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Management of Rumex dentatus (Toothed Dock) by Fungal Metabolites under Field Conditions.

Byline: Muhammad Akbar and Arshad Javaid

Abstract

A field trial was conducted to investigate the herbicidal potential of culture filtrates of four Drechslera spp. viz. D. australiensis (Bugnicourt) Subramanian and Jain. D. biseptata (Sacc. and Roum.) Richardson and Fraser D. hawaiiensis M.B. Ellis and D. holmii (Luttr.) Subramanian and Jain against toothed dock (Rumex dentatus L.) a problematic dicotyledonous weed of wheat (Triticum aestivum L.). These culture filtrates were used either alone or in combination with half dose of a commercial herbicide Bromoxynil+MCPA (2-methyl-4-chlorophenoxyacetic acid). Herbicidal application either alone or in combination with fungal culture filtrates completely controlled the weed growth. All the four fungal culture filtrates when used alone significantly reduced the weed biomass by 3058% over weedy check. Consequently grain yield in wheat was increased by 622%. Culture filtrates of D. australiensis and

D. hawaiiensis exhibited the highest herbicidal activity resulting in 58 and 57% reduction in weed biomass over weedy check. Application of culture filtrates of D. australiensis and D. hawaiiensis resulted in Rs. 11632 (28%) and Rs. 10413 (25%) more profit than conventional weedy check respectively. However the highest net profit of Rs. 70100 was obtained in hand weeding treatment followed by Rs. 69128 in half dose of herbicide which was Rs. 28444 (68%) and Rs. 27472 (66%) higher than weedy check respectively. It is concluded that culture filtrates of D. australiensis and D. hawaiiensis contain herbicidal metabolites and can be used as alternatives to herbicides for the management of R. dentatus in organic wheat production.

Keywords: Alternative management; Drechslera spp.; Rumex dentatus; Weed of wheat

Introduction

In agro-ecological systems weeds are important because these compete for the same resources as for cultivated crop and ultimately deteriorate the quality and yield of crops (Khan et al. 2012). R. dentatus is an important weed growing in wheat field in Pakistan (Siddiqui and Bajwa 2001). Yield losses in wheat have been documented as high as 55% when R. dentatus was growing in 1:1 ratio with wheat (Siddiqui et al. 2010). At present the most reliable mean to control such weeds is the use of chemical herbicides such as Bromoxynil+MCPA fluroxypyr +MCPA and traisulfuran+terbutryn (Abbas et al. 2009; Naseer-ud-Din et al. 2011). However there are some environmental consequences of these synthetic agro- chemicals such as evolution of herbicide resistance in weeds and health hazards (Rashid et al. 2010; Singh et al. 2012). There is thus need to replace these synthetic agro-chemicals with eco-friendly herbicides based on natural compounds from plants and microbes (Javaid 2010).

The literature abounds with examples of herbicidal fungal metabolites used as weed control agents. These fungal metabolites have been proved as potential herbicides both in crude and purified forms (Javaid 2010). Culture filtrates of different species of Trichoderma are known to exhibit herbicidal activity against little seed canary grass (Phalaris minor Retz.) and R. dentatus (Javaid and Ali 2011). Similarly culture filtrates of various fungal species exhibited significant herbicidal activity against parthenium (Parthenium hysterophorus L.) weed (Javaid and Adrees 2009; Javaid et al. 2011). Phytotoxic fungal metabolites namely leptosphaerodione elsinochrome A and cercosporin have been isolated from different isolates of phytopathogenic fungal species Stagonospora convolvuli Dearn. and House. These metabolites have been shown to be toxic to Convolvulus arvensis L. and Calystegia sepium (L.) R. Br. (Ahonsi et al. 2005).

Evidente et al. (2008) isolated four oxazatricycloalkenones namely phyllostictines A-D from Phyllosticta cirsii Desm. and tested for their herbicidal activity. Phyllostictine A was proved highly phytotoxic against canada thistle [Cirsium arvense (L.) Scop.].

