maize program drew short and long term strategies with the help of a CIMMYT Expert. As an immediate short term action to contain GLS, systemic fungicide Tilt 25 EC (active ingredient propiconazole) was supplied free of cost to the farmers. A longer term strategy pursued was the introduction, evaluation and selection of GLS tolerant genotypes for the highland ecosystem. Over 100 GLS tolerant genotypes were introduced from CIMMYT Colombia, Mexico, Zimbabwe and Nepal. These materials were initially evaluated in a disease hotspot sites and then further tested in multi-location trials in GLS affected areas across the country. Farmers were engaged for Participatory Variety Selection by organizing farmer’s field days at the trial sites. Finally, in 2011 considering the need of GLS tolerant varieties for farmers, two GLS tolerant genotypes ICAV305 and S03TLYQAB05 were provisionally released. In the 2011 season, these two provisionally released genotypes were put under large scale demonstration in the GLS affected areas in nine districts across the country. In 2012, the two genotypes were formally released by the Technology Release Committee of the Ministry of Agriculture and Forest. Rapid seed increase of the new varieties was initiated through farmers from Community Based Seed Production groups and so far 75% seed replacement of GLS affected farmers has been accomplished.
Key words: Gray leaf spot, hotspot, yield loss, participatory variety selection, community based seed production and seed replacement.
1. Introduction 92°10 ′E. The country has a total geographical area of
38,394 km 2 with a population of 745,600 people [1]. Bhutan is a small landlocked mountainous country The forest (tree) cover of the country is about 70.46%, located in the southern slopes of eastern Himalayas. It arable land 2.93%, meadow land 4.10%, shrub land is sandwiched between the two great Asian 10.43%, snow cover land 7.44% and bare areas 3.20% civilizations, China to the north and India in the east, of the total geographic area [2]. Agriculture is the west, and south. The country lies between latitudes mainstay of the people with an estimated 69% of the 26°45 ′N and 28°10′N, and longitudes 88°45′E and population engaged in farming. Rice, maize, wheat,
barley, buckwheat and millets are the major cereal Corresponding author: Tirtha Bdr. Katwal, M.Sc., research fields: tropical agriculture development, crop production.
crops cultivated in Bhutan and rice is by far the most E-mail: tirthakatwal@gmail.com.
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
important and preferred food crop of the Bhutanese. attained epidemic scale in 2007. It affected 4,193 Maize (Zea mays L.) is a major food crop cultivated
maize growing households. The total area affected by 69% of the rural households for subsistence. The
was 4,821.89 acres and the total production loss due to total area under maize in 2010 was 61,476 acres; the
the disease was 6,504.12 Mt [3]. The estimated total production was 57,666 Mt with a national
production loss of the affected farmers ranged from 50% average yield of 2.38 t·ha -1 [3]. Maize ranks first in the
to 70% [3]. In Zambia, when similar GLS epidemic extent of area cultivated amongst the food crops.
occurred in the mid 1990s, the average yielded losses Maize cultivation in the country ranges from less than
ranged from 28% to 54% with an average of 33.5% 300 m to nearly up to 2,800 m asl owing to its
[9]. The impact of this disease, farmers crop versatile capacity to adapt to different environments. It
husbandry practices, disease management strategies plays a critical role in ensuring the household food
and the disease reaction on the GLS resistant security. It is estimated that 80% of the total
genotypes introduced from CIMMYT Colombia are production is consumed at the household level by the
discussed in this paper.
farmers which is valued at Nu. 353 Million (1 USD =
2. Materials and Methods
54 Ngultrum (Nu.)) annually [4]. About 6% of the total production is sold which is an important source
An initial report of premature drying of maize was of household income. The rest of the production is
received from 12 maize growing districts in 2006. used as seed, processed into different products and fed
Such reports mostly came from maize growing areas to the livestock. The maize production environment in
above 1,500 m asl. Accordingly, field visits and rapid the country is broadly categorized into three zones
surveys were conducted in the affected areas by the mainly based on the altitude. The three production
researchers from the NPPC (National Plant Protection zones are: sub-tropical maize production zone I (<
Centre) and the RNR RDCs (Renewable Natural 1,200 m asl) or low altitudes; sub-tropical maize
Resources Research and Development Centers). The production zone II (1,200-1,800 m asl) or mid
crop was in the late maturity stage and most of the altitudes; and the highland maize production zone (>
lower leaves had completely dried. Some lesions of 1,800 m asl) [5]. These different production zones
TLB were observed in the few upper leaves which vary widely in their production potentials and
were partially green. TLB was suspected to have constraints.
