the n m o x o x i c m , trahslocatiqn ahd fate o f
DIMETHYL 2,3,5,6-TETRACHLOROTKREFHTUALATE (DCFA)
II RESISTANT AID SUSCEPTIBLE WEED SPECIES
A THESIS SUBMITTED TO THE GRADUATE SCHOOL OP THE
UNIVERSITY OP HAWAII IN PARTIAL FULFILLMENT
OP THE REQUIREMENTS FOR THE DEGREE OF
MASTER OP SCIENCE
IN HORTICULTURE
SEPTEMBER 1966
»r Robert Verson Osgood
Thesis Coeaitteei
Reese R. Roeenovekl, Chairman Janas L. Bravbaker Richard E. Green We certify that we have n e f this thesis sad that in our opinion It is satisfactory in scope end quality ee e thesis for the degree of Master of Science in Horticulture.
TBJESZS COtOIITTSE
Chainaan ACXJfOWLEDCEMEOT’S
The author would like to express hie appreciation to hr* L. K.
Llapel of Boyce Thompson Institute for float Research Inc. end to hr* h* X. Stallard of Plaaoud Alkali Co* for their helpful suggestions aad for the supply ef chealeals necessary for the reaearcb. Ia addition the writer gratefally acknowledges hie wife, Petti, for hor help Is typing the rough drafts of the aanuscrlpt. t m $ of c o n r m w
P«*« fix m m jm m m m ...... ♦ ...... m a s s t s u c t ...... iv
LIST OF m s s ...... v u u s t OF j x s b u s ...... v i i i n m m m cn m • * , * ...... 1 uxmxnuc im n ...... • • ...... 3
fOUBASCE AMD SttiCEPTHIlLm 10 DGPA ...... 3
STAGE OF FLAUT DLVELQJHEMT AMD TBS fSRBroxicm of dcfa ...... 4
use $ m o f w m ic jm uptake Dt m m on 10 FKROSOXXCm • • ...... 4
DIFFERENTIAL UPTAKE* TBA&&U)CATX0fi» AMD ADSOSmOS IS RZLAHOM 10 FHWOlOXlCITT . . . «
SWrtCMS OF DCFA XSJISOf ...... U
KATmL'LS MB MEWSDO...... 14
w m m n ia x t stu ou s ••«•••*.«...••••• 14
UPTAKE AMD EUUKSUXMXSXI STUD IES...... 15
IS Vim > METABOLISM STUDIES ...... 19
STATISTICAL METHODS ...... 20
USQLaB - SD U S O B tS X ...... 22
FBTTOTOXIcm STUDIES...... 22
8PBKS MB mmLXATJOS STUDIES ...... 32
15 V m O METABOLISM STUDIES ...... 39 s m m m ...... 40 AFPEHDXE ...... 42 aSFESKSCES ...... 52 the sBYiomicm* t S k M s w c s s m A m m m of D d S O T L 2»3,5»4*TinB/4mOSOTESEnt1SS/XATE (OCPA) m i i s s u m a m > s t s c o m s ;eed s v m m
ABM
Satisfactory bloasaays were develops* sbr the determination of d m ! resistance or susceptibility to DCPA vs lag hypocotyl and root
growth measurement* as crltaxis, the hypocotyl# of susceptible
species, pure Lane Cl&fttiflSft 2 & m m ) « * *F*«r amaranth spiooaus). were inhibited by 50 percent «t 45 end 2 ppn, respectively, while *fcs res ieteat species, floret# paintbrush (halite iayaajc*). required 7,000 ppm for equivalent inhibition. Spanish needle (Btdaaa pllasal hypocotyle were sot inhibited at concentrations up to 10,000 ppn. Soots were, is ell cases, were tolerant to DGPA then hypocotyle. Purslane end spiny ansranth roots were inhibited by 50 percent at 161 sad 40 ppn, respectively; however, flora*# paintbrush end Spanish needle roots wore not inhibited at concentrations up to 10,096 ppn. DCPA bed a pronounced effect on the elongation of hypocotyl epidermal colls end root heirs of spiny suursdh. In addition, the root heirs of the sens species wars observed to he hi^ly distorted la the presence ef DCPA. Autoradiographic studies shewed that radioactive DCPA was translocated to approximately the sans extant in hoth res latent and susceptible weed species at the cotyledon-stage cf development. The hypocotyl was shewn to ho the primary sits fron which DCPA was teens located in #11 species studied. Roots absorbed DCPA, but did not translocate appreciable amounts. By we ana ef thin layer chromatography and atrip count lot, it «ai found that DCTA-C1* wee net hydrolyand in vitro hy either aneeoptible or realatent need apeelea. The radioactive regions on the chroaatograne were detected hy weans of a strip senator. It was eenelwdnd that selective treeslocation was probably not responsible for the difference in acii selectivity in the species studied at the cotyledons tags ef decs lap- sat, k limited study ef S C M metabolism indicated that this compound one net netohellaed in extracts ef reslatent or susceptible species. LIST OF TABLES Tag® TABLE I EFFECT OF DCPA OH THU HYTOCOTYL GROWTH OF SGSCEFTIIUi A»» RESISTANT WEED SPECIES ...... 27 TABLE IX EFFECT OF DCPA OH SOOT GKW1R OF SUSCEPTIBLE AND USXSXAIff WEED SPECIES ...... 29 table m m m w G A t m of dcfa-c14 eh fqbolabi mt> G/LIMSOG 24 HOOKS AfTHR TREATMENT ...... 33 table vr mmwcAtxm or scpa-c1* id spiny M u u m AND FLORA’S PAXBTBE038 24 BOORS AflD 34 HOOKS AFIHR XRSAUGCiT ...... 34 THE PHTTOTOXICITT, TRAMSLOCATION AMD FATS 07 DIMETHYL 2,3,5,6-TETRACKLOROTHREPHTHALAl* (DCPA) IM RESISTANT AMD SUSCEPTIBLE WEED SPECIES IMTBODUCTZOM In Hawaii ona of tha aajor deterrents to the establishment of n •uccaaaful wood control progran in vegetable cropa in tha broad spectrum of wood spoclaa. Roaanowskl (1966) roportad that approximately 60 apaclaa of major importance hare boon encountered in tho ocrooalng trials conducted in tha Department of Horticulture at the University ef Saveli* Many of these speclea ere easily killed by the herbicides presently in one, but others ere highly tolerant. Growers who use a single herbicide often experience n shift in weed population to tolerant species in as few ee two cropping cycles. Dimethyl 2,3,5,6-tatrachloroterephthalato (Dac-893, DCPA) is a pre-emergence herbicide recommended for tho control of many grasses end bread-lesved weeds in vegetable crops is Bewail. DCPA has given excellent weed control In n number of locations, but has been non-tonic to n few troublesome weed species, e.g., flora's paintbrush (Stills 1evenlea), galinsaga (Calieeoxa parvlflora), Spanish needle (Bideas pilose). svinecress (Coroaopus dldvnws). jlnson wood (Datura stramonium) and other commonly found wood speclea. If tho resistant species era present in e field where DCPA le used, e shift in weed population to those species can be expected due to their resistance tod to lock of other wood competition. Tho purpose of tho present study was to investigate the phytotoxielty, tranalocation and fete of DCPA la resistant end 2 nwtptlblt weed species in order Co gain ea Insight into the selective action action of this herbicide. LITERATURE EEVIKV Scheldt sad Limpel (1962) reported thet the berbleidal properties of dimethyl 2,3,5,6-tetrachloroteraphthalata (DCPA) were discovered es e result of e