Some species of genus Drechslera are known for the production of herbicidal metabolites. Evidente et al. (2006a b) isolated four herbicidal constituents viz. ophiobolin A 6- epi-ophiobolin A anhydro-6- epi-ophiobolin A and ophiobolin I from Drechslera gigantea Heald and Wolf which were very effective against several grassy and dicotyledonous weeds including animated oat (Avena ludoviciana Dur.) annual canarygrass (Phalaris canariensis L.) lambsquarters (Chenopodium album L.) and common sowthistle (Sonchus oleraceus L.). In the recent past few studies conducted in Pakistan showed that metabolites of Drechslera species have the potential to be used against problematic broad-leaf weeds namely R. dentatus and parthenium (Parthenium hysterophorus L.) as well as grassy weeds of wheat namely Avena fatua and Phalaris minor (Javaid et al. 2011; Akbar and Javaid 2012a b 2013).

However all these earlier studies regarding the herbicidal activities of fungal metabolites were carried out either in Petri plates or in pots. Such studies under field conditions are lacking. The present study was therefore carried out to evaluate the herbicidal effects of culture filtrates of four Drechslera spp. from Pakistan namely D. australiensis D biseptata D. hawaiiensis and D. holmii against a problematic broad-leaf weed of wheat namely R. dentatus under field conditions.

Materials and Methods Selection of Test Fungal Isolates

Four species of Drechslera viz. D. australiensis D. hawaiiensis D. biseptata and D. holmii were obtained from Fungal Culture Bank of Pakistan Institute of Agricultural Sciences University of the Punjab Lahore Pakistan. Single spore isolates of all these fungal species were maintained on malt extract agar medium.

Selection of Weed of Wheat and Wheat Variety

Seeds of the test weed species were collected from wheat fields of Lahore Pakistan at the end of growing season of wheat in May 2009. A commonly cultivated wheat variety in Punjab Pakistan namely Sehar 2006 was selected for the present study. Certified seeds of the wheat variety were procured from Federal Seed Certification Department Lahore Pakistan.

Preparation of Culture Filtrates of the Test Fungi

Minimal medium (M-1-D) was prepared in distilled water as described by Evidente et al. (2006b). This medium consisted of 1.2 mM Ca(NO3)2 0.79 mM KNO3 0.87 mM KCI 3.0 mM MgSO4 0.14 mM NaH2PO4 87.6 mM sucrose 27.1 mM ammonium tartrate 7.4 M FeC13 30 M MnSO4 8.7 M ZnSO4 22 M H3BO3 and 4.5 M KI. The pH was adjusted to 5.5 with 0.1 M HCl. Medium was poured into 500 mL conical flasks at 200 mL medium in each flask. Flasks were autoclaved at 121C and 103425 P cm-2 pressure for 20 min and cooled to room temperature. Flasks were individually inoculated with agar discs (5 mm) of each of the four test fungal species from the margins of actively growing fungal colonies. Inoculated flasks were incubated at 252C in an incubator for 28 days. Cultures were filtered through four layers of muslin cloth centrifuged at 4000 rpm for ten minutes followed by filtration through sterilized Whatman filter paper No. 1. These filtrates were stored at 4C in a refrigerator.

Sterilized distilled water was added to the original filtrates (100%) to prepare dilution of 50% (Javaid and Adrees 2009).

Field Preparation

Field trial was carried out during the wheat growing season of 20092010 at Dhing Shah District Qasur (30 56' N and 74 13' E) 80 kilometers from Lahore Pakistan. All the recommended agronomic practices from preparation of field till harvesting were followed. Soil was sandy loam in texture having available potassium 43 mg kg -1 EC 2.4 mScm 2.4 pH 7.7 organic matter 0.84% and available phosphorous 4.5 mg kg

Experiment was laid out in randomized complete block design with three replications. Each plot measured 1.54 A- 1.54 m2. Nitrogen (N) was applied at 160 kg ha-1 as urea P2O5 at 110 kg ha-1 as single super phosphate and K2O at 60 kg ha as sulphate of potash. Full doses of P2O5 and K2O and half-dose of N were applied as basal while rest of the N was top-dressed at initiation of flowers.

Sowing of Seeds

Three wheat seeds per hill were sown at 22 cm inter and intra row spacing accommodating seven rows in each plot with 7 plants per row. Wheat seeds were sown on November 15 2010. After germination thinning was carried out at the stage of full emergence of first leaf to maintain one wheat seedling at one place for uniformity in different treatments. First irrigation was carried out twenty days after sowing and subsequent irrigations were carried out according to the requirement of the crop. A total of six irrigations with tube well water were carried out as dry spell was observed through out course of the present study. Seeds of R. dentatus were sown in the field at the time of first irrigation. Weed species belonging to different plant genera along with R. dentatus emerged in the field after first irrigation. All weed species except R. dentatus were removed manually and 1:1 ratio of R. dentatus and wheat plants was maintained.

