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Investigation of Seed Damaging Pathogens Associated with Wheat Crop in Bhimber Azad Kashmir, Pakistan and Their Managements.

Byline: Tanveer Hussain, Muhammad Ishtiaq, Shehzad Azam, Mehwish Maqbool and Waheeda Mushtaq

Summary: Mycopathogens were explored from wheat germplasm cultivars from District Bhimber of Azad Kashmir. In this study, 10 different seed-borne pathogens were isolated from District of Bhimber, Azad Jammu and Kashmir, Pakistan. The Agar Plate Method (APM) and Towel Paper Method (TPM) were used for detection of seed borne pathogens. The disease incidence (percentage) and disease severity of fungi varied with respect to type of pathogen and seed sampling sites. Kernel bunt caused by Tilletia indica showed highest incidence (67.25%) and severity (7.0) on 0-9 rating scale. Fusarium graminearum showed the highest infection rate in three sub-divisions of district Bhimber as compared to others. The fungal attacking pathogens on wheat crop were control through fungicides treatment and treatments with plant extracts. Maximum germination rates were calculated in three sub-divisions of Bhimber after treatment of Tilt fungicide.

As 86% germination rate in Samahni, 87.5% in Bhimber and 84.5% in Bernala was calculated. Antifungal activity of five plant extracts (Acacia nilotica L., Azadirachta indica L. Juss., Eucalyptus citriodora Hook, Ficus bengalensis L. and Allium sativum L.) were evaluated in four different solvents. Highest minimum inhibitory concentration (MIC) was calculated of all plants in methanolic extracts. Maximum MIC (57.38 mcg/ml) exhibited by extracts of Acacia nilotica leaves against ten fungi. Azadirachta indica extracts in different solvents against wheat-seed fungal pathogens was shown more antimicrobial activity as compared to other four plants. Azadirachta indica extract in methanol showed the highest mean of antifungal activity (62.20 mcg/ml) against ten different fungal pathogens. Antimicrobial activity (MIC) of Ficus bengalensis in different solvents against nine wheat-seed fungal pathogens was also investigated.

Highest MIC was measured against B. graminis (57.50 mcg/ml) and S. macrospora (57.00 mcg/ml) by using methanolic extract of Ficus bengalensis. Allium sativum extracts isolated from methanol solvent showed an excellent antifungal activity (49.57mcg/ml) against ten wheat attacking fungal pathogens. The antifungal activity of Eucalyptus citriodora in methanol indicated a better control of different wheat-seed fungal pathogens.

Keywords: Tilletia tritici, Seed-borne pathogens, Fungicides, Plant extract, Seed Germination, Antimicrobial Activities

Introduction

Pakistan is a very fertile country for wheat production and indigenous communities mostly rely on this domestic crop for food and fodder necessities. Pakistan falls in top ten wheat-producing countries of the world and at ninth position in terms of wheat cultivated area. Pakistan is an agricultural country. Therefore, its economy depends on agriculture sector. Total cultivated area under wheat production is 8650 ha with the production rate of 23473 tons with an average yield of 2714 Kgs per hectares [1]. It added 13.1% value in agriculture and 2.7 % indicated to the Gross Domestic Product (GDP). Wheat was cultivated on 8805 thousand hectares, presenting a reduction of 3.6% over previous year area of 9132 thousand hectares [2]. The state of Azad Jammu and Kashmir lies in the northeast of Pakistan under the foothills of Himalayas. Wheat is an important staple food crop of Azad Jammu and Kashmir [3-7].

Wheat (Triticum aestivum) is being attacked by several fungal disease, many of them are caused by seed-borne pathogens. The fungal species were attacks leaves of mature plant, affects its photosynthetic process, and hence reduce yield of starch quantity and quality. Most of the infectious diseases are seed-borne in nature that causes many losses both in storage as well as in the field.

The major causes of reduction of wheat yield in Pakistan include seed-borne Stagonospora, common bunt (stinking smut) and loose smut. Leafand head blight diseases are Powdery mildew, leaf rust, leaf blotch (Septoria tritici), Stagonospora (Septoria) nodorum leaf, glumes blotch and Fusarium head scab [8, 9]. Some of these diseases are also commonly present in district of Bhimber, Azad Kashmir.