joint research program between Boyce Thompson Institute for Plant Research and Diamond Alkali Co. Screening work with DCPA was initiated by Diamond Alkali Co. in the spring of 1938 with special aphasia placed on crab grass control in turf. The early field screening was so successful thet the compound was teat-marketed under the name Dacthal in I960. DCPA is presently cleared for use with 28 vegetable crops (USDA Summery of Registered Agricultural Pesticide Chemical Decs, October, 1965). In addition, it is raco— ended for use In turf, ornamentals, end some agronomic crops grown for seed, e.g. cotton, alfalfa and rice (Diamond Alkali Co.). DCPA is a white, crystalline, essentially odorless compound with a melting point of 150° C. and a solubility in water of less then 0.9 ppm (Diamond Alkali Co.). TOLIRAHCB AMD SUSCEPTIBILITY TO DCPA DCPA has exhibited a wide range of selectivity on weeds end crops. Schuldt and Llmpel (1962) reported that it has not been possible to predict which weeds or crops would be tolerant or susceptible to DCPA, thet is, DCPA toxicity was not correlated with taxonomic relationships of plants. Ronaaewskl (1966) observed, in Hawaii, thet the weedy monk are of tho Composite folly found la herbicide screening plots were especially tolerant of DCPA, e.g. gallasoga (Callaaota oervlflora). 4 flora’s paintbrush (Emilia lavaniea. formally called E. sonchlfolla, e f. Fosterg, 19*8), Spanish needle (Sitlecs pilose). and cock*1bur (Xaathlun saccharatue). Swlneereaa (Cornopus didyeua) la tha Cruelferae and jlmson weed (Saturn stramonium) In the Selanaceae are also highly tolerant. Many grasses and broad leafed weeds found in vegetable crops are susceptible to DCPA, e.g. crabgraas (Mgltarla sp.). sand bur (Ceochrus echlnatua), spiny aaaranth (Aaaranthus splnosus) and and purslane (Fortulac* oleracea) (Sea Appendix Table 1 for a awnury of weede tolerant and susceptible to DCPA in Hawaii). Sealed crepe have alee exhibited e wide range of tolerance to DCPA. In general, however, even thoee crope which ere susceptible as geminating seeds, ere highly resistant when past the cotyledon stege of development. Schuldt, Limpel and Lemont (I960) reported DCPA to be relatively inert when applied to foliege thet la, it had little or no post-eaergenee activity, making this herbicide especially useful for lay-by and over the plant treatment. STACE OF PLANT SEFBLOPMEHT AMD THE PHTTOTOXICXTT OF DCPA The affects of herbicides on pleats of the sane apaelas at various stages of develepeeat are often strikingly different. Bleeknen, Holly end Heberts (1949) reported that tbe genus Psrsvsr wss IOCS killsd by e .21 solution of KCPA bstween gemination and the production of the first leaf; however, in the period between the production of leaves end flevering, there wee a sharp decrease in susceptibility; there was complete resistance to HCPA at flowerleg. Aberg (1944) reported thet dormant and non-gsmlnated seeds are usually tolerant of herbicides, 5 •ad that herbicides tr« generally most effective when applied to plants w h m they «ro actively groving or if they hove baas weakened by active growth which exhausted their food reserves. DCPA is a pre-emergence herbicide which la applied to the soil before the weed seeds germinate. McKinley (1965) reported that DCPA did not inhibit the germination of annual ryegrass (Lollun multlflonm). but reported that it was highly phytotoxic to the coleoptlles of emerging seedlings of the same species* Komanovski (1966) observed that DCPA showed me post-emergence activity on a wide range of weeds and vegetable crops in Hawaii, however, Brown and Purtlck (1963) demonstrated some post-emergence activity of DCPA on dodder (a parasitic weed). They postulated that the dodder was being controlled systemlcally by DCPA which wee absorbed by the elfelfe. Beyer, Hoffman and Fey (1965) found DCPA to have some peet-energence activity on alfalfa, but did not conclude that the toxic reaction of dodder was due to DCPA absorbod by tho alfalfa; rather, it waa reported that tha DCPA absorbed by the dodder itself was rssponsibla for tho phytotoxicity observed. Xt should be noted that 10 pounds par acra (active ingredient) of DCPA were necessary to kill tho doddar tendrils. At tho 5 pound rata, DCPA Induced yellow-green foliage; however, growth vac not stopped. Melfeoly, ot cl. (1966) reported that no practical systemic (poot-cmcrgenee) control of dodder may bs obtained with DCPA with or without surfactant. However, tha sans authors reported that fair to good pre-emergence dodder control could be obtained with 7.5 pounds (active ingredient) per acre, but that tha tins of application was vary taper Cant, Scheldt awl L Impel (1962) reported that DCPA m u aa kighljr effective pre-emergence herbicide, hut that it had so poet emergence activity. la summery, thea, DCFA la highly toxic to susceptible meeda after t * n d M t l M hat before eaergeaee from the Mil. It la slightly toxic er non-toxic to meeda which are peat the eetyledea stage of development. He practical post-emergence control of wood apoclea h u haea reported with the peeelhle exception ef dodder (Arena end furtick, 1963) and (Sayer, Hoffman and fey, IMS). hartley ( 1 9 6 4 ) cone laded that the root ia the neat important entry point far noa-voiatila herbicide# applied to the toil, ha fawn* that braeelca alba eoald emerge without injury after 19 pound* par acre ef the iodine aalt of miOC (Siaox) m u applied to the aoil awrfaee end held there by sub-irrigation while only 2 pounds per u r e applied helew the aeed gave 190 percent kill, Siam apparently n u t cone in eeateet with the roots ef this species for phytotexieity to occur, hartley postulated that sheet entry ia important probably only far thou herbicides which ere voletlie, such u EPTC. he pointed out that the hypeeetyl is not an important pathway ef entry to* non-volatile herbicide entry bee su e it in not euily matted u it emerge* from the aetl due to unfavorable angle* of emergence. In contrast, the root ia in intimate contact with the M i l sad ia nuily matted. Bartley stated that herbieidu to the vapor state may enter the sheet even though contact with the M i l is net mad*. Appleby (1962) and Appleby, far tick 7 and Fang (1963) showed w r y good