Treatments and Experimental Layout

There were twelve treatments comprising of a weed free control a weedy check four treatments with the application of culture filtrate of D. hawaiiensis D. holmii D. biseptata and D. australiensis two treatments comprised of application of synthetic herbicide (Bromoxynil+MCPA) at recommended and half dose. Remaining four treatments comprised application of culture filtrates of all the four test Drechslera species mixed in 1:1 ratio with half dose of synthetic herbicide.

Schedule of Foliar Sprays

A total of three sprays were carried out with fungal culture filtrates. First spray was carried out when R. dentatus was at three to four leaves stage (40 days after sowing of wheat). Two subsequent sprays with fungal culture filtrates were carried out with intervals of 7 days. Culture filtrates were sprayed at 100 L ha-1. Only a single spray of synthetic herbicide Bromoxynil+MCPA 200/200EC either alone or mixed with fungal culture filtrates was carried out at three to four leaf stage of the weed using Knapsack hand sprayer with 4T-jet nozzle.

Harvesting and Data Collection

Wheat crop was harvested at maturity on April 15 2011. Data regarding plant height dry biomass number of fertile tillers per plant total grain yield and hundred grains weight were recorded. Whole weed biomass from each plot was harvested at the time of harvesting of wheat. Weed plants were cut at the soil surface level and completely dried in an electric oven at 60C and dry biomass was measured. Wheat dry biomass and grain yield as well as weed biomass were calculated on per hectare basis.

Statistical and Economic Analyses

All the data were subjected to analysis of variance (ANOVA) followed by Duncan's Multiple Range Test to separate the treatment means at 5% level of significance using computer software COSTAT. In order to find out comparative benefits of different treatments economic and marginal analyses were carried out on the basis of mean values of grain yield in different treatments following the procedure given by CIMMYT (1988).

Results

Effect of Fungal Culture Filtrates on Weed Biomass

Both recommended and half doses of herbicide Bromoxynil+MCPA completely checked the growth of the target weed species. Similarly weed growth was also completely checked when half dose of the herbicide was used in combination with culture filtrates of different Drechslera spp. In general culture filtrates of all the four Drechslera spp. significantly reduced biomass of R. dentatus as compared to weedy check. However variability among the herbicidal activity of the culture filtrates of different Drechslera spp. was evident. Among the four Drechslera spp. culture filtrates of D. australiensis and D. hawaiiensis were found to be more effective against R. dentatus than the culture filtrates of the other two Drechslera species. There was 58 57 31 and 30% reduction in dry biomass of R. dentatus due to foliar application of culture filtrates of D. australiensis D. hawaiiensis D. biseptata and D. holmii respectively (Table 1).

Effect of Fungal Culture Filtrates on Wheat Growth and Yield

Maximum and significant reduction of 14% in height of wheat plants was recorded due to R. dentatus in weedy check as compared to weed free control. Plant height was also significantly reduced due to presence of the weed in treatments where only culture filtrates of different Drechslera species were used as foliar spray. There was 6.3 7.7 10.7 and 13.3% reduction in plant height due to foliar spray of D. australiensis D. hawaiiensis D. biseptata and D. holmii respectively as compared to weed free control. The effect of weed competition on height of wheat plants was insignificant over control where synthetic herbicide Bromoxynil+MCPA was sprayed either alone or in combination with the culture filtrates of the four Drechslera species (Table 2). The response of number of tillers and total above ground dry biomass to the weed competition and foliar spray applications was generally similar to that of the response of plant height.

Maximum and significant reduction of 30% and 28% in tillering and above ground dry matter respectively was recorded in weedy check as compared to control. Similarly there was 921% and 1223% reduction in number of tillers and above ground dry matter respectively due to weed competition in various treatments where only the fungal culture filtrates were used as foliar spray. In contrast in all the synthetic herbicide treatments either alone or in combination with fungal culture filtrates the effect of R. dentatus interference on the two studied parameters was nonsignificant as compared to control (Table 2).