Seed health is a basic requirement for agricultural science for desired good population and a better quantity of wheat crop. Successfully managing wheat diseases involves appropriately integrating resistant varieties, high-quality seed, fungicide treatments, proper planting time and site, crop rotation, tillage, high fertility, and other cultural practices. Seed treatment by various antifungal plant extracts is the best, environmentally safe and economical way to keep good seed health condition. Chemical fungicides and eco-friendly methods are found to be more effective in controlling fungal diseases of wheat seeds before sowing than other conventional methods [10, 11]

Firstly, this research work was depicted that the screening of pathogenic fungi from seeds of wheat crop. Secondly, the disease incidence, severity and infection was measured from seed samples of wheat crop. Thirdly, two main management strategies were applied on wheat seeds for the eradication of fungal diseases as; i) Different fungicides were applied on wheat seeds before sowing for the control of fungal pathogens and ii) Different five plant extracts are used against pathogenic fungi assessing their antifungal activity that attack on wheat crop..

Experimental

Sampling of Seed

Seeds of wheat cultivars (Glaxy-13, Punjab-11, Aas-11 and Faisalabad-08) were collected randomly from three tehsils (sub-divisions) of district Bhimber, Azad Kashmir. Sampling of wheat grains were taken at the time of harvesting [12]. All the used equipments washed with distill water and detergent before using. All the seeds were kept intended for sowing separate from larger grain bulks. The collected seeds were persevered in Laboratory of Department of Botany, Mirpur University of Science and Technology (MUST), Bhimber Campus, Azad Kashmir for further fungal pathogens analysis. For this purpose, seeds were surface sterilized by the treatment of 5% Sodium Hypo-chloride before starting experimental trails. After that seeds were inoculated on culture media and blotter sheets for 5 to 7 days for mycofloral analysis and identification [13, 14].

Measurement of Disease Incidence and Disease Severity

Measurement of disease incidence and disease severity were carried out by the agar plate method (APM). The collected wheat seed lots were surface sterilized with 0.5 % Sodium Hypochlorite. Four hundred seeds were taken from each sample in Petri plates having already potato dextrose agar (PDA) media in such a way that 11 seeds were placed all around and one at the center of plate. The Petri plates containing seeds were kept in the laboratory for seven days at 26 +- 1degC and light 12 hours after that petri plates of seed samples were examined under low power stereomicroscope. Pathogenic fungi were counted and identified under different magnifications (7X to 30X) of a stereomicroscope (Nikon SMZ-1 stereo zoom microscope has a 100mm working distance). Some fungal pathogens identified by morphological characters of colonies, past experience and by the help of mycological literatures [15, 16, 17, 18]. The percent incidence and severity (0-9 rating scale) of disease were measured by the index:

(Equations)

Where; S (axb) = Sum of the symptomatic plants and their corresponding scoring scales. n = Total number of sampled plants and z= Highest Score Scale.

The disease severity (DS) of wheat varieties were measured by using 09 rating scale in experimental trails [19, 20]. Where: 0= no symptoms, 1= 10-20% spots on leaves, 2=20-30% spots, 3= 30-40% spots, 4= 40-50%, 5= 50-60% spots, 6= 60-70% spots, 7=70-80% spots, 8= 80-90% spots and 9= 90-100%.

Measurement of Seed Germination

Towel paper method (TPM) is a better for measurement of germination of wheat seeds. Seeds were germinated between two layers of moist towel paper and incubated under conditions that promote fungal growth. Line the base of sterilized Petri dishes with three layers of absorbent paper moistened with sterile water. Drain off excess water and placed 20-25 seeds manually with sterilized forceps. The seeds of wheat were evenly distributed on blotter sheets to avoid mutual contact. Then, the petri dishes having seeds were incubated under near ultraviolet light in alternating cycles of 12 hours light/darkness for seven days at 20+-2degC. The Petri dishes were examined under a stereo-binocular microscope for fungi developing on the seeds. Profuse seedling growth may make interpretation difficult. This may be overcome by adding 2, 4-D sodium salt to provide a 0.2% moistening solution [21]. The germination rate was calculated by the following formulae:

iii. Germination rate (%):

No. of seeds containing a particular fungus/Total seeds used x 100

Chemical Treatment

Seeds of each wheat variety were treated with six fungicides: Benlate, Difenoconazole, Strobilurins, Thiabendazole, Thiram and Triticonazole at the rate of 2 g/kg. The fungicides were applied on seeds of each collected sample in sterile conical flasks separately. After all treatments, seeds were placed on blotter sheets in petri plates @ 10 seeds / per plates and then moistened with distill water. Then the sterile seeds were grown as described above by TPM method [22].

