Shading led to changes in the leaf histological characteristics of Mikania micrantha H.B.X., leaves at higher

light intensity being thicker than those at lower light intensity. There was a significant increase in the area of

spread of imazapyr droplets and retention on the upper leaf surface at lower light intensity, suggesting that

shading had changed the upper surface topography and the amount of epicuticular wax of the leaves

Ipor, Isa

Tawan, Cheksum

Universiti Putra Malaysia Institutional Repository

PertanikaJ. Trap. Agric. Sci. 18(3): 163-168(1995) ISSN: 0126-6128© Universiti Pertanian Malaysia PressThe Effect of Shade on Leaf Characteristics ofMikania micrantha (Compositae) and Their Influence onRetention of ImazapyrI.B. IPOR and C.S. TAWANFaculty of Resource Science and TechnologyUniversiti Malaysia Sarawak94300 Kota Samarahan, Sarawak, MalaysiaKeywords: Mikania micrantha, shade, imazapyr, leaf surfaceABSTRAKLindungan telah mengakibatkan perubahan terhadap ciri-ciri histologi daun Mikania micrantha H.B.X.,dimana daun yang berada pada intensiti cahaya yang tinggi adalah lebih tebal daripada daun dari intensiticahaya yang rendah. Peningkatan dengan kadar yang bermakna bagi luas semburan dan retensi irnazapyr dipermukaan atas daun dalam intensiti cahaya yang rendah menunjukan lindungan telah menukarkan topografipermukaan atas dan kuantiti lilin daun.ABSTRACTShading led to changes in the leaf histological characteristics ofMikania micrantha H.B.X., leaves at higherlight intensity being thicker than those at lower light intensity. There was a significant increase in the area ofspread of imazapyr droplets and retention on the upper leaf surface at lower light intensity, suggesting thatshading had changed the upper surface topography and the amount of epicuticular wax of the leaves.INTRODUCTIONMikania micrantha H.B.Ie is a pernicious weed incrops such as rubber, cacao, oil palm, coconut,banana, pepper and tea. It usually grows profuselyin places receiving high rainfall or in humidhabitats (Holm et at. 1977). The obnoxiouscharacter of this weed is mainly due to its rapidgrowth and spread, which smothers neighbouringplants, as well as its ability to root at the nodeswhen the stem comes in contact with soil(Macalpine 1959). Ipor (1991) reported that M.micrantha is a shade-tolerant species and persistsin sites receiving 25% of full sunlight.Imazapyr (isoproypylamine salt of 2-(4-isopropyl-4-methy1-5-oxo-2-imidazoin-2-yl)nicotic acid) is a broad spectrum herbicidecommonly used for controlling both annualand perennial weeds (Fine et al. 1983). It is asystemic herbicide and is readily absorbed andtranslocated in plant tissues (Mallipudi et al.1986). The uptake, translocation and activityof imazapyr increases significantly as lightintensity decreases (Ipor and Price 1990).It is well known that level of light intensitiesduring growth can markedly alter themorphological, anatomical, physiological andbiochemical properties of leaves. Ipor (1989)found that growth pattern, rate of expansion,final leaf area and specific area of individualleaves of M. micrantha were greatly influencedby shade or light intensities.Daubenmire (1970) reported thatepicuticular wax and distribution of crystallinewax had a significant effect on herbicidepenetration. Hence, the objective of this studywas to determine the role of light intensity inaltering the topographical characteristics of wax,epidermal cell size and morphological structureof M. micrantha, which are likely to influencethe retention of imazapyr.MATERIALS AND METHODSMidrib Sectioning for Light MicroscopyPlants were grown by using the proceduresdescribed by Price and Ipor (1990). Themiddle part of the second youngest lamina ofI.B. IPOR AND C.S TAWANM. micrantha of plants grown under