Data regarding the effect of weed competition and various types of foliar spray applications on grain yield and 100 grains weight are presented in Table 2. Maximum grain yield (2.320 t ha-1) was obtained in weed free treatment. The highest reduction in grain yield (43%) was recorded in weedy check over control. Similarly significant reduction in grain yield was also recorded due to the weed competition over con trol in treatments where only fungal culture filtrates were used in the foliar spray application. There was 21 % 22% 35% and 36% reduction in grain yield over weed free control in treatments of foliar spray applications of culture filtrates of D. australiensis D. hawaiiensis D. biseptata and D. holmii respectively. R. dentatus interference significantly reduced the weight of 100 grains by 30% 25% and 27% in weedy check and in treatments where culture filtrates of D. biseptata and D. holmii respectively were used in the foliar spray application respectively.

The effect of R. dentatus on grain yield and 100 grains weight was non-significant in Bromoxynil+MCPA treatments either alone or in combination with fungal culture filtrates.

Table 1: Effect of foliar spray of herbicide Bromoxynil+MCPA and metabolites of Drechslera spp. on biomass of Rumex dentatus

Treatments###Weed dry biomass (t ha-1)###Reduction over weedy check (%)

Weed free (Hand weeding)###0 d###-

Weedy check###3.557 a###0

Drechslera hawaiiensis###1.520 c###57

D. holmii###2.496 b###30

D. biseptata###2.449 b###31

D. australiensis###1.492 c###58

Bromoxynil+MCPA (Recommended dose)###0d###100

Bromoxynil+MCPA (Half dose)###0d###100

Bromoxynil+MCPA (Half dose) + D. hawaiiensis###0d###100

Bromoxynil+MCPA (Half dose) + D. holmii###0d###100

Bromoxynil+MCPA (Half dose) + D. biseptata###0d###100

Bromoxynil+MCPA (Half dose) + D. australiensis###0d###100

Table 2: Effect of foliar spray with recommended (RD) and half dose (HD) of herbicide Bromoxynil+MCPA and metabolites of Drechslera spp. on growth and yield of wheat

Treatments###Plant height (cm)###No. of tillers/plant###Dry biomass (t ha-1) Grain yield (t ha-1)###100 grains weight (g)

Weed free (Hand weeding)###90.0 a###11 a###5.637 a###2.320 a###4.12 a

Weedy check###77.1 d###8d###4.082 c###1.333 e###2.89 b

D. hawaiiensis (Dh)###83.1 bc###10 b###4.926 a-c###1.799 cd###3.73 a

D. holmii (Dhl)###78.0 d###9c###3.366 bc###1.477 de###3.01 b

D. biseptata (Db)###80.3 cd###9c###4.498 bc###1.515 de###3.07 b

D. australiensis (Da)###84.3 b###10 b###4.966 ab###1.838 b-d###3.76 a

Herbicide (RD)###86.3 ab###11 a###5.392 a###2.137 a-c###3.99 a

Herbicide (HD)###89.3 a###11 a###5.493 a###2.254 ab###4.10 a

Herbicide (HD) + Dh###88.7 a###11 a###5.598 a###2.227 ab###4.11 a

Herbicide (HD) + Dhl###88.7 a###11 a###5.470 a###2.149 a-c###4.06 a

Herbicide (HD) + Db###88.3 a###11 a###5.365 a###2.203 a-c###3.96 a

Herbicide (HD) + Da###89.3 a###11 a###5.520 a###2.254 ab###4.11 a

Economic and Marginal Analyses

All the treatments showed higher net profit than weedy check. Among the various fungal culture filtrate treatments filtrates of D. australiensis gave the highest net profit of Rs. 53288 followed by Rs. 52069 due to spray of filtrates of D. hawaiiensis which were Rs. 11632 (28%) and Rs. 10413 (25%) higher than the net profit in weedy check. The highest net profit of Rs. 70100 was provided by hand weeding followed by Rs. 69128 in by half dose of herbicide which were Rs. 28444 (68%) and Rs. 27472 (66%) greater over weedy check respectively (Table 3). Hand weeding was the best treatment according to marginal analysis with a 3077% marginal rate of return followed by half dose of herbicides with a 2097% marginal rate of return. Rest of the treatments were dominated because of either increasing cost that vary or lower net profits (Table 4).