Treatment with Plant Extract

Antifungal activity was estimated by measuring minimum inhibitory concentration (MIC) taking into consideration through agar well diffusion technique. Moreover, among the current strategies, an alternative strategy was used for wheat crop disease management, there has been great success achieved using plant-derived products.

The collected wheat seeds were treated with different plant extracts for reducing fungal contamination. This treatment method is more reliable as compared to fungicides because this method has few/no side effects [23].

Fresh, healthy and disease free leaves of Acacia nilotica, Azadirachta indica, Eucalyptus citriodora, Ficus bengalensis and Allium sativum were collected from district of Bhimber, Azad Jammu and Kashmir. The collected leaves were washed by distil water thoroughly to separate from debris. The leaves were sliced and shade dried for 20 days. The dried leaves of different plants were grind coarsely and stored for further use.

Maceration of leaves was adopted for obtaining crude extract. During the procedure, powdered plant material was extracted in non-polar solvent with gradual shifting to polar solvents. Two hundred grams of powder of leaves of each plant was weighed and soaked first in petroleum ether for eight days then it is filtered, dried and re-soaked in chloroform for eight days and then filtered, dried and re-soaked in methanol and distilled water in same way. Finally, four extracts of each plant were obtained. The polarity indices of each solvent selected for investigation.

For the antifungal activity, crude extracts and their fractions were tested against different isolated fungi by using the Agar Well Diffusion (AWD) Method. Minimum inhibitory concentration (MIC) was measured after incubation. The least concentration that added at successfully obviated microbial growth was treated as MIC.

The AWD technique dilute inoculums of fungi were spread on Petri plates contains desired media, standard hole was made in the all petri plates using cork borer and 2-3 ml of crude extracts was filled in the hole under laminar flow and then each plate was labelled, covered and placed at 35 degC for a day-night (24 hours) rotation for investigation of antimicrobial activities [24, 25].

Statistical Analysis

All the data regarding seed-borne fungi were statistically analyzed by applying Duncan Multiple Range Test (DMRT). Significance was calculated at p< 0.05 and p< 0.01 levels of probability. Each value is the mean of three independent replicates [26].

Results and Discussion

In current study, ten different seed-borne fungal pathogens were isolated from wheat crops of district of Bhimber, Azad Jammu and Kashmir in Pakistan. The disease incidence (percentage) and disease severity (on 0-9 rating scale) varied with respect to type of disease and seed sampling sites.

Kernel bunt caused by Tilletia indica showed highest incidence (67.25%) and highest severity (7.0). Similarly, loose smut (Ustilago nuda) was observed on second number. The minimum incidence and severity was shown by a pathogen Pyrenophora tritici-repentis (Tan Spot). Other observed diseases were also shown a significance reduction of wheat crop in district Bhimber, Azad Jammu and Kashmir (Table 1). Khan [27] observed similar findings. He reported 17 genera and 45 species of seed-borne fungi of wheat seeds from Pakistan. Kyali et al. (2010) was also observed the frequency of different wheat seed fungi [28]. Dawar and Ghaffar (1991) have made such similar results on seed of sunflower and Rasheed et al., (2004) on groundnut seed [29, 30]. Wheat yield losses up to 25% have been reported due to powdery mildew. Spot blotch occurs commonly in the Great Plains of the United States [31].

Spot blotch caused by Septoria tritici also can occur as epidemic form [32]. Yield losses of up to 50% have been documented to be caused by these spot diseases in winter wheat [33]. Significance difference was also measured. Many investigated species of fungi were significantly different as indicated in Table 1. Although, some fungal species did not show significant difference. It means that they show similar incidence and severity significantly.