three levelsof shade (0, 50 and 75%) was cut into a pieceof 7 x 7 mm and fixed in formalin-acetic acid(FAA) under reduced pressure for a week. Thesamples were dehydrated in a graded ethylalcohol series. After dehydration, the tissue wasinfiltrated and embedded in paraffin, whichallowed to solidify. Sections 10 J.Lm thick werestained with alcian blue and safranin (50%alcohol) (Sass 1958).For stomata counting, the middle portionof the preserved leaves was cut into pieces 10 x10 mm. The tissues were thoroughly washedwith distilled water then gently boiled in 45 mlof 15% nitric acid until the adaxial and abaxialsurfaces separated. The tissues were gentlytransferred into a petri dish and washed withtwo changes of distilled water. The remainingmesophyll was brushed away with a fine brush.The cuticles were then treated with 5% aceticacid for 30 sec and immediately transferred intosodium hypochlorite (NaGCl) for 30 min untilthe tissues became clear. They were then washedtwice with distilled water before being transferredinto 50% ethanol for 2 min followed by stainingwith 50% safranin for 10 min. After dehydration,the cuticles were mounted in Canada balsam oreuparal and the slides dried on a hot plate fortwo weeks. The stomata were counted under alight microscope.Scanning Electron Microscopy of Upper Leaf SurfaceLeaf pieces 0.75 x 0.75 cm were taken from themiddle portion (avoiding the midrib) of thesecond youngest leaf of plants grown at thethree levels of shade mentioned above as well asfrom plants growing naturally in an open habitatin an oil palm plantation in Malaysia. The leafpieces were affixed to aluminium stubs withcolloidal silver adhesive and immediately cooledin liquid nitrogen. Specimens were then freezedried between at -40 - -60°C for 48 h.Specimens were coated with gold andexamined and photographed using a Jeol T20scanning electron microscope (SEM) at 3200xmagnification.Wax Deposits on the Leaf SurfaceThe amount of epicuticular wax was estimatedgravimetrically in a chloroform extract, using amethod similar to that of Souza and Williams(1986). Fully expanded leaves from the threeshade regimes were excised and their surfaceareas determined using a photomax tracer. Theleaves were then dipped in chloroform twice for10 sec, which was then filtered through WhatrnanNo.1 paper into a pre-weighed test tube. Testtubes with extracts were placed in a fume hoodto evaporate the chloroform and then in a forcedair oven at 45°C before being transferred into avacuum desiccator and dried to constant weight.Droplet Spread on Leaf SurfaceA Buckard microapplicator was used to apply0.2 JLI droplets of lisamine red formulations onthe upper leaf surfaces (Mabb and Price 1986).The eighth and second youngest leaves of plantsat the ten-leaf growth stage were used. Thediameter of the droplet deposit was measuredafter 24 hours with a calibrated graticuleeyepiece and the area calculated. Theexperiment was carried out with thirty replicatesand repeated twice.Spray Retention of lmazapyrFifty-leaf stage plants were used. The plants weresprayed with solutions containing the solubledye lisamine (1% w/v) with imazapyr (0.3 kga.i/ha) and distilled water. A maldrive sprayingsystem was used to deliver 211 1/ha.After the sprayed deposits had dried, theleaves were detached born each plant and thesprayed deposits were washed off with 25 ml ofdistilled water. The lisamine concentration wasmeasured in a spectrophotometer atwavelength 460 J.Lm. The value of the peakpoint was compared with a standardconcentration curve for the calculation of theequivalent amount of herbicide in the sprayeddeposit. Data were expressed in J.Lg herbicideper cm2 of leaf. The experiment was repeatedtwice with thirty replicates.RESULTSHistology ofM. micranthaThe effect of shading