evidence for shoot uptake of EFXC as wall ae other carbonate herbicides which had previously been thought to be active primarily through roots. Appleby, et al. pointed out that the studies which lead to the conclusion that the carbamate, propham, wee absorbed primarily by roots were carried out with plants which were beyond the cotyledon stage of development. This is a criticise which can be aade of aany herbicide absorption studies involving other pre-eaergance herbicides. Additional evidence has shown that the carbaaate herbicides are active prlaarily through the shoot. Freisaa, Banting and Walker (1962) reported that the effectiveness of 2,3-DCDT (di-allate) depended upon shoot uptake end that negligible damage resulted when only roots were exposed. Dawson (1963) end Barker (1966) concluded the shoot wee primarily responsible for the uptake ef KFTC. In addition, Barker showed CDEC end CDAA to bo active through the shoots of energing seedlings. Utter (1960) proposed thet DCBA night be absorbed by the celeeptllee rather then the roots ef emerging grass seedlings. Indeed, McKinley (1963) shewed that DCBA tree phytotoxic primarily to the eoleoptiles of emerging annuel ryegrass seedlings. And Limpel (1965) confirmed that the major aits of DCBA entry into susceptible annual rye grass and Italian millet la through tho shoot as it emerged through treated soil. The evidence now et hand indicates thet DCBA, ee EBTC, depends upon uptake by emerging, serial plant parts in order to eater the plant » and Indue* « phytotoxic response. Grasses have boon studied in neat of tho DCPA phytotoxicity experiments reported thus far* DlFFg&OiTIAL OFTAKB. T1AMSL0CATI0B. METABOLISM AMP ADSORPTIOM IM RELATION TO PHTTOTOXICITT Optaks and transXoestloa. Differential uptake and translocation eon account for differences In species' tolerance to a particular barbieIds, and autoradiographic atethoda provide a useful tool for tho study of these processes, Tang and Butts (1954) shoved that carboxyl C** labeled 2,4-D was absorbed at a slower rate by corn and wheat than by beans, asking the beam store susceptible to this herbicide. Ashton (1956) found that sugar cane and bean absorb slailar amounts of 2,4-D5 however, the bean plants were wore susceptible because of a store rapid rate of translocatlon. Yaaaguchl (1961) found that ETTC-S-*5 moved primarily in the transpiration stress, that is, movement was apop lastlc. Movement of EPTC was limited when foliar application was made, but was general when applied to roots. Translocatlon patterns of EPTC were similar la resistant and susceptible species. It should be noted that Yaaaguchl'a study was carried out with plants which were beyond the cotyledon otage of development and thet no application* were made to the hypoeotyla or coleoptllea of omorging seedlings. EPTC is e pre-emergence herbicide, designed primarily to kill weeds emerging through tho soil and not plants which have reached tha trua leaf stage of development. Appleby, Furtlek end Tang (1965) reported that EPTC-C** waa readily absorbed by roots and shoots of oat seedling* as they emerged through treated soil 9 and It was translocated to aa equal extant both acropetally and baalpatally, Tha absorption and translocatlon of DCPA has boon studied at Tho Boyce Thompson Institute for Plant Research, Inc. and at The University of California* Llnpel (1965) summarised tha translocatlon studies carried out with DCPA-C1* and DCPA-Cl^* on tolerant crop species at Boyce Thostpson, Mien DCPA was applied either pro- or post-eaergenee to tha sell little, If any, was found In tho aerial parts of tolerant crop plants* DCPA remained where applied when placed on foliage either as drops of aqueous suspensions or when dissolved In nethyl cellosolve* DCPA was absorbed by tha roots of plants growing la treated soil. Crafts and Yaaeguehl (196A, p. 75) reported that DCPA moved only slightly In bean plants via the synplaat or apoplest when It was applied to the foliage( therefore, only traces were found In the stena and roots after foliage treatment* These Investigators also reported that DCPA was only slightly mobile In barley when applied to leaves with only traces being found In the roots* Crafts and Yanaguehi (1964) reported that bean roots accusmlated DCPA, but that little was translocated beyond the hypocotyl* Barley roots neither absorbed nor translocated DCPA In appreciable quantity* Fey and Yaaaguchl (1965) reported that bean foliage absorbed and moved only trace amounts of DCPA-C** to other plants parts* In the same study, when DCPA was applied to the roots of beans, small amounts (slightly greater than trace amounts) were translocated to the hypocotyl and epleotyl, and trace amounts wore found la the leaves. 10 Bayer, Hoffman, aad Toy <1965) studied the absorption and translocatlon of DCPA-C** and DCPA-C1*6 la realstaat alfalfa aad auseaptlbla dodder (Cuscata Indecora Cholay). Applications vara made to mature alfalfa leaves, steas, aad roots aad to the tendrils of dodder. The autoradiographs indicated that there vas a United movement of radioactive tracer la the alfalfa plant with almost no movement out of tha alfalfa loaf. Thera was no evidence of movement into the stem nor wee there eecunuletloa et tha leaf margin or tip. When DCPA was applied te alfalfa stems, there wee alight movement of about 2 laches in an aeropetal direction hut thore waa no evidence of DCPA accumulation in alfalfa leaves or attached dodder; however, whan DCPA waa applied to dodder tendrils there was movement of 1 to 2 Inches largely in a baaipetal direction. When DCPA vas applied te alfalfa roots there waa no aeropetal tracer movement after 1 day and only limited aeropetal movement after 7 days. There waa no accumulation of DCPA in aerial parts of alfalfa or dodder; there wee, however, accumulation of DCPA in root tipe of alfalfa. Metabolism. Shaw at al (I960) proposed that differential uptake and translocatlon of horblddos do not oatlroly explain tho broad range of tolorance ef various plant speelas to thooo compounds. Another selective aw chanlee, that of differential metabolism, is known te occur in plants of different species aad even between different varieties; it has bean proposed as a selective mechanism of major