caused the damage. To assist the maize program in Bhutanese maize farmers in the Highland and
further confirming this problem, technical assistance Sub-tropical Zone II are facing a new, extremely
of CIMMYT (International Maize and Wheat Center), serious problem of GLS (Gray Leaf Spot), fungal
South Asia Regional Office, Kathmandu was sought. disease which was previously never reported in the
The CIMMYT office immediately recommended the country [6]. According to [7], the incidences of GLS
input of Dr. Carlos De Leon, a former plant has increased during past two decades and today it is
pathologist of CIMMYT for two weeks. The technical one of the greatest threats to global maize production
expert made extensive field visits in the major disease [8]. In Bhutan, GLS and TLB (Turcicum leaf blight)
affected areas to determine the level of disease have become economically important diseases incidences and identify the disease. Informal especially in areas above 1,200 m asl. Serious
discussions were held with the affected farmers to outbreaks of these diseases were reported from 12
understand their field and crop management practices. major maize growing districts in 2006. GLS spread
Infected leaf samples were collected for laboratory rapidly in the highland maize growing areas and
investigation. The collected samples were incubated
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
and observed under the microscope and finally GLS four entries had enough seed and one which was a and TLB were confirmed in 2007. GLS was observed
hybrid was dropped from seed increase. To screen the to be more serious and damaging as compared to TLB.
introduced materials under disease pressure, a disease Immediately after the confirmation of the disease, the
hotspot site at Chaskar (1,960 m asl), in Mongar expert recommended an immediate and a long term
district where the disease occurs naturally and in strategy for the management of the disease abundance due to continuous cropping of maize was considering the serious impact of the disease on the
identified. Verma [9] reported that new materials were household food security of the maize dependent
screened for tolerance to GLS in the Zambian Seed farmers. All the traditional maize varieties including
Company’s farm at Lusaka that had turned into a good the most popular improved variety Yangtsipa (Suwan
“hotspot” for GLS due to continuous cropping of GLS
1) were found to be highly susceptible to GLS. susceptible maize varieties. The screening and The immediate and short term strategy adopted was
evaluation for tolerance to GLS in Bhutan started from the spray of systemic fungicide Tilt 25 EC (active
March 2008. To facilitate the screening of the ingredient propiconazole). The longer term and a more
materials, two acres of land was leased at Chaskar sustainable strategy was the introduction, evaluation
from a farmer by the maize research program. After and selection of GLS tolerant maize genotypes as the
initial evaluation, promising materials were promoted majority of Bhutanese farmers cannot effort expensive
to the nationally coordinated trials for evaluation in fungicides. Moreover, repeated use of fungicides are
multi-location trials across the country through the not feasible both economically and environmentally
Regional Research Centers. At the trial sites, farmer’s [10] besides physical constraints due to steep terrain
field days were organized to engage farmers for PVS slopes more than 50%. The use of genetic resistance
(Participatory Variety Selection). As GLS was new to has been noted as the most sustainable means to
the country, on the job training were provided to the prevent maize production losses from GLS in Africa,
researchers and extension staff in monitoring the especially for subsistence farmers who cannot afford
disease. In the trial sites, GLS was constantly to purchase expensive fungicides [11].
monitored by the researchers. At the disease hotspot To initiate the development and selection of GLS
trial site in Chaskar, the introduced materials were tolerant genotypes for the country, GLS tolerant
planted in the third week of March and harvested in germplasm were requested from CIMMYT late September. GLS was scored based on the scale of International. Accordingly in 2007, 45 GLS tolerant
1-5 where 1 = no lesions visible, 2 = few lesion seen genotypes from CIMMYT Colombia, 23 from Mexico,
on two lower leaves, 3 = lesions visible on most eight from Zimbabwe and six from the National Maize
leaves below the ear, 4 = many lesions visible on Program of Nepal were introduced to the country. As
leaves above the ear and 5 = all leaves dead. All other the quantity of seeds was small, the first step was the
agronomic parameters were recorded at the time of off season increase of the seed at Lingmethang
harvest. Existing improved and released variety research farm (640 m asl) through controlled Yangtsipa and a local variety from Chaskar were used pollination. The first batch of materials to arrive in the
as the check varieties in the trial site. country was 45 entries from CIMMYT Colombia.
3. Results and Discussions
From the 45 entries, 39 entries were planted at Lingmethang Research Sub Centre (640 m asl) in 3.1 Disease Confirmation and Conditions That
Favoured Disease Development
October 2007 as winter nurseries for seed increase through controlled pollination. From the 45 entries,
The observation of symptoms in the field and the
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
subsequent observation of spores under a simple Majority of Bhutanese farmers use locally made microscope confirmed the disease to be GLS (Fig. 1)
ploughs drawn by the bullocks which do not penetrate caused by the fungus Cercospora zeae-maydis (Tehon
very deep in the soil. The plough does not adequately and Daniels 1925). Another fungal disease TLB
incorporate the crop residues deep in the soil. Farmers caused by Exserohilium turcicum (Synonymous;
in the highlands (> 1,800 m asl) normally practice Helminthosporium turcicum ) locally known as continuous cultivation of maize without crop rotation Songsongma was also confirmed. TLB was present in
due to the limited land holdings, short growing season the country, but serious infection was noticed only
and lack of other suitable optional crops. As a result of after 2006. GLS was previously never reported in the
the disease, the maize production has substantially country and was confirmed for the first time. Severe
declined from 2005 (Fig. 2).
incidences of GLS were observed at elevations nearly All these cropping practices like continuous and above 1,500 m asl. At these elevations, GLS was
mono-cropping of maize [12-14] and use of minimum found to be causing the most damaging effects with
or conservation tillage practices [12, 15, 16] have been some farmers losing almost 100% of their crop [6]. At
found to increase the incidence of the disease. In the elevations 1,500-1,800 m asl, both GLS and TLB
were prevalent. Below 1,700 m asl, TLB was more prevalent but with incidences of lesser economic significance. GLS symptoms were also seen at 600 m asl, however, the impact was much lesser as compared to areas above 1,500 m asl.