The infection (percentage) produced by fungal pathogens were measured by TPM and APM in the three sub-divisions of district of Bhimber, Azad Jammu and Kashmir. The comparative analyses among the three sub-divisions and between the two methods were indicated in Table 2. The percent infection of pathogen Fusarium graminearum in Samahni was 28.35% calculated by TPM and 39.19% infection by APM. In Bhimber 25%, infection was calculated by TPM and 34.54% infection was measured by APM. Similarly, in Barnala Fusarium graminearum had shown 26.17% infection by TPM and 33.26% by APM. This means that pathogen Fusarium graminearum had shown highest infection rate in three sub-divisions of district of Bhimber as compared to others two. The second infectious pathogens Tilletia indica was observed in all the study area. Only Tilletia controversa was appeared less on wheat crop and shown minimum infection rate.

These all results were also revealed that TPM indicates low infection rate as compared to APM technique (Table 2). Similar methods were used for measurement of infection by Khattak et al. (1993) that reported that higher percentage of mycoflora isolated by APM as compared to towel paper method in soybean. Solanke et al., (1997) also described that APM showed more fungi than TPM [13]. Mehta (1993) also reported that seed infection by fungal pathogens adversely affected seed germination and losses of seed viability when severity of infection is high. Hence, seed infection with different fungi was adversely affected on rain-fed wheat cultivation areas [14]. These results and supported studies showing that climatic conditions influence on the growth and infections of fungal pathogens to wheat crop. Fungi grow more in moist and humid conditions. Therefore, in rainy season, fungal pathogens have more chances of production and induced infection on wheat crop.

Chemical Treatment

In Table 3, seed germination was calculated after treatment of seed dressing fungicides. Fungicide Tilt was shown better effects as compared to others. As 86% germination rate in Samahni, 87.5% in Bhimber and 84.5% in Bernala was calculated. These maximum germination rates were calculated in three sub-divisions of Bhimber after treatment of Tilt. It means that this fungicide was more effective against fungal pathogens. Siddiqui and Arif (2008) measured similar results [34]. They explains that seed borne fungi can be controlled by treated with fungicides. They used different fungicides for measurement of their efficiency in laboratory against the seed mycoflora of wheat crop. Fungicides were used @ 2g/kg seeds gave the best result with an optimum does of 1.5 g/kg seeds, significantly increased the seedling emergence, and reduce the mycoflora as compared to untreated seeds (control).

In Denmark, fungicide application to control powdery mildew and Septoria diseases resulted in yield increases of 400-2700 kg ha-1 with margin over cost varying from 500 kg ha to 2000 kg ha [35, 36]. All treatments with fungicides were indicated significant increase of germination rate as compared to control. It means that fungicides treatments are helpful for better growth and more yields of wheat crop.

Treatment with Plant Extracts

The purpose of this investigation was to highlight the antimicrobial efficacy of different plants and their potential against virulent pathogens. The antimicrobial appraisal encompassed antifungal evaluation of the respective plants. Comparative analysis of antifungal activity of five collected plants (Acacia nilotica, Azadirachta indica, Eucalyptus citriodora, Ficus bengalensis and Garlic extracts) in four different solvents were summarized in Figure 1. Highest MIC was calculated in all plants methanol extracts.

Table-1: Disease incidence and disease severity of pathogens caused different seed diseases of wheat crop in district Bhimber, Azad Kashmir.

S/N###Seed-Diseases###Causal-Agent###Incidence (%)###Severity (0-9)

1###Kernel bunt###Tilletia indica###67.25a###7.0a

2###Loose smut###Ustilago nuda###65.50a###5.7b

3###Powdery mildew###Blumeria graminis f. sp. tritici###45.00c###3.9c

4###Stinking Smut###Tilletia caries and T. foetida###52.70b###5.6b

5###Glume blotch###Phaeosphaeria nodorum###34.62cd###2.8cd

6###Sooty Moulds###Alternaria spp. and Cladosporium spp.###55.95B###6.0a

7###Dwarf Bunt###Tilletia controversa###30.06e###2.3d

8###Ear Blights(Scab)###Fusarium graminearum###50.47b###5.2b

9###Tan Spot###Pyrenophora tritici-repentis###24.00f###2.0e

10###Downy mildew###Sclerophthora macrospora###37.50d###3.0c

Table-2: Comparative analysis of seed borne fungi (percentage infection) through different techniques of wheat crop in three sub-division of district Bhimber, Azad Kashmir