Discussion

In the field study herbicidal activity of culture filtrates of four Drechslera species was assessed and compared with a commercial herbicide Bromoxynil+MCPA. The chemical herbicide was used either alone as recommended and half dosages or its half dose was applied in combination with culture filtrates of various Drechslera species. R. dentatus reduced wheat grain yield by 43% over weed free control. Application of recommended dose of chemical herbicide completely killed the weed plants. Although none of the fungal culture filtrates treatments completely eliminated the weed however these markedly reduced the weed biomass by 3058% over weedy check. Similar decline in weed biomass has also been reported by Javaid et al. (2011) where culture filtrates of Drechslera spp. were exploited to manage parthenium weed in pot experiments. Wheat grain yield losses in treatments where foliar spray of culture filtrates of D. australiensis D. hawaiiensis

D. biseptata and D. holmii was carried out were 21 22 35 and 36% respectively as compared to 43% losses in weedy check. This variation in herbicidal activity of culture filtrates of different Drechslera spp. could be attributed to the variation in chemical constituents of different fungal species (Evidente et al. 2005; Zhou et al. 2008; Eneyskaya et al. 2009; Yang et al. 2009). Previous studies conducted regarding the effect of foliar spray of culture filtrates of different pathogenic fungi including species of Fusarium Alternaria and Drechslera against parthenium weed support the findings of the present study and suggest that fungal culture filtrates can be exploited as herbicides (Idrees and Javaid 2008; Javaid and Adrees 2009; Javaid et al. 2011).

Table 3: Economic analysis for the use of fungal metabolites for management of Rumex dentatus in wheat

###T1###T2###T3###T4###T5###T6###T7###T8###T9###T10###T11###T12 Remarks

Grain yield###2.320###1.333 1.799###1.477###1.515###1.838 2.137###2.254###2.227###2.149###2.203###2.254 t ha-1

Gross income###72500 41656 56219 46156 47344 57438 66781 70438###69594###67156###68844###70438 wheat grain price@

(PKR ha-1)###PKR 1250 40 kg-1

Cost of labour for 2400###PKR 300 man-1

hand weeding###(8 men day-1 ha-1)

Cost of fungal###3750###3750###3750###3750###1250###1250###1250###1250 PKR 5 L-1 of fungal

metabolites ha-1###culture filtrates

Cost of herbicide###1820###910###910###910###910###910 PKR 1820/560 g

ha-1###active ingredients

Spray application###1200###1200###1200###1200###400###400###400###400###400###400 PKR 400 man-1 (one

cost ha-1###man day-1 ha-1)

Cost that vary###2400###0###4150###4150###4150###4150###2220###1310###2560###2560###2560###2560 PKR ha-1

Net profit###70100 41656 52069 42006 43194 53288 64561 69128###67034###64596###66284###67878 PKR ha-1

T1###Weed free (Hand weeding)

T2###Weedy check

T3###Drechslera hawaiiensis @ 250 L3S ha-1

T4###D. holmii @ 250 L3S ha-1

T5###D. biseptata @ 250 L3S ha-1

T6###D. australiensis @ 250 L3S ha-1

T7###Bromoxynil+MCPA (Recommended dose)###@ 560 g a.i. 1S ha-1

T8###Bromoxynil+MCPA (Half dose) @ 280 g a.i. 1S ha-1

T9###Bromoxynil+MCPA (Half dose) + D. hawaiiensis @ 280 g a.i. 1S ha-1 + 250 L1S ha-1

T10 Bromoxynil+MCPA (Half dose) + D. holmii @ 280 g a.i. 1S ha-1 + 250 L1S ha-1

T11 Bromoxynil+MCPA (Half dose) + D. biseptata @ 280 g a.i. 1S ha-1 + 250 L1S ha-1

T12 Bromoxynil+MCPA (Half dose) + D. australiensis @ 280 g a.i. 1S ha-1 + 250 L1S ha-1

Table 4: Marginal analysis for the use of fungal metabolites for management of Rumex dentatus in wheat

Treatments###Dose (ha-1)###Variable cost Net profit Marginal cost Marginal net profit Marginal rate

###(PKR ha-1)###(PKR ha-1) (PKR ha-1)###(PKR ha-1)###of return (%)