Importance. Vain (1964) and Shew et el (1960) stated that moat chemicals are metabolised in plants, but et different rates; therefore, plants nay be 11 tolerant or susceptible to a particular herbicide depending upon tha relative rate at which the herbicide is metabolised. Luckwill and Lloyd-Jonas (1960a) found the apple variety, Cox, which ia resistant to 2,4-0, to astabolix* 57X of applied 2,4-0 in 92 hours while Braaleys Seedling, a susceptible variety, metabolised only 2 per cent* The sane authors (1960b) reported that the tolerant red currant metabolised 2,4-0 to a greater extent than the susceptible black currant* However, when Luckwill end Lloyd-Jones tested their hypothesis of differential metabolism on other species, they found low rates of 2,4-0 Mtabollam in both resistant end susceptible species. They concluded, then, thet metabolism of 2,4-0 may account for tho resistance of tolerant speciaa, but that it ia not tho only mechanism in operation. Shaw at al (1960) aad Crafts (1961) have made extensive reviews of the proposed mechanisms for the metabolism of various groups of herbicides. Evidence has bean presented for the metabolism of DCPA. Skinner, Stallard end Prlddle (1964) reported that CCPA under went a two-step hydrolysis under field conditions to monomethyl 2 ,3,5,6- tetrechloroterephthalate (%-eeld font) end 2,3,5,6-tetrachlorotere- phthalic acid (acid form)* However, they reported thet the metabolism of DCPA takes place primarily in the soil with subsequent uptake of the metabolites by the pleats* Only very email amounts of DCPA have boon shown to ha metabolized by plants. Satisfactory procedures for the detection of DCPA in plsnts and sell have boon reported. Schuldt et al (1961) reported a colornetric method for the determination of DCPA* And Priddle, Stallard and Skinner (1964) compared the colormetrlc method with a gas chromatographic 12 method. Skinner, Stallard and Priddle (1964) davalopad a gas chroma to graphlc method for tha determination of tha metabolitaa of DCPA. Limpel (1966) raportad that D. £• Skinner had davalopad a thla layer chromatographic aatbod for tha reparation of DCPA aad ita metabolite*. Skinner used pre-coated thin layer chromatography aheeta with a fluorescent indicator. The solvent system vas butanol, water and acetic acid Still v/v/v, and tha apota vara detected under ahort vava ultraviolet raya. Adsorption. In addition to those selective mechanisms already mentioned, plants may inactivate herbicide molecules by adsorbing them on altaa which do not produce a physiological response, e.g. a protein molecule. Brian (1958. 1960) obtained a broad correlation between tho ability of plant extracts to adsorb MCPA and tolerance to this herbicide, that is. those species whose extracts adsorbed MCPA to tha greatest extant ware tha least susceptible. To tha author's knowledge, there have bean no published reports dealing with tha adsorption of DCPA to plant components. SYMPTOMS OP DCPA IMJUKT McKinley (1965) observed that DCPA induced enlargement of tha basal node of tha shoots of annual ryegrass as wall aa causing tha production of short enlarged roots. These "cancerous" enlargements, ha reported, vara probably due to call proliferation in tho merlotenatlc region. McKinley observed that tha symptoms of DCPA Injury are like thoee produced from an excess of a compound such as 3-lndoleacetle acid (1AA). 13 Bo reported thet DCPA et eub-lethel doses stinulsted shoot end root growth, end et lethel doses It wee more toxic to ehoote then roots. A eery fine line of division wee reported between doeee which stlmuleted end those which inhibited shoot end root growth. Komenowskl (1966) reported thet even those susceptible weeds end crops which lnltielly eseepe DCPA Injury In the field remsln stunted ere ttsuslly killed before they resch neturlty. Sesskl end Koslowskl (1963 e,b,c,d) reported thet DCPA did not effect the gernlnetloe, photosynthesis or resplrstiea of Plnus reslnoee seedlings. MATERIALS AMD METHODS The phytotoxicity, translocatlon sod metabollsa ef DCPA la both resistant end susceptible eotyladoa-stage weeds were studied la the following experiments. FHTTOTPXICITT 8TPDIIS Screening. DCPA was screened in the laboratory against susceptible and resistant weed species. The Latin aaaes of the weed species used la the studies ere given now te avoid repetition later. The following coaaoa naaes will be used throughout the text: Aaaranthus snlnosea (splay amaranth), Bidens pilose (Spanish asedls), Emilia ievaalca (flora's paintbrush), Cellneoee aarviflora (galiasoga), aad Portulece olsracaa (purslane). The effects of DCPA on two susceptible species, spiny amaranth and purslane end two resistant species, flora's paintbrush end Spanish needle, were determined in four eoperato experiments under comparable conditions. Seeds were pre-germiaated la covered Petri dishes on flltor paper which contained 5 ml of half-strength Uoeglend's solution, (Roaglend end Arraon, 1950) la order to reduce the variability in the amount of elongation of hypocotyla end roots. Just after the amargeaee of the primary roots, ten seedlings were transferred to each of 5 Petri dishes which were lined with e double layer ef filter paper. Tha susceptible species were treated with 0,1,5, 10 end 50 ppm (active ingredient) of DCPA, while resistant species were treated with 0, (1,000 or 3,000), 5,000 7,000 end 10,000 ppm (active ingredient) of 15 DCPA. Sine* DCPA is insoluble in water (solubility less then 0.5 ppm), five al of aqueous suspensions of the above treatments were applied to the Petri dishes by n e w s of an atoslser. Each treatment was replicated three times In a randomised block design. The Petri dishes were placed In the dark at 24° +2° C. for 72 hours aad immediately thereafter the seedlings were fixed la FAA. In order to assay the phytotoxicity of DCPA to the susceptible and resistant weed species, hypocotyl and root measurements were made of the test seedlings. The data were expressed as percent Inhibition according to tho following formulat poreeat inhibition ■ 100 - x 100 check average Percent inhibition of tho roote end hypocotyle ves plotted on a probability acala against the Log^0 ef the DCPA concentration, thet la e problt transformation was