The cause for outbreak of these two diseases in Bhutan is attributable to the farming practices which are congenial for the development of the diseases. The predominant farming practices adopted by the maize growers in the country are: (1) mono-cropping of maize with occasional rotation with potato; (2) use of
maize stalks and crop residues as livestock bedding,
Fig. 1 GLS conidia as observed under simple microscope,
and subsequently as FYM (farm yard manure)—a
major source of nutrients for the crop; (3) reduced or minimum tillage due to steep terrain. 93.97 100.00
Apparently, the main source of inoculum for GLS 90.00
tonnes) 80.00 71.06
was the infected crop residues especially the leaves
and the leaf sheath left on the soil surface. Bhutanese
metric
farmers in the highlands practice the continuous
cultivation of maize, use maize stover for feeding the 40.00
cattle as animal bedding and also collect and heap the
stover in the field to be spread in the field at the time
Production (Mt,
of planting. The leftover residues from the feeding
2005 2006 2007 2008 2009 2010 stalls go into the compost yard which is later spread in Year
the maize field. These practices seemed to greatly favor the development of the disease.
Fig. 2 Maize production trend in Bhutan [3].
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
Uganda, Africa where maize is one of the main crops, from Zimbabwe and six from the National Maize leaving the disease infected stover in the soil surface
Program of Nepal were the primary source for and planting of susceptible variety were identified as
adapting and releasing GLS tolerant genotypes. In the key factors responsible for the perpetuating of
2008 season, most of the genotypes appeared to be GLS [17]. Further, in most maize growing areas of the
promising with reasonably good level of field country, cloudy weather with high humidity and
tolerance to GLS and TLB. In the first season, the extended period of wetness prevails with the onset of
main selection parameter was the tolerance to GLS monsoon from late May till September, which favours
and TLB and not too much emphasis was given to the growth and development of the pathogens [18].
other agronomic traits. Among all the genotypes, the genotypes from CIMMYT Colombia were most stable
3.2 Immediate Management of the Disease and exhibited better tolerance to GLS while genotypes
Since the diseases had a serious threat on the from CIMMYT Mexico appeared to be inbred lines household food security of the farmers, spraying of
with poor plant type. All the materials from CIMMYT systemic fungicide Tilt (active ingredient Zimbabwe were white maize which is less preferred propiconazole) was recommended as an immediate
by farmers and the ones from Nepal were all measure. Tilt 25 EC (active ingredient propiconazole)
susceptible to GLS. From the 45 genotypes from was imported from India and was recommended to be
CIMMYT Colombia, 15 genotypes which received an sprayed at 2 mL per litre of water with minimum of
average GLS and TLB score of less than 2.5 [19] were one spray, two weeks before flowering. The DoA
selected for further evaluation (Table 1). (Department of Agriculture) with fund support from
In 2009 maize season, the 15 selected entries were the EU-ASSP (EU-supported Agriculture Sector evaluated in five different locations above 1,500 m asl Support Project) supplied 1,650 L of fungicide Tilt
where GLS occurs in abundance. The results indicated (propaconizole) worth Nu. 1.65 Million the affected
significant difference on GLS and TBL incidences and farmers free of cost. However, spraying was yield among the different genotypes evaluated (Table cumbersome and ineffective due to steep terrain where
2). The results also showed that there was no large water had to be fetched from downhill, lack of
difference on disease tolerance and yield among the appropriate spraying machines and accessibility of far
different genotypes as compared to the check varieties. flung maize fields.
This is mainly because the introduced genotypes are new to the highland maize ecosystem and are still
3.3 Introduction, Evaluation and Selection of Disease undergoing adaptation whereas the check varieties are Tolerant Varieties well adapted to the highland ecosystem.
The use of genetic resistance has been noted as the To engage farmers and extension as active research most sustainable means to prevent maize production
partners in evaluating and selecting suitable GLS losses from GLS, especially for subsistence farmers
tolerant genotypes, farmer’s field was organized for who cannot afford to purchase expensive fungicides
the PVS (participatory variety selection) at the disease [11]. Currently, there are no GLS tolerant varieties
hotspot trial site in Chaskar. A total of 34 farmers and released for the highland maize growing areas in the
extension staff who participated in the PVS ranked country and therefore, the introduction, evaluation and
Cap. Miranda 99Bact1F-1 (Entry No. 1) as their first selection of GLS tolerant maize genotypes were given
choice, S03TLYQAB05 (Entry No. 35) as second, the highest priority. The 45 GLS tolerant genotypes
GLSIY01/SPMAT (Entry No. 6) as third and from CIMMYT Colombia, 23 from Mexico, eight
Villavicencio 03Asp1C (LET-EARLY) (Entry No. 21)
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
Table 1 The 15 promising GLS tolerant genotypes selected from CIMMYT Colombia materials, 2008.
Si No. Entries No.