on the leaf characteristicsand anatomy are presented in Plate 1 andTable 1. Leaves grown at 0% shade in bothglasshouse and the open area in the field weresignificantly thicker than those at other lightregimes. The number of cells between the upperand lower leaf surfaces increased with increasein light intensity.164 PERTANlKAJ. TROP. AGRIC. SCI. VOL. 18 NO.3, 1995THE EFFECf OF SHADE ON LEAF CHARACTERISTICS OF MlKANIA MICRANTHA•Plate 1: Transverse sections ofMikania micrantha leavesgrown under (A) 0%; (B) 50%; (C) 75% shade;(D) open areas; (E) in mature oil palm plantation.(Scale bar = 6Omm)The size of epidermal cell of leaves grownunder low light intensity increased appreciably.Palisade cell diameter also increased significantlywith light intensity.Upper Leaf Surface ofM. micranthaThe width of epidermal cells on the uppersurface decreased appreciably with increasinglight intensity (Plate 2). The epidermal cells ofleaves from 0% shade in the glasshouse andopen areas in the field were fewer than of leavesfrom 75% shade (Table 1). The wax on theupper leaf surface was generally flattened.Wax Deposits on Leaf SurfacesThe average amount of chloroform-soluble waxfrom leaves under different shade levels ispresented in Table 2. The quantity of epicuticularwax per unit area of leaves increased as the levelof shade decreased: the amount from leavesunder 0% shade was more than twice that from75% shade.Droplet Spread on Upper Leaf SurfaceThe area of spread of droplets on both youngand old leaves at 75% shade was significantlygreater than that at 0 and 50% shade (Table 3),TABLE 1Effect of shading on the histological characteristics of leaves of Mikania micranthaGlasshouse FieldLeaf cell characters 0% 50% 75% Open ShadedNumber of stomata per mm2Upper surface 142a 73c 10d 101b 12dLower surface 597a 381b 295c 298c 234dLeaf thickness (IJ-m) 207a 194b 136c 210a 105dNumber of cellsfrom upper to lower 7.8a 6.1bc 5.5cd 6.4b 4.8dleaf surfaceAverage width of:Epidermal cell (IJ-m) 32.6b 38.5a 42.7a 28.1b 22.5cXylem (IJ-m) 13.4b 20.9a 18.9a 19.8b 13.3bPhloem (x 10-3 IJ-m) 7.0a 8.5a 9.1a 7.7a 8.8aMesophyll cell (IJ-m) 25.2b 32.8a 25.4b 31.1a 23.3bPalisade cell (IJ-m) 60.6a 54.4b 40.0d 46.3c 25.6eWithin each row, values sharing the same letter are not significantly different at 5% level, accordingto Duncan's multiple range test.PERTANIKAJ. TROP. AGRIC. SCI. VOL. 18 0.3, 1995 165I.B. IPOR AND C.S TAWAN*Means with the same letter within the column arenot significantly different (P>0.05) according to DMRTWithin columns means with the same letter are notsignificantly different (P>0.05) according to DMRTTABLE 4Amount of imazapyr retained on leaves of Mikaniarnicrantha grown under different shade levels75%62.50a12.52c3.16a0.95b1.17b50%2.43d31.01bShade levelImazapyr spray retained(fLg cm·2) *Area of droplet spread(mm2)0%2.87d4.44do5075Shade level(%)TABLE 3Effect of shade levels on area of spread ofimazapyr drops on the upper surface ofMikania mircrantha leavesOldYoungLeaf stageTABLE 2Effect of shading on the quantity of epicuticulat-wax deposits on the leaf sUli"ace ofMikania micranthaPlate 2: EM micrographs showing leaf surface of Mikaniamicrantha at different levels ofshade (A) 0%; (B)50%; (C) 75 % shade; (D) open areas; (E) inmature oil palm plantation: (F) without epicuticularwax after stripping with cellulose acetate.