made. Cytologies! Studies. Some cytologies! affects of aqueous suspensions ef DCPA were Investigated. Splay amaranth aeede were pre-germiaatod then placed In Petri dishes lined with filter paper to which 0, 1, 5, 7, end 10 ppm of DCPA suspensions had bean added Is 5 ml of dlatlllod water. After 40 hours et 35° C. the seedlings were harvested, stained In aqueous aafranln, and mounted on microscope slides In 5 per cent glycerine. Beth the hypocotyl epidermal cells and root haIra wars observed for aboomslitlas. UPTAKE Am T&AM8LOCATIOM STUDIES Two studios were made ef the site of entry end translocatlon of 16 DCPA ia r«tiiMRt and susceptible used species at tba cotyledon-stage of development. DCPA-C14 was utilised in eech experiment and the results v a n baaad on interpretation of autoradiographs * Preparation of Radioactive PCPA Stock Solution. A 73 ag sample of methyl eater-labeled dimethyl 2,3,5,6-tatrachloroteraphthalata (DCPA-C1*) having a specific activity of X.Yl/i c/og was received from Boyce Thompson Institute for Plant Research Inc. The intensity of the beta ray emission vas 830 cpm/dg. The entire 73 ng sample was placed la solution la 3,3 al of methyl eellosolve (moaoaetbyl ether of ethylene glycol). This stock solution containing ,0283 c/Al vas used ia all traaslocstlon and metabolism studies reported herein. Translocatioa Experiment dumber 1. The purpose of this esperiasat vas to determine the site of uptake sad pattern of translocatioa ef DCPA ia susceptible purslane end resistant gsllaaoga. Saads of oach of these species vara sown on filter paper la Patri dishes. When the hypocotyle of the seedling* were approximately 1% cm long they vara transferred to individual 9,4 x 2,1 cm glass vials which vara supported by aaaas of aluminum capo and lanolin plugs (Pig* Z), The viola, containing one plant each, vara filled with 33 al of one-half strength Boatload's solution (Besglaad sad Arroa, 1930)and placed ia a growth chamber far 24 hears before treatment. The chaaber wee oat for so 8-hour day at 29° C. sad a 16-hour night ot 21°C. 14 DCPA-C was applied to the cotyledons and hypocotyle by aaaas of s syringe sad to the roots with o micro-pipette. The cotylodoao sad 17 hypocotyl* were treated with l«s* than 1/x 1 of tho DCPA stock solution rad tho roots wot* trsstod with 5 U 1 of tho stock solution through tho nutrient solution* Methyl cellosolve-treated control plants wars also grown. Kach trostnrat was repeated four tines. Tho plants wore harvested 24 hours after trostnrat and wero prepared for autoradiography* The plants wore renewed fron the vials and tho roots, hypocotyl* and cotyledons wore separated so that no translocatlon would occur during tbs drying process. After separation of tho parts, tho plants wore placed between two layers of waxed paper and two 3" x 5” Index cards. Tho pressed plant speclneas wore than placed In an oven sat at 60° C. for approximately 12 hours. Upon renewal fron tha oven, the plants wore carefully nouated on white paper backed with cardboard. The shoots containing the nouated eeparated plant parts were autoradiographed against Kodak no-screen X-ray flln for 10 days. Subsequently, tha fllns were developed for two nlautea, fixed aad than dried. The latenaltlse of tha Inages on the flln wore analysed subjectiwely as follows! 0-no Image, 1-trace Image, 2-low Intensity lasge, 3-noderate lnage, and 4-lntense image. Translocatlon Experiment Somber 2. Tha site of uptake aad tho tranalocatloo ef DCPA-C** ware studied In susceptible splay ansraath aad realataut flora's paintbrush. Plants of tha abewe species, at the cotyledon-stage of devalopnent, were attached to tho rlns of 9.4 x 2.1 co glasa wlals by naans of lanolin (Pig. XX). The vlala wore filled with 33 cc of one-half strength Boatload's solution. Three plants were supported on the rln of each wlsl aad the plants were treated 24 hours 18 cotyledon treatment Figure 1 - Location of DCPA-C-*-^ treatments in experiment 1. Figure 2 - Location of DCPA-C^ treatments in experiment 2. 19 >ft« u t « b U « h M B t la tha vlaii. Tha treatments vara applied to tha cotyladons, hypocotyls aad roota aa la Experiment Number 1. Tha hypocotyl* aad eotyladoaa vara topically traatad vith lass than l/ll of DCPA-C14 stock solution vhlla tha roota vara traatad vlth 5 A 1 of stack solution. Tha DCPA-C1^ applied to tha hypocotyl vas hold la plica by aaans of a lanolin ring aad each traataent vas replicated 6 times vlth each spades. Tha plants vara grown under lov Intensity fluoraacant light at a temperature of 24° ±2° C. Three replicates vara harvested at 24 aad 36 hours* respectively, after treatment. Control plsata vara traatad vlth aathyl callosolva aad harvaatad aa tha treated plants. The plants vara prepared for autoradiography as in Experiment Humber 1; however, tha flln waa exposed 7 days Instead of 10. Tha sane subjective rating system was used as In Experiment Number 1. 18 VITRO METABOLISM STUDIES Tha fata of DCPA vaa studied In vstar extracts of tvo susceptible weed species, splay amaranth and purslane aad tvo resistant weed species. flora's paintbrush and Spanish needle. Seeds of tha above spadaa vara germinated la aaparata Petri dishes on a doable layer of filter paper aad grova to tho cotyledon stage of development. One gram of plant notarial* including all plant parts* vas collected from each Petri dish. Tha material vaa ground, extracted la 10 ml of distilled water* divided Into two 3 ml portions aad placed la glass vials. One 3 al portion received 20 /tfl of the DCPA stock solution aad 20 a 1 of aathyl callosolva (the solvent used to put DCPA Into solution) wero added te the eecoad 3 al portion. TweatyA1 of DCPA stock solution were also added to a 20 vial containing distilled water. The vials were incubated at 35° C. aad 1 al saaplas ware drawn froa each at 30 and 72 hours after treatment. The 1 al saaples were evaporated to dryness ia watch glasses ia a hood and 1 al of aathyl cellosolve vas added. This solution was evaporated to .4 al to increase the concentration of DCPA and/or aetabolltes. Aliquots of lOjill were spotted on 1 inch stripe of pre-coated Eastman Chronogram TLC sheets (type K 301 ft, silica gel with fluorescence indicator). The strips were developed in a amyl alcohol (n-pentanol)~glaclal