###Samahni###Bhimber###Barnala

###S/No.###Pathogens

###TPM###APM###TPM###APM###TPM###APM

###1###Tilletia indica###26.45###35.62###24.00###33.15###25.00###32.27

###2###Ustilago nuda###13.08###20.40###09.46###16.25###10.54###19.00

###3###Blumeria graminis f. sp. tritici###21.00###29.35###20.37###29.00###20.00###28.50

###Tilletia caries and Tilletia

###4###24.23###32.00###21.50###31.00###22.45###30.00

###foetida

###5###Phaeosphaeria nodorum###25.38###34.00###22.06###32.07###23.79###31.05

###6###Alternaria spp. and Cladosporium spp.###16.00###21.35###12.00###22.00###11.47###23.16

###7###Tilletia controversa###10.60###17.00###06.50###14.43###07.36###12.94

###8###Fusarium graminearum###28.35###39.19###25.00###34.54###26.17###33.26

###9###Pyrenophora tritici-repentis###23.00###31.00###22.00###30.46###21.00###29.24

###10###Sclerophthora macrospora###20.00###25.00###16.35###24.03###17.04###25.90

Table-3: Calculation of different seed dressing fungicides effects on seed germination of wheat crop in district Bhimber, Azad Kashmir.

S/No.###Fungicides###Samahni###Bhimber Barnala Means

1###Sterilized Water

###(Control)###35.00###38.75###36.50###+-36.75

2###Thiram###69.15###71.00###64.00###+-68.05

3###Thiabendazole###75.06###77.35###74.65###+-75.69

4###Tilt (Triticonazole)###86.00###87.50###84.50###+-86.00

5###Difenoconazole###41.00###44.62###42.00###+-42.54

6###Benlate###82.50###83.00###76.20###+-80.57

7###Strobilurins###52.00###54.00###45.90###+-50.63

###Means###62.96###65.17###60.53###+-62.89

The MIC verified the antifungal potential of Acacia nilotica. Methanolic extracts had shown a better antifungal activity against five selected plants. Highest MIC (57.38 mcg/ml) exhibited by methanolic extracts of leaves against ten fungi. Moreover, least potential (35.59 mcg/ml) was concluded by distilled water extracts of Acacia nilotica leaves (Fig. 1).

The antifungal activity of many plantderived products against a wide range of phytopathogens has frequently been documented. The efficacy of the antifungal properties of some herbaceous and medicinal plants against wheat seedborne mycoflora was recorded. Sukirtha and Lali [37] conducted similar study. They observed the effect of aqueous leaf extracts of 8 plant species viz. Acacia nilotica, Alstonia scholaris, Azadirachta indica, Eucaylptus citriodora, Ficus bengalensis, Mangifera indica, Melia azedarach and Syzygium cuminion against seed borne mycoflora of wheat [37]. All medicinal plants were showed positive antifungal activity.

Pretorius et al. (2002) tested crude extracts from thirty-nine plant species for their antifungal potential against seven economically important plant pathogenic fungi [38]. Hussain et al. (2009) reported the antifungal activity of methanolic, ethanolic and boiling water extracts of Barringtonia racemosa leaves, sticks and barks [39]. Pawar (2011) assayed the antifungal activity of stem extracts of nine plants towards Azadirachta indica, Lantana camera, Callistemon rigidus, Capsicum annum, Datura inoxia, Terminalia thorelii, Citrus aurantifolia, Lawsonia inermis, and Santalum album against 5 seed-borne pathogenic fungi [40]. These all studies supported to our current antimicrobial analysis against fungal pathogens.

The antimicrobial activity (MIC) of Azadirachta indica (leaf extracts) in different solvents against wheat-seed fungal pathogens were evaluated in Figure 1. Methanol solvent was shown highest antifungal activity (62.20 mcg/ml) against different fungal pathogens. Petroleum ether ranked at second number according to antifungal activity (45.86 mcg/ml). While chloroform indicated less antifungal activity (44.47 mcg/ml) as compared to methanol and petroleum ether. Similar findings were obtained by many other researchers [12, 41].