(Magnification = 3200x, Scale bar = 25cm)Shade Level Mean Weight of EpicuticularWax (f.Lm cm·2)but there was no significant difference betweeno and 50% shade on young leaves.Within each column, values sharing the same letterare not significantly different according to Duncan'smultiple range test.Spray Retention of Imazapyr on M. micranthaLeavesThere was a trend towards increased retentionof imazapyr with shade (Table 4). Plants under75% shade retained more than three times thatfrom 0% shade (Table 4).0%50%75%8.2a5.2b3.4aDISCUSSIONPlants grown under high light intensities showeddifferences in leaf surface structure from thosegrown at low light intensities (Table 1). Highlight intensity reduces leaf expansion resultingin thicker leaves. Boardman (1977) found thatleaves of Atnplex patula grown at 20 mw cm-2were seven cells thick compared with three orfour cells in leaves grown at 2 mw cm-2. Themesophyll cells grown under low light intensitywere smaller and more densely packed and therewere fewer vascular strands. The size of theepidermal cell of M. rnicrantha was considerablysmaller under high light intensity.Shade was observed to play an importantrole in the development of the epicuticular waxesof M. rnicrantha. A greater deposit of epicuticularwax was found under higher light intensities166 PERTANlKAJ. TROP. AGRIC. SCI. VOL. 18 NO.3, 1995THE EFFECT OF SHADE ON LEAF CHARACTERISTICS OF MlKANIA MICRANTHA(Table 2). Skoss (1955) found that shaded leavesof ivy (Hedera helix) had less cuticle and waxthan those exposed to full sunlight. Martin andJuniper (1970) also reported that wax productionon soybean (Glycine max) leaves increased withlight intensity. Significant increase in wax offield bindweed (Convolvulus aruensis) leaves wasalso reported under high light intensities(Steward et al. 1986).A significant increase in the droplet spreadwas observed on leaves from shaded plants(Table 3). Dorschner and Buchholtz (1956)found that shading by companion cropsincreased the wettability of alfalfa (Medicagosativa) Jeaves. In the present study, wettabilitywas significantly correlated with differences inquantity and density of the epicuticular waxcrystals, and to the size of the epidermal cellsof M. micrantha.There was a trend toward greater retentionof imazapyr on M. micrantha leaf surfaces grownunder shade (Table 4). Differential sprayretention is dependent on leaf surfacecharacteristics and the angle of incident of thespray droplet to the leaf (Ennis et al. 1952;Brunskill 1956; Blackman 1958). Ennis et al.(1952) reported that the waxy layer on the leafwas an important characteristic affecting spraydroplet repulsion. In this study, the waxy layerof M. micrantha leaves grown at high lightintensities repelled spray droplets more effectivelythan those grown at lower light intensity. Inheavily waxed leaves, Brunskill (1956) showedthat spray droplets bounced off the leaf becausethe angle of incidence had decreased. This maybe the main explanation why more imazapyr isretained on the leaf surface of M. micranthagrown under lower light intensity and which areless waxy. In addition M. micrantha at 0 and 50%shade levels had leaves which were slightly erectand facilitated runoff, whereas at 75% shade,the leaves were oriented horizontally, biggerand proportionally longer. Less bounce ofdroplets should occur at 75% shade, which maycontribute to the greater spray retention observedat 75% shade.Changes in morphology, surface structureand histological characteristics influenceretention of imazapyr on plants exposed todifferent shade levels. Price and Ipor (1990)reported that leaves of Paspalum conjugatumgrown under low light intensity (75% shade)had significantly increased uptake andtranslocation of imazapyr, and ascribed this tothe thinner leaves with higher permeability.Uptake and translocation have repeatedly beenshown to account for the effectiveness ofherbicides Uensen 1982). The greatersusceptibility of plants growing under highershade levels means that a smaller quantity of theherbicide and less frequent application ofherbicide are needed.REFERENCESBlACKMAN, G.E. 1958. Differential spray retentionand selective action of herbicides. In Proceedings1st African Weed ControL Saffron Walden: BritishCrop Protection Conference.BOARDMAN, N.K. 1977. Comparative photosynthesisof sun and shade plants. Annual Review ofPlantPhysiology 28: 355-377.BRUNSKILL, RT. 1956. Physical factors affecting theretention of spray droplet on' leaf surfaces.Proceedings of the British Weed Control Conference3: 593-603.DAUBENMIRE, R. 1970. Steppe vegetation ofWashington. Bulletin of the WashingtonAgricultural Experimental Station 62: 80-82.DORSHCHNER, K.P. and K.P. BUCHHOLTZ. 