acetic acld-water (8:1:1 v/v/v) solvent system. The TLC strips were spotted with DCPA-traated extracts froa resistant and susceptible weeds. DCPA-G^* standard aad a solvent standard were scanned with a Packard radiograa scanner Model #7200. The linear range of the machine was 1,000 with a tins constant of 30 and a scanning speed of 1 as per minute. The else of the collimstor wss 1 cm2. In a preliminary experiment, the Rf values were determined for pure non-radioactive saaples of DCPA and its metabolites, aononethyl 2,3,5,6-tetrschloreterephtbalate ana 2,3,5,6-tetrachloroterephthallc acid. The spots were scanned under short wave ultraviolet (uv) rsys. DCPA and its aetabolltes sbsorbsd uv. in this region, hence on the TLC strips with fluorescence indicator thou showed up ss dark spots against an orange background. STATISTICAL METHODS Analysis of variance was used to determine the significance of troataent affects in the DCPA phytotoxicity study and Duncan's multiple 21 range cast vaa used to daternine If tha treatment means vara significantly different. Double asterisks Indicate significance at the 1 percent level and a single asterisk Indicates significance at the 5 percent level. Means vhleh are connected by lines are not significant at the 5 percent level. Probit analysie vas used to transform the sigmoid inhibition response of weed seedlings to a atraight line* the eye-fit method vas used to plot the dosage response line as described by Coving (1959) and Roberts (1965). RESULTS ADD DISCUSSION PHYTOTOXICITY 8TUDIES Screening. Using root end hypocotyl elongation aa criteria for toxicity, DCPA vaa screened against tvo susceptible vend species, spiny aaaranth and purslane, and tvo resistant veed species, flora's paintbrush and Spanish needle. The effects of DCPA on hypocotyl and root elongation of the above susceptible aad resistant veed species are given in Tables I and IX, respectively aad the results are statistically analysed ia Appendix Tables 2-8. It should be noted that DCPA vas screened against each species in separate but comparable experiments. The resulte indicated thet there ves e very broed range of tolerance to DCPA exhibited by the epecies tested. Those species vhlch have been termed susceptible es e result of field screening required e relatively lev concentration for 50 percent inhibition of roota end hypocotyla while the epeclee vhlch have been termed resistant required ouch higher concentrations. The percent inhibition of hypocotyl end root elongation, on a probability seals, vaa plotted against tha Log1Q of tha DCPA concentration (figs. 3 and 4). A probit analysis vas mada of tha rasulting doaaga response llnaa. By either interpolation or extrapolation, tho doaaga necessary for 50 parcant lahlblton of hypocotyl* or roots vas determined from tha doaaga response lines. Tha hypocotyl* of tha tvo susceptible species, purslane and spiny amaranth, were inhibited by 50 parcant at DCPA concentrations of 45 and 2 ppm, respectively, while the raalstant flora's paintbrush required 7,000 ppm 23 for an equivalent amount of inhibition (Fig. 3). Spanish needle hypocotyl* vara not significantly inhibited at 10,000 ppea (Table I). Boots of all the species tested vers sere tolerant to DCPA than vara hypocotyl*. The roota of the susceptible species, purslane aad spiny aaaranth, were inhibited by 50 percent at 101 and 40 ppn, respectively (Fig. 3). The roots of flora's paintbrush and Spanish needle vara not inhibited at rata* up to 10,000 ppn (Table III). In feet, stimulations vara observed in the roota of resistant species which probably would have bean significant at tha 5 percent level had mere replications bean made. Spiny amaranth vas shown to bo nor* susceptible to DCFA than purslane, a result that la surprising in light of field results which indicate that tha opposite is usually tha case. Amaranth any, indeed, be inherently more susceptible, but under field conditions it nay be more tolerant. Tha larger nunber of amaranth escape* that ara observed in DCFA-treeted fields as compared to puralan* escapes nay be due to a larger number ef aaaraath seeds on the surface of tho soil. McKinley (1965) shoved that annual ryegrass (Lollun nultlflorun) could emerge from treated soil if the seeds war* placed on the noil surface aad postulated thet thla species escaped DCFA Injury because it* coleoptlles vara not ia contact with tha soil. McKinley shewed, ia another experiment, that relatively low concentrations of DCFA could kill emerging annual ryegrass if tha coleoptlles ware in contact with tha soil while much higher concentrations vara necessary to kill this species if only tho roots wars exposed; however, the slightly higher 24 toliruca of spiny m n a t b la tho (laid nay also bo daa to nriatal roslotonco. Aaaranthua is known to hybridise freely end ouch norphologlcel variation of this genua has been observed Is Bewail by the author. Cllaatologlcal end edaphic factora undoubtedly also ploy e role in selectivity of DCFA to veed speciea in the field. Much useful infornetion eea be obtained fro* the dosage response lines (Figs. 3 and 4). DCFA vas screened against the faypoeotyls of purslane aad aaaraath seedlings ever a relatively wide concentration range, that is fro* 1 ppa to 30 ppa; however, increase la inhibition per wait ef concentration Increase vas rather sea11. that is ths slopes of tha dosage response lines vers shallow. Coving (1959) pointed eat that if a dosage response line had a shallow slope, the addition of higher concentrations would sot bring about a worthwhile increase in the percent ef inhibition, end therefore would not bo ocoaeaicel. On tho other heed, if the elope of tho dosage response line wee eteep. the addition ef higher concentrations would bring about e disproportionate aaouat ef inhibition and would bo oeonoaical. The dosage response line far flora's paintbrush vaa nuch ataapor than those for aaaraath and purslane; however. It should bo noted that tha high concentrations necessary to Inhibit tho growth of this species probably would not bo oeononlcal under any eurewaatanees. Cvtolosical swptoaa. Tha aplderaal calls of hypocotylo ami tha root hairs of spiny aaaraath seedlings were observed after they had boon grown for 60 hours in 0. 