T2 Weedy check###-###-###41656

T8 Bromoxynil+MCPA###280 g a.i.1S###1310###69128###1310###27472###2097

T7 Bromoxynil+MCPA###560 g a.i.1S###2220###64561###-###-###D

T1 Weed free (hand weeding)###-###2400###70100###180###5539###3077

T12 Bromoxynil+MCPA + D. Australiensis###280 g a.i.1S + 250 L1S 2560###67878###-###-###D

###1S###1S

T9 Bromoxynil+MCPA + D. hawaiiensi###280 g a.i. + 250 L###2560###67034###-###-###D

T11 Bromoxynil+MCPA + D. biseptata###280 g a.i. 1S + 250 L1S 2560###66284###-###-###D

T10 Bromoxynil+MCPA + D. holmii###280 g a.i. 1S + 250 L1S 2560###64596###-###-###D

T6 Drechslera australiensis###250 L3S###4150###53288###-###-###D

T3 D. hawaiiensis###250 L3S###4150###52069###-###-###D

T5 D. biseptata###250 L3S###4150###43194###-###-###D

T4 D. holmii###250 L3S###4150###42006###-###-###D

Moreover a number of herbicidal compounds have also been isolated from different species of Drechslera. As an example culture of D. siccans (Drechsler) Shoemaker is reported to yield a phytotoxin named as 6 8-dihydroxy-3- (2'-hydroxypropyl) isocoumarin (de-o-methyldiaporthin) and found very effective against common oat (Avena sativa L.) barnyardgrass [Echonocloa crus-galli (L.) P. Beauv.] and spiny amaranth (Amaranthus spinosus L.) (Hallock et al. 1988). Capio et al. (2004) isolated two phytotoxic compounds namely cytochalasin B and dihydrocytochalasins from culture filtrates of D. wirreganensis Wallwork Lichon and Sivan. and D. campanulata (LACopyrightv.) B. Sutton. Similarly another metabolite namely drazepinone with broad spectrum herbicidal activity has been isolated from D. siccans (Drechsler) Shoemaker. This compound was characterized as 3512a-trimethyl-255a12a-tetrahydro-1H naphtha [20 30:4 5] furo [23-b] azepin-2-one (Evidente et al. 2005).

In the present study original culture filtrates were applied as foliar spray. It is very likely that efficacy of these filtrates can be enhanced markedly if these are applied in a concentrated form because generally quantity of active herbicidal constituents in fungal culture filtrates is very low (Evidente et al. 2006a b).

In the present field trials half dose of synthetic herbicide also completely killed the R. dentatus plants. Consequently the effect of combined application of fungal culture filtrates and half dose of herbicide could not be assessed. Further studies are required in this regard using lower concentrations of the herbicide in combination with fungal culture filtrates. In the present study no adverse effect of the fungal metabolites on growth of wheat was noted. This differential response of the weeds especially R. dentatus and wheat to fungal metabolites can be best exploited in the management of R. dentatus and possibly other broad-leaf weeds by the culture filtrates of Drechslera spp. under field conditions. To the best of our knowledge the present study is the first report of using fungal metabolites as herbicidal agents in true field conditions. Generally earlier experiments were carried out in Petri plates trays or pots (Vurro et al. 2001; Javaid et al. 2011).

The usefulness of any treatment depends on the cost involved and its economic return as well as its impact on the environment. In the present study an economic benefit of Rs. 11632 and 10413 was achieved by the application of culture filtrates of D. australiensis and D. hawaiiensis that was 28% and 25% higher than the net profit obtained in weedy check. Although the highest net profit and economic return was obtained with hand weeding followed by application of 280 g a.i. ha-1 of Bromoxynil+MCPA however fungal culture filtrates are more environment friendly than herbicides and can be used as an alternative for weed management in organic farming systems.

Conclusion

The present study concludes that culture filtrates of D. australiensis and D. hawaiiensis contain potent herbicidal constituents for the management of R. dentatus as they reduced biomass of this weed by 58% and 57% consequently increased the wheat grains yield by 22% and 20% respectively as compared to weedy check. Recently Akbar et al. (2014) have identified holadysenterine as potent herbicidal metabolite from culture filtrates of D. australiensis that may be used as an alternative to chemical herbicides in production of organic wheat.

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Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Feb 28, 2015
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