Pedigree
GLS score*
TLB score*
Yield t/ha
1 1 Cap. Miranda 99Bact1F-1
2 13 Menegua 01 Phaeo
32 Granada 01Phaeo1AS2
Villavicencio 03Phaeo1A(SA4)
S03TLYQ AB05
66 GLSIY01/SPMAT
ICA V305
Villavicencio 03Asp1C(LET-EARLY) 2.5 1.3 7.58
Cimcali 03HCG1A
10 3 Turipana 01DMR 1D(1)
GLSIY01HG"A"
Granada 03Poly1A(SA4)
Villavicencio 03Asp1C(QPM)
Villavicencio 03Phaeo1A(Elites)
ACROSS S9624-1
2.0 2.0 4.05 16 Yangtsipa (Improved Check) 3.1 4.0 4.74
*: disease score based on scale of 1-5, where: 1 = No lesions are visible; 2 = Few lesion seen on two lower leaves; 3 = Lesions visible on most leaves below the ear; 4 = Many Lesions visible on leaves above the ear; 5 = All Leaves dead.
Table 2 GLS, TLB and yield of 15 selected genotypes from CIMMYT Colombia at five different locations, 2009.
Mean of 5 locations
Entry Pedigree
GLS* TLB* Yield (t·ha -1 )
1 Cap. Miranda 99Bact1F-1
2 Granada 01Phaeo1AS2
3 Turipana 01DMR 1D(1)
1.9 1.7 5.4 5 GLSIY01HG"A" 2.0 1.6 4.16 6 GLSIY01/SPMAT 1.9 1.7 4.76
9 ACROSS S9624-1
13 Menegua 01 Phaeo
15 Granada 03Poly1A(SA4)
17 Villavicencio 03Asp1C(QPM)
21 Villavicencio 03Asp1C(LET-EARLY)
23 Villavicencio 03Phaeo1A(SA4)
25 Villavicencio 03Phaeo1A(Elites)
33 Cimcali 03HCG1A
35 S03TLYQ AB05
38 ICA V305
1.9 1.9 5.35 Yangtsipa 2.0 1.7 5.57 Local 2.2 1.8 5.45
Location (L)
Entry (E)
S.E.D
as fourth choice. Farmer’s selection criteria included
flint gains and size of the cobs. Based on the results
plant height, husk cover, tolerance to GLS, yellow
and farmer’s preferences, three genotypes namely
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
Entry No. 1, 35 and 21 were selected for further much belter as compared to the check varieties. The evaluation. The genotypes Entry No. 6 which was
results from multi-location trial of four selected preferred by the farmers was dropped due to less
genotypes in four locations in 2010 revealed that new quantity of seed. One additional genotypes, ICA
genotypes showed much higher tolerance to GLS as V305 (Entry No. 38) that was not selected by the
compared to the local check variety (Table 3). There farmers during the PVS, was included for further
was significant difference in yield and the highest evaluation based on the advice of the CIMMYT -1 yield of 5.33 t·ha was recorded for Yangtsipa, the
experts. This genotype ICA V305 is a stable variety improved check variety. Among the new genotypes, released in Colombia and one of the parents is Suwan -1 the highest yield of 5.27 t·ha was recorded for Cap.
1 (Yangtsipa) which is a popular variety in Bhutan. Miranda 99Bact1F-1 (Entry No. 1) (Table 3). This All the selected genotypes showed higher tolerance to
genotype has semi dent grains and higher percentage GLS than the check varieties (Table 2).
of open husk which is less preferred by the farmers. In 2009 season, a very close monitoring of GLS
The mean data of three years indicated that this was done for the four selected genotypes and the two
genotypes had high incidence of Turcicum Leaf blight check varieties. The GLS incidence starts by the last
and yield was comparable to that of genotypes week of July and reaches the peak in the last week of
S03TLYQ AB05 (Entry No. 35) and ICAV305 (Entry August. All the four new genotypes including the
No. 38) and therefore was not considered for check varieties were infected by GLS, however, the
immediate release (Tables 4 and 5). Although the incidences of GLS was much higher in the two check
yield of ICA V305 (Entry No. 38) is lower than that of varieties right from the initial stage and continued to
the check varieties, it had better tolerance to GLS and increase until all the leaves were dead by end August
good husk cover. Due to the urgency for GLS tolerant (Fig. 3).
varieties for maize growing areas above 1,500 m asl, This strongly indicates that in the event of an early
the two genotypes S03TLYQ AB05 (Entry No. 35) incidence of GLS, the new genotypes can tolerate
and ICAV305 (Entry No. 38) were selected for large
Entry # 33 Entry # 35
2.0 Entry # 38 GLS Score (1-5)
Yangtsipa Local
Assessment Time (Days after planting)
Fig. 3 GLS progression at Chaskhar, 2009.
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
Table 3 Mean GLS, TLB score and yield t/ha for 2010 season (mean of four locations). Entry No.
Pedigree
GLS, score*
TLB, score
Yield (t·ha -1 )
1 Cap. Miranda 99Bact1F-1
21 Villavicencio 03Asp1C(LET-EARLY)
35 S03TLYQ AB05
38 ICA V305
Yangtsipa 2.0 2.2 5.33 Local 2.5 2.4 3.96
Table 4 Other agronomic traits of selected varieties, 2010 season.