1. S. 7. and 10 ppa of DCFA. It was observed that DCFA had a pronounced inhibitory affect on tho elongation of 25 hypocotyl epidermal colls end root hair* of spiny nartnth. In additon, tho root hoiro of splay amaranth produced ia the presence of DCPA vere observed to be distorted, thet Is, they vere twisted end often spiralled instead of straight (Fig. 5). The shortened, distorted root hairs produced in the presence of DCPA ere certainly impaired functionally. It le possible, therefore, thet pert of DCPA'e tonicity to spiny amaranth aeedlings is due to impaired uptake of nutrients due to distortion of root heirs* The distortion end inhibition of root heirs tree very such greater et 5 ppn then et 1 ppn, but concentrations greater then 5 ppn Induced little added distortion or inhibition (Pig. 5). McKinley (1965) observed thet DCPA caused e proliferation of cells la the epical aerIstenet1c regions ef both root end shoots of susceptible annuel ryegrass (Lollun aultlflorua). This ‘'cancerous growth,** he reported, caused the laaf sheath to rupture aad exposed the internal proliferated area, McKinley reported thet the symptom of DCPA injury to annuel ryegrass were such like those of growth regulators such as IAA. In tho dicot pleats studied herein there wee no obvious proliferation of cells in the epical nerlstenetic regions; hovevsr, bulgss wars observed In the region of the hypocotyl just above the junction with the root in spiny aaaraath. When this region was observed microscopically, it appeared thet the bulges vere not the result ef cell proliferation; on the contrary, the lateral ealergeaent ef Individual cells seeaed responsible. It is possible thet DCPA acts on 26 monocots differently than on dlcot*; In any ease, it ia highly toxic to certain species of both sub-classes of higher plants. Other species of weeds, (flora's paintbrush aad Spanish needle) were shown to be highly resistant to DCPA when hypoeotyls and roots were used as indicators of toxicity (Tables I and II). Roots were observed to be more tolerant to DCPA than hypoeotyls in all the species tested. In fact concentrations as high as 10,000 ppa of DCPA did not Inhibit the roots of flora's paintbrush and Spanish needle. These results are in agreement with McKinley (1965) who reported DCPA to be more toxic to the coleoptlles of emerging annual ryegrass than to the roots. McKinley observed that the coleoptlles of annual ryegrass were stimulated (18%) at 72° P. ia soil at a concentra tion of 1.6 ppm DCPA and inhibited at 2 ppm (76%). Roots were stimulated (40.2%) at 1.6 ppm aad inhibited (671) at 2 ppm. McKinley's results indicated that there waa a very narrow dividing line between stimulation and inhibition of the roots and shoots of annual ryegrass by DCPA. Since some growth regulator properties of DCPA have been demonstrated, this chemical might be experimented with in tissue culture media or in propagation studies. The results of the phytotoxicity studies offer some interesting areas of speculation as regards the possible mechanisms by which DCPA kills plants. Dicot weed seeds which germinate under the surface of the soil depend upon hypocotyl elongation ia order to emerge and begin to phyotsynthesIsa. Since DCPA was shown to inhibit hypocotyl elongation, it can be hypothesized that tha prevention of hypocotyl 27 TABLE I. EFFECT OF DCFA OH THE HYPOCOTYL GROWTH OF SUSCEPTIBLE AMD RESISTANT WEED SPECIES. * DCPA Average Percent Species Concentration Hypocotyl of Percent (ppn) Length (on) Check Inhibition b 0 (check) 19.75 100.0 0.0 Spiny 1 12.20 61.7 38.3 Aaaraath (S) 5 7.36 c 37.3 62.7 10 6.80 34.4 65.6 50 5.60 28.4 71.6 0 (check) 11,13 100.0 0.0 1 8.16 73.2 26.8 Purslane (S) 5 6.77. 60.7 39.3 10 6.46 57.9 42.1 50 3.7*1 51.9 48.1 0 (check) 30.2 100.0 0.0 Flora's 3000 25.2, 83.4 16.6 Paint- 5000 22.01 72.8 27.2 brush (R) 7000 16.8, 55.6 44.4 10000 12.8' 42.4 57.6 0 (check) 52.2 100.0 0.0 Spanish 1000 49.5 94.8 5.2 Needle 3000 50.0 95.8 4.2 7000 45.0 86.2 13.8 10000 43.3 83.0 17.0 a - Separate experiments under coeparahle conditions, b - Percent inhibition equals 100 less percent of check, c - Means connected by lines are not significant at 5 percent level. (Duncan's Multiple Range Test) (S) - Susceptible species. (K) - Resistant species. INHIBITION (PROBIT) I C ACNETAIN PM ) PPM ( DCPACONCENTRATION 29 TABLE ZI. EFFECT OF DCFA ON ROOT GROWTH IN SUSCEPTIBLE AND RESISTANT WEED SPECIES. * DCPA DCPA Percent Species Concentration Root of Pereeat <«*> Length (os) Check Inhibition® 0 (check) 10.28 100.0 0.0 Splay 1 8.62 83.8 16.7 Aaaraath (S) 5 6.96 67.7 32.3 10 5.99. c 58.3 41.7 50 5.371 52.2 47.8 0 (check) 5.56, 100.0 0.0 1 4.941 88.8 11.2 Purslane (S) 5 4.21 76.6 22.4 10 4.0 0 (check) 24.4 100.0 0.0 Spanish 1000 23.9 98.0 2.0 Needle 3000 26.5 108.6 - 8.6 7000 25.6 104.0 - 4.9 10000 26.7 109.4 - 9.4 a • Sap&rat:* experiments under comparable conditions, b * Percent inhibition equals 100 leas percent of check, e - Means connected by lines are not significantly different at 5 percent level. (S) - Susceptible species. (R) - Resistant species. Vo INHIBITION (. PROBIT ) CA CONCENTRATION DCPA (PPM) 31 A. B. C. Figure 5 - Root hairs of spiny amaranth seedlings grown for 60 hours in A. Hoagland's solution (check) B. 1 ppm DCPA C. 10 ppm DCPA. The lengths are approximate values from the uppermost region of the root. 32 growth M y bo o primary (actor la the herbicldal activity of DCFA. Tha pussling oaeapoa of aaacoptlbla wood apaclaa la tho DCFA treated (laid arc probably due, ia oomo cocoa, to sooda which have germinated oa or near the surface of the coll aad have had little or no hypocotyl-coil cobtact. Indeed. McKinley (1965) shoved a direct correlation between DCFA injury to annual ryegrass and the depth of aeed cowing, that la, the aeeda which were cloae to the aurface of the aoll vere injured least by DCFA. Those species which escape DCFA Injury because they M y be Inherently resistant, e.g. flora's paintbrush, gallasega, Spanish needle, probably would not be controlled under any circuMtances. If a farmer who uses DCFA notices a build-up of resistant species be should either use a different herbicide or mix DCPA vlth other chemicals so that a broad spectrum of weed species will be controlled. The laboratory