1956. Wettingability of aqueous herbicidal sprays as a factorinfluencing stands of alfalfa seedlings. AgronomyJournal 48: 59-63.ENNIS, W.B., R.E. WILLIAMS and K.P. DORSCHNER.1952. Studies on spray retention by leaves ofdifferent plants. Weeds 1: 274-286.FINE, RR., T.R PEOPLES and D.R CIARLANTE. 1983.AC 252925 - A new broad spectrum herbicide.Proceedings of the 9th Asian Pacific Weed ScienceConference, p. 436-439. Asia Pacific Weed Society.HOLM, L.G., D.L. PLUCKNETT, J.V. PANCHO and J.P.HERBERDER. 1977. The World's Worst Weeds:Distribution and Biology. University Press ofHawaii.IpOR, I.B. 1989. Action of herbicides on Mikaniamicrantha H.B.K. and Paspalum conjugatum Berg.PhD thesis, University of London.IpoR, I.B. 1991. The effect of shade on the growthand development of Mikania micrantha H.B.K.Malaysian Applied Biology 20(1): 57-63.PERTANlKAJ. TROP. AGRIC. SCI. VOL. 18 NO.3, 1995 167LB. IPOR AND C.S TAWANIpOR, LB. and C.E. PRICE. 1990. Effect of shading onthe uptake and translocation of 14C- paraquatand 14C- imazapyr in Paspalum conjugatum Berg.In Proceedings of 3rd Tropical Weed ScienceConference. Kuala Lumpur: Malaysian PlantProtection Society.JENSEN, K.I.N. 1982. The roles of uptake,translocation and metabolism in the differentialintraspecific responses to herbicides. InHerbicide Resistance in Plants, ed. H.M. Lebaronand J. Gressel, p. 133-162. New York: Wiley-Interscience.MABB, L.P. and C.E. PRICE. 1986. Fluazifop-butylactivity of Imperata cylindrica (L) P. Beauv. (1)'Studies on phytotoxicity, spray adhesion andherbicide uptake. Weed Research 26: 301-305.MACALPINE, R. 1959. A note on Mikanta scandens.Two and a Bud 6: 6-8.MALLIPUDI, B.M., BA KNOLL, A.L. HORNG and EJ.ORLOSKI. 1986. Absorption, translocation andsoil dissipation of imazapyr under fieldconditions. In Proceedings of the Weed ScienceSociety, North Tennessee. 20th - 22nd january.Weed Science Society North Tennessee.MARTIN, J.T. and B.E. JUNIPER. 1970. The Cuticle ofPlants. London: Edward Arnold.PRICE, C.E. and LB. IpOR. 1990. The effect of shadeon the activity of paraquat and imazapyr onPaspalum conjugatum. In Proceedings of the 3rdTropical Weed Science Conference, Kuala Lumpur,4th-6th December, 1990. Malaysian PlantProtection Society.SASS, J.E. 1958. Botanical Microtechnique. 3rd edn.Ames: Iowa State University Press.SKOSS, J.D. 1955. Structure and composition ofplant cuticle in relation to environmentalfactors and permeability. Botany Gazette 117:55-72.SOUZA, LF. and J.L. WILLIAMS. 1986. Chloroform-soluble fraction from soybean [Glycine max(L.) Merr.] leaves following herbicide applica-tions. Weed Research 26: 181-183.STEWARD, L.S., A.H. HARVEY and F.D. HESS. 1986.Effect of adjuvants and environment duringplant development on glyphosate absorptionand translocation in field bindweed(Convolvulus arvensis). Weed Science 34: 811-816.(Received 15 October 1991)(Accepted 3 November 1995)168 PERTANlKAJ. TROP. AGRIe. SCI. VOL. 18 NO.3, 1995

The effect of shade on leaf characteristics of Mikania micrantha (Compositae) and their influence on retention of Imazapyr

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The effect of shade on leaf characteristics of Mikania micrantha (Compositae) and their influence on retention of Imazapyr

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