Average plant height Average ear height Husk cover**
Ear aspect ***
Entry No. Pedigree
1 Cap. Miranda 99Bact1F-1
21 Villavicencio 03Asp1C(LET-EARLY) 222.8
35 S03TLYQ AB05
38 ICA V305
** Husk cover based on scale of 1-5 where 1 = best (fully covered) and 5 = open husk; *** Ear aspect based on scale of 1-5 where 1 = best quality with uniform ear size and good grain filling 5 = poor quality.
Table 5 Mean GLS, TLB and yield of four selected genotypes.
Mean of three years
Entry No. Pedigree
GLS TLB Yield (t·ha -1 )
1 Cap. Miranda 99Bact1F-1
21 Villavicencio 03Asp1C(LET-EARLY) 2.2 2.1 3.80 35 S03TLYQ AB05
38 ICA V305
scale demonstration and provisional release in 2011
released genotypes were put under large scale
season considering the mean performances of three
demonstration in the GLS affected areas in nine
years (Table 4). Although the yield of ICA V305
districts across the country. The mean yield reported
(Entry No. 38) is lower than that of the check varieties,
from the large scale demonstrations for the two
it had better tolerance to GLS and good husk cover
genotypes S03TLYQ AB05 (Entry No. 35) and
(Tables 4 and 5).
ICAV305 (Entry No. 38) were 3.73 t·ha -1 and 4.43
In 2011, the TRC (Technology Release Committee) -1 t·ha , respectively. In order to facilitate the rapid seed of the MOAF (Ministry of Agriculture and Forest) in
replacement of the GLS affected farmers, seed
its 15th meeting endorsed the provisional release of
production was done using the concept of CBSP
the two GLS tolerant genotypes S03TLYQ AB05
(Community Based Seed Producers) group [20] and
(Entry No. 35) and ICAV305 (Entry No. 38). The
nine CBSP groups have been formed in five districts
urgency of GLS tolerant variety was one important
for seed multiplication of the two new GLS tolerant
consideration for the release.
genotypes. Finally, in 2012 based on the overall
Further in the 2011 season, the two provisionally
performance in 2011 season and general acceptability
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the Highland Maize Production Eco-systems of Bhutan
of the farmers, the two provisionally released genotypes were proposed for final release to the TRC of the MoAF. The TRC finally endorsed the release of the two genotypes in April 2012. After the release the two genotypes were given local names as Chaskarpa for genotype ICAV305 (Entry No. 38) and Shafangma Ashom for genotype S03TLYQ AB05 (Entry No. 35).
The main justification for the release were that GLS epidemics has gained the status of a national emergency as all the available maize varieties were found to be highly susceptible to the disease. The two new genotypes have shown much higher field tolerance to GLS as compared to the existing varieties. Further, in the event of early outbreak of GLS, the two new genotypes will perform much better in terms of disease tolerance and production. Both are open pollinated varieties for which seed production is easy and the two genotypes have a higher yield potential and have shown comparable yields with the check varieties despite the fact that they are relatively new to the highland ecosystem and are undergoing adaptation. Both have yellow flint grains, and Shafangma Ashom S03TLYQ AB05 (Entry No. 35) is a QPM (Quality Protein Maize) which is more nutritious than the traditional maize varieties. It is the first QPM maize variety released in the country and will immensely contribute to the nutritional requirement of the maize farmers particularly that of the children [21].
The two new varieties are recommended for the maize production zone II (1,200-1,800 m asl) and Highland maize production Zone (> 1,800 m asl) where the two disease GLS and TLB have severely affected maize production. Both the varieties also perform well in Sub-tropical maize production zone I (< 1,200 m asl) or low altitudes. By 2013 season, the maize program has accomplished around 75% seed replacement of the GLS affected farmers with the two new varieties mostly with the supports of DRDP-WB, DoA MoAF (World Bank through the Decentralized Rural Development Project).
4. Conclusion
The high altitude farmers in Bhutan are facing the serious problem of GLS and TLB. Although, use of fungicide Tilt 25 EC seems to be quite effective, yet the use of chemicals will not be sustainable. The small and subsistence Bhutanese farmers cannot afford expensive fungicides, besides the difficulty to spray manually in steep slopes where most of the maize is grown and the detrimental impact of continuous use of fungicides on environment does not make the use of fungicide a suitable disease management option. The release of two GLS and TLB tolerant maize varieties has come as a big respite for the Bhutanese maize famers particularly those above 1,500 m asl. With rapid seed increase through the CBSP groups, 75% seed replacement of the affected farmers with the two GLS tolerant varieties has been accomplished by 2013 planting season. The seed increase and replacement of the affected farmers is rigorously being pursued in collaboration with the National Seed Center and the district extension services.