It has been previously studied that the plant extracts, Calotropis procera, Eucalyptus globulens, Jatropha multifida Azadirachta indica, Allium sativum were significantly reduced mycelial growth of fungal pathogen. Similar antifungal properties of ethanolic leaf extracts of M. azedarach against Rhizoctonia bataticola, Fusarium chlamydosporum, Alternaria alternata, Trichoderma viride and Aspergillus niger have recently been reported by Aqil and Ahmad [41]. Khaleduzzaman (1996) obtained 73.0 - 86.3% control of seed-borne infection of A. tenuis, B. sorokiniana, C. lunata and Fusarium spp. in wheat by seed treatment with Neem leaf extract. Thus based on findings of the present study, Neem leaf extract appears to be better in controlling seedborne infection of some specific fungi [12]. Therefore, Neem leaf extract may be advocated for controlling seed-borne infection of fungi in sorghum based on proper seed health test.

The MIC of Eucalyptus citriodora (leaf extracts) in different solvents against wheat-seed fungal pathogens was investigated (Fig. 1). These results were also shown highest activity in methanol extracts. The MIC calculated against all fungi was 58.54 mcg/ml. The leaf extracts of Eucalyptus citriodora in methanol solvents was highest against S. macrospora. Similar findings were also investigated by Aqil and Ahmad [41]; Sukirtha and Lali [37]. They analyzed antimicrobial effects of plant Eucalyptus citriodora and observed the significance results.

Antimicrobial activity of Ficus bengalensis (leaf extracts) in different solvents against wheatseed fungal pathogens was also investigated. Highest MIC was measured against B. graminis (57.50mcg/ml) and S. macrospora (57.00mcg/ml) by use of methanol. Leaf extracts in petroleum ether, chloroform and distilled water was shown less MIC against all investigated fungal pathogens respectively (Figure 1). Similar investigations were obtained by Sukirtha and Lali [37]. They were used extracts of leaves of Ficus bengalensis and proved that this plant indicated antifungal activity.

Allium sativum extracts in different solvents against wheat-seed fungal pathogens were also used for investigation of antimicrobial activity as management strategies. Garlic extracts isolated from methanol solvent shown excellent antifungal activity against 9 wheat attacking fungal pathogens. MIC measured high (49.57 mcg/ml) against analyzed fungi. Petroleum ether and chloroform was shown less MIC value as compared to methanol Allium sativum extracts respectively. It means that methanol solvent was more suitable agents for antifungal activity against different plants as indicated in Figure 1. Analia et al. (2013) conducted similar research work. As Allium sativum extracts is used for the control of seed borne fungi of wheat crop. Generally, garlic is very promising considering its highly significant antifungal activity and antimicrobial properties against seed-borne mycoflora [42].

Conclusions

In current research work, ten different seedborne fugal pathogens were investigated from wheat crops of district of Bhimber, Azad Jammu and Kashmir in Pakistan. Agar Plate Method (APM), Towel Paper Method (TPM) and Agar Well Diffusion (AWD) methods were used for pathogens assessment and their management. The incidence (percentage) and severity (on 0-9 rating scale) of fungi varied with respect to type of disease and seed sampling sites. Fusarium graminearum was shown highest infection rate in three sub-divisions of district Bhimber as compared to others. The fungal attacking pathogens on wheat crop were control through fungicides treatment and biological treatments. Moreover, the efficacy of the antifungal properties of some medicinal plants against wheat seed-borne mycoflora was recorded.

Antifungal activity of five collected plants (Acacia nilotica L., Azadirachta indica L. Juss., Eucalyptus citriodora Hook, Ficus bengalensis L. and Allium sativum L. extracts) were evaluated in four different solvents. Highest minimum inhibitory concentration (MIC) was calculated of all plants in methanol extracts. The plant Azadirachta indica (leaf extracts) in different solvents against wheat-seed fungal pathogens was shown more antimicrobial activity as compared to other four plants. Azadirachta indica (leaf extracts) in methanol solvent was shown highest mean antifungal activity (62.20mcg/ml) against nine different fungal pathogens. All applied management strategies were reduced or minimized the fungal infection. Thus, we have improved the yield of wheat crop in the study area by use of fungicides and plant extracts (biocontrol) treatments. However, fungicides indicated hazardous impacts on the surrounding environment.

Therefore, in this current study biocontrol strategy is recommended for future control of wheat seed attacking mycoflora.

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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Feb 28, 2017
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