Acknowledgments
The National Maize Program of the Department of Agriculture, Ministry of Agriculture and Forests, Bhutan acknowledges the support of CIMMYT International for providing the valuable maize germplasm and technical guidance; the SDC (Swiss Intercooperation) Helvetas funded RNR (Renewable Natural Resources) Research Systems Project for the financial support in technical capacity building; the EU-ASSP/DoA/MoAF Bhutan for research funds; the Biodiversity Use and Conservation Asia Program (BUCAP-NBC/MoAF/Bhutan) for financial support to undertake PVS and capacity development and the Royal Government of Bhutan for supporting the maize research program. The Decentralized Rural Development Project (DRDP-World Bank) of the DoA, MoAF has supported the seed production and replacement through the promotion of Community Based Seed Production Programs. It has immensely
Evaluation of Gray Leaf Spot Tolerant Genotypes from CIMMYT in the
Highland Maize Production Eco-systems of Bhutan
contributed in improving the seed production system [10] F.M. Latterell, A.E. Rossi, Gray leaf spot of corn: A disease on the move, Plant Disease 67 (1983) 842-847.
and technical capacity building of researchers, [11] A. Menkir, M. Ayodele, Genetic analysis of resistance to
extension and farmers. We also acknowledge the gray leaf spot of midaltitude maize inbred lines, Crop Sci. financial support of CIMMYT International and the
45 (2005) 163-170.
SLMP (Sustainable Land Management Project)-NSSC [12] P.M. Beckman, G.A. Payne, External growth, penetration, and development of Cercospora zeae-maydis in corn
(National Soil Services Center) for supporting the leaves, Phytopathology 72 (1982) 810-815.
participation of maize researchers in the 11th Asian [13] J.C. Rupe, M.R. Siegel, J.R. Hartman, Influence of Maize Conference at Nanning, China, where an
environment and plant maturity on gray leaf spot of corn abstract of this paper was presented as a poster on
caused by Cerscopsora zeae-maydis, Phytopathology 72 (1982) 1587-1591.
GLS management. [14] S.I. Harlapur, Epidemiology and management of
References Turcicum leaf blight of maize caused by Exserohilum
turcicium (Pass.) Leonard and Suggs, Ph.D., University [1] MOIC, Bhutan Portal [Online], available:
of Agricultural Sciences, Dharward, India, 2005. http://www.bhutan.gov.bt/government/abt_geography.ph
[15] C.W. Roane, Observations on gray leaf spot of maize in p (accessed Jan. 15, 2013).
Virginia, Plant Disease 58 (1974) 456-459. [2] LCAR, Land cover assessment report, in: Agriculture,
[16] N.R.X. de Nazareno, P.E. Lipps, L.V. Madden, Survival The National Soil Service Center and the Policy and
of Cercospora zeae-maydis in corn residues in Ohio, Planning Division, Thimphu, Bhutan, 2010.
Plant Disease 76 (1992) 560-563. [3] DOA, Agriculture Statistics, in: Agriculture, Thimphu,
G. Bigirwa, R.C. Pratt, P.E. Lipps, E. Adipala, Farming Bhutan, 2010.
components for gary leaf spot disease severity in districts [4] T.B. Katwal, P. Dem, G.B. Chhetri, L. Bockel, M.
of contrasting incidence, in: Seventh Eastern and South Punjabi, Maize commodity chain analysis, A working
Africa Regional Maize Conference, 2001. document, Department of Agriculture, Ministry of
[18] P.A. Paul, G.P. Munkvold, Influence of temperature and Agriculture, Thimphu, Bhutan, 2006.
relative humidity on sporulation of Cercospora [5] S. Shrestha, T.B. Katwal, B.B. Ghalley, Adoption and
zeae-maydis and expansion of gray leaf spot lesions on Impact Assessment of Improved Maize Technologies in
maize leaves, Plant Disease 89 (2005) 624-630. Bhutan, Council of RNR Research of Bhutan, RNR RDC
[19] T.B. Katwal, D. Wangchuk, N.B. Adhikari, N. Wangdi, S. Wengkhar, 2006.
Wangdi, P.B. Biswa, First year report on maize breeding [6]
C. de Leon, Report of activities and recommendations on and selection for tolerance to gray leaf spot and turcicum a consultancy to Bhutan, August 12-26th, 2007, RNR
leaf blight diseases: A working document, RNR RDC Research Centre, Bajo, Wangdiphodrang, Council of
Wengkhar, Field Crops Sector, Mongar, 2009. RNR Research of Bhutan, Bhutan, 2007.
[20] T.P. Tiwari, G. Ortiz-Ferrara, C. Urrea, R.B. Katuwal, [7] J. Wang, M. Levy, L.D. Dunkle, Sibling species of
K.B. Koirala, R.C. Prasad, et al., Rapid gains in yield and Cercospora associated with gray leaf spot of maize,
adoption of new maize varieties for complex hillside Phytopathology 88 (1998) 1269-1275.
environments through farmer participation. II. Scaling-up [8] J.M.J. Ward, E.L. Stromberg, D.C. Nowell, F.W. Nutter,
the adoption through community-based seed production Gray leaf spot: A disease of global importance in maize
(CBSP), Field Crops Research 111 (2009) 144-151. production, Plant Disease 83 (1999) 884-895.
[21] K. Mbuya, K.K. Nkongolo, A. Kalonji-Mbuyi, [9] B.N. Verma, Gray leaf spot disease of maize-loss
Nutritional analysis of quality protein maize varieties assessment, genetic studies and breeding for resistance in
selected for agronomic characteristics in a breeding Zambia, in: 7th Eastern and Southern Africa Regional
program, International Journal of Plant Breeding & Maize Conference, 2001.
Genetics 5 (2011) 317-327.
May 2013, Vol. 7, No. 5, pp. 453-458
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Old Drug for New Use: Searching for MEK1 (Mitogen- Activated Protein Kinase Kinase 1) Inhibitor by the Computer Aided Drug Design
Po-Yuan Chen 1 , Hong-Jye Hong 2 , Mien-De Jhuo 1 , Tzu-Ching Shih 3 , Yu-Chi Wu 1 , Chia-Hsing Cheng 1 , Yen-Yu Huang 1 and Tzu-Hurng Cheng 1
1. Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan 2. School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan 3. Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung 404, Taiwan
Received: November 27, 2012 / Accepted: January 24, 2013 / Published: May 30, 2013.
Abstract: An old drug with a new use can significantly reduce the cost and time for new drug research and development. MAPK (Mitogen-activated protein kinase) plays a very important key role in signal transduction pathways of cell proliferation and
differentiation. According to the statistics, there are about 30% persons who suffered from cancers related to the MAPK signal transduction pathways. Therefore, many researchers are focused on blocking these pathways in cancers therapies. Ras/Raf/MEK/ERK, however, is one of very important pathways among MAPK message transduction pathways. More and more information about MEK protein inhibitors are unveiled in several recent years. In the present study, the authors utilized MEK inhibitors which were already published and their activities were available to construct 2D-QSAR model by using CADD (multiple linear regression). Then, the authors searched certified FDA drugs (Drugs@FDA 6184 drugs) making preliminary screening. The secondary screening on 3D structures were followed by using Docking, Scoring and Pharmacophore analysis to find out most suitable MEK inhibitors to become a fundamental database in drug discovery. The results are shown the ALogP, number of aromatic rings, number of hydrogen bond acceptors and number of hydrogen bond donors are all in positive correlation. According to the equation from 2D-QSAR model, the results conform to the previous description.
Key words: MEK (Mitogen-activated protein kinase kinase), MAPK (mitogen-activated protein kinase), pharmacophore, QSAR (quantitative structure-activity relationship), PHP (hypertext preprocessor).
Abbreviations
the protein activation, suppression all together with MAPK—Mitogen-activated protein kinase
cell behavior and surviving. Ras/Raf/MEK/ERK MEK—Mitogen-activated protein kinase kinase
MAPK (Mitogen-activated protein kinase) is the most ERK—Extracellular signal-regulated kinase
crucial pathway that controls cell proliferation and QSAR—Quantitative structure-activity relationship
differentiation among all message transduction CADD—Computer aided drug design
FDA—Food and drug administration pathways [1, 2]. It is inevitable to think of tumor
PHP—Hypertext preprocessor while mentioning the function of MAPK that can
control cell proliferation. Thus, many related
1. Introduction
researches even point out that there are about 30% The message transduction routes often influence the
persons who suffered from cancers that are related to downstream gene expression inside cells thus affect
the MAPK signal transduction pathways [3] which includes pancreatic cancer, colon cancer and lung
Corresponding author: Po-Yuan Chen, Ph.D., assistant professor, research field: bioinformatics. E-mail: cancer [4]. pychen@mail.cmu.edu.tw.
Old Drug New Use
In recent news, an old drug with new use can dramatically reduce the cost and time for new drug development. A new drug released on market in America needs 15 years and 0.8 billion USD in average and the average number of drugs that can pass the FDA certification to release on market is only around 30. But the use of old drug for new use only need two years and 17,000,000 USD, compared with new drug developing, the cost is extremely low. Nobel Prize winner James Black said: “start from old drug to develop new is the most effective way”.
The main purpose of this study is to search for possible MAPK message transduction pathway inhibitor with fast and effective way for cancer therapy. MEK phosphorylation can enhance ERK
phosphorylation in MAPK message transduction
Fig. 1 MAPK message transduction pathway chart. MEK
pathway. While MEK stop expressing, ERK is also
will not activate downstream protein ERK and ERK could stopped to express, and the downstream gene not further express the downstream gene if MEK inhibitor
suppresses MEK expression thus achieve the inhibit effect.
expressions are also suppressed (Fig. 1) [2, 3].
Additionally, MEK inhibitor PD184352 already had clinical trial in 1999 [5] and great amounts of research data have been accumulated during recent year. Thus, the use of known drug activity statistical data to test new unknown drug (old drug new use) is applicable.
The main series of transduction pathway among all MAPK message transduction pathways compose of three: (1) the c-jun kinase pathway which regulates many transcription factors; (2) the p-38 pathway which manly activates inflammatory response; (3) the most important pathway is the Ras/Raf/MEK/ERK
pathway which influences cell proliferation, Fig. 2 Famous MEK inhibitor recently and PD184352
differentiation, living and apoptosis. The MEK protein analogues: (a) PD184352; (b) PD318088; (c) PD98059;
(d) U0126.
plays a critical role in the pathway: firstly, the MEK will be affected by the upstream Raf and and MEK2 in 1999. The main function of this drug is phosphorylate at serine, and then induce the to make a non-competitive effect between MEK and downstream ERK to phosphorylate at specific tyrosine
MgATP. The drug is also proved to have the function to complete message transduction. MEK contains two
of inhibiting cell proliferation and already had clinical kinds of protein MEK1 and MEK2 which have 79%
trail. MgATP and MEK combination can similarities. Both of them can activate ERK [2, 3, 6].
phosphorylate Ser298 and Tyr300 to further activate MEK inhibitor, PD184352 (a.k.a. CI-1040) downstream ERK protein [7-9]. The PD184352
(Fig. 2a), already has very high selectivity over EMK1 analogues will penetrate deeply into MEK to produce
Old Drug New Use
Van der Waal force between the hydrophobic area of Met143, Ile141, Leu118 and Phe209, electrostatic force on the hydrogen atom on Vall27 and finally the H-bonding on Ser212 then block the MEK phosphorylation by ATP (Fig. 3) [3].
More analogous medicines (PD318088 (Fig. 2b)) based on the same backbone structures were developed for activity tests in order to study more about the related mechanism between PD184352 and MEK [3]. Recently, there are more and more medicine that have been synthesized for MEK like PD98059 (Fig. 2c) (MEK1 Inhibitor, Cell Signaling Technology), U0126 (Fig. 2d), and etc.. This kind of
medicine used the same mechanism as PD184352 to
Fig. 3 Illustration of 3D structure of amino acids
block MEK. neighboring PD318088 and MEK protein (PDBid:1S9J).
Green: MgATP; Blue arrow: entrance of MEK protein for
2. Materials and Methods PD318088; Purple: fluorine; Red: oxygen.
PubMed from other researcher is applicable for next This research used 2D-QSAR (2D-quantitative step. There are some limitations for using these structure-activity relationships) as reference for drug materials and keeping the literature from sample screening. 2D-QSAR is a drug design method to build experiment, same author, same method etc., and to up the model of relationship between chemical
reduce error. structure and activity (pIC 50 ). The theory is to take the
The drugs are divided into two groups: overall structure of known active molecular as
The purpose of first group is to build up 2D-QSAR parameters and input the test value for regression
model and take it as Training Set. The drug
parameters such as molecular weight, H-bond donor, the drug activity. Once the real activity is in linear
analysis (this research topic: active pIC 50 ) to estimate
H-bond acceptor, AlogP and molecular solubility will relation with estimate activity and with acceptable
be calculated with multiple linear regressions for correlation, the result can be applied on unknown
linear regression curves before the model is built up. active molecule to predict the real activity. Computer
The purpose of second group is to build up calculations with rapid in speed and great amounts of
2D-QSAR efficacy and take it as Test Set. The molecules are the merits for this method. Reversely,
2D-QSAR needs to be reconstructed if the predicted the predicted results are only data after calculation that
activity and real activity have enormous difference (or needed further verification.
with non-linear relation). Those parameters involve in Refer to Fig. 4 for experiment procedure:
calculation which needed to be modified till linear The 2D-QSAR model is constructing under the
relation established in order to go through as many Windows OS with multiple linear regression in drug
MEK inhibitor test as possible. The ongoing test also design package software, Discovery Studio 2.0, to
includes famous clinical MEK inhibitor such as build up the regression equation. The MEK inhibitor
PD184352, PD98059, U0126, CI-1040, and etc.. with known activity is required for building up the
Rapidly screen the FDA drug database for drugs QSAR model specifically for MEK protein. Collecting
that receive FDA certification (Drugs@FDA 6184 of those activity test results of known MEK inhibitor on
drugs) and transform the name into 2D structure with
Old Drug New Use
Fig. 4 Research flow chart and estimate schedule.
chemACX chemical molecular database after the calculate the docking score. The docking score can calibrated 2D-QSAR built. This step is accomplished
firstly be the secondary screening condition which can by using PHP (hypertext preprocessor) program
eliminate those drugs with lowest scores. Those drugs design to search for FDA drug database and download
with the lowest score indicate the sterostructures do the 2D structure within chemACX. Calculate the
not match with the binding site space in MEK proteins. character value and input into 2D-QSAR for Observe the relationship between drug potency group preliminary screening.
and molecules surround proteins after molecular This primary screening is just the result from 2D
docking. The observation process is accomplished by plane which could not be applied on stereoscopic
Pharmacophore program in Discovery Studio 2.0 to MEK protein and inhibitor. The Ligand Fit program in
analyze the molecule properties surround MEK Discovery Studio 2.0 drug design software is used for
protein binding sites and drug molecule characteristics. molecular docking. This program can calculate the
As previously mentioned, PD184352 and its stereo structure change of drugs, simulate the binding
analogous can go deeply into the hydrophobic region site while drug enters proteins (as mentioned of Met143, Ile141, Leu118 and Phe209 of MEK. It previously, the MEK inhibitor binding site is nearby
also produces electrostatic force with oxygen atoms in Met143, Ile141, Leu118 and Phe209) and also
Val127 and forms H-bond boding on Ser212 [3]. Take
Old Drug New Use
the similar properties to perform secondary screening that can be able to exclude those unqualified drugs.
3. Results
The primary 2D-QSAR model (Fig. 5), since the model has not been tested, therefore the result may still have some changes which includes Training Set reselection or parameters change, and etc..