bioassay of DCFA toxicity reported herein Is a fast Mthod for determining the relative tolerance of plant species to this chemical. This assay should be used to establish the tolerance of otbor plant species te DCFA. Perhaps e eyetoMtlc study of this typo produce o phylogenetic pattern of DCFA toxicity. UPTAKE AMD TRAM8LOCATION STUDIES Tho dote depleting the extent of DCPA-C14 translocatlon at 24 hours after treatment In the susceptible speclaa, purslane, and tbs resistant species, gsllnsogs, 24 hours after treatment, are given In Table III. DCFA-C** was applied either to the hypoeotyls, cotyledons, or roots of tho experimental plants and the extant of Its translocatlon from each 33 type of application waa rated subjectively. The ratings were baaed on the Intensity of images on autoradiographs (See page 17 for rating system). The purslane data indicate that all tha treated plant parts absorbed equal amounts of tracer, but tha DCPA-C1* which was absorbed through the hypocotyl moved to other plant parta more readily than that which was absorbed both through the roots and the cotyledons. The pattern of DCPA-C** translocatlon in gallnsoga was similar to that in purslane under the conditions of the experiment. The patterns of DCPA-C** translocatlon in susceptible amaranth and resistant flora's paintbrush are given in Table IV. In this experiment one group of plants of each species vas harvested at 12 hours after treatment and another at 36 hours after treatment. In flora's paintbrush, at both harvest times, DCPA-C1* vas absorbed to the same extent by hypoeotyls and cotyledons; however, the DCPA-C** absorbed by the hypocotyl readily moved into tha cotyledon while that which was absorbed by the cotyledon moved only in trace amounts to the hypocotyl. DCPA-cl* absorbed by the root did not move to either the hypocotyl or the cotyledon. The translocatlon of DCPA-C** in flora's paintbrush is illustrated in Appendix Pigs. 1-4. DCPA-C1^ was absorbed equally by hypoeotyls and cotyledons of spiny amaranth at both 12 aad 36 hours. Unlike flora's paintbrush, only trace amounts were translocated froa hypocotyl to cotyledon. Similarly, the root moved only trace amounts to the cotyledon. In one case a trace amount of radioactive tracer waa found in one of the amaranth control plants, therefore, trace amounts found in treated plants might be Dimethyl 2,3>5.>6-tetrachloro Monomethyl 2,3,5,6-tertachloro 2}3,5,6-tetrachlorotere terephthalate terephthalate phthalic acid (DCPA) (^~-acid form) (acid form) Fig. 6 The two-step hydrolysis of dimethyl 2,3,5,6-tetrachloroterephthalate 35 TABLE 111, TBANSLOCATION OF DCPA-C14 IH PURSLANE AMD GALINSOGA Subjective Ratings ^ (24 hours sftsr treatment) Species Psrt Treated Psrt Rated Hwocotyl Cotyledon Root Purslane hypocotyl 4 b 3 3 cotyledon 2 4 0 root 1 1 4 check 0 0 0 Gallasoga hypocotyl 4 3 2 cotyledon 1 4 0 root 1 8 4 cheek 0 0 0 a - Subjective Retlugs: 0 - so image, 1 * trace image, 2 - lev intensity image, 3 - moderate image, and 4 - intense image. b - Average ef 4 replications, rounded to nearest whole number. 36 TABLE XV. TRAHSLOGATIOH OF DCPA-C14 IK SPINY AMARANTH AND FLORA’S PAINTBRUSH Subjective Ratings * (12 hours after treatment) Species Part Treated Part Rated RzKSftfjrA Cotyledon Root Flore’s Hypocotyl 4 b 3 0 Paintbrush Cotyledon 1 4 0 Root 0 0 3 Cheek 0 0 0 Splay hypocotyl 4 1 0 Amaranth Cotyledon 0 4 C Root c 1 1 Cheek 0 0 0 (36 hours after treatment) Flora’s Hypocotyl 4 3 0 Paintbrush Cotyledon 1 4 0 Root 0 0 3 Chock 0 0 0 Splay Hypocotyl 4 1 0 Amaranth Cotyledon 0 4 0 Root 0 1 2 Chock 1 0 0 a - Subjective Ratings: 0 - no image, 1 - trace image, 2 - low Intensity iaage, 3 - moderate imago, and A - intense imago. b - Average oC 3 replications, rounded to nearest whole number. 17 artifact*. D m root* of splay amaranth absorbed lass DCPA-C14 from tho nutrient solutioa than did roota ef flora's paintbrush. Since the susceptible and raaiatant apaclaa in both experiments had similar absorption tranalocation pattarns, it appears that those processes are set important nochsnlsws ef selectivity. It sheaid ha noted, however, that no attempt was made to identify the radioactive compound after it wee absorbed and treaalocated by the pleat* Since methyl eater labeled OCPA-C1^ wee need, hydrolysis within the plant could yield radioactive nethanol which by iteeif or in combination with pleat chemical, components could result in e positive tracts location test (fig. I), therefore, the radioactive c o m p o u n d within the plant any net he OOPA; rather, it any he n metabolite* However, experimental evidence la presented later which indicates that tin DCPA is net neteheliasd in water extracts ef reals teat and susceptible eeed spec lea. DCPA has been reported to be very ianeblle in plants by several investigators, Crafts and Yanaguohi (1964), fey and Yamagecbi (1965), Bayer, Hoffman and Toy (1965), and LImpel (1966). However, ia all ef these studies, plants were used which ware beyond D m cotyledon stats sf development end so treatments were mode to hypecetyls* In D m present study, OCPA-C1* was found to be nehile in D m eeropetnl direction when applied to the hypeeetyls ef ell the species studied with ths exception ef spiny amaranth. In this species trams amounts wars translocated from D m hypoeotyl. McKinley (1965) shewed that DCPA which wee taken by emerging eeleeptiiee ef grasses wee mere phtotexic than that which was taken up by the roots. 38 In the present study, roota were ohowu to absorb aad accumulate DCPA bat vary little traaslocation occurred, There appeared to be a bleak at the point of Junction of the root and hypocotyl which would not allow the passage of DCPA to the aerial parts through tha aylas. It la not known whether tha block waa produced la response to DCPA treatment or If It sue an inherent characteristic. It wee shown In the purslane and galincogs experiments (Table IV) that DCPA could move freely Into tha roota frou tha hypoeotyls; hence, this is good evidence for phloem move- sent. However, the bulk ef DCPA applied te the hypocotyl was translocated la the xylea to the cotyledon. The lack of DCPA movement frou the rests night explain tha lack of phytotoxicity that McKinley observed in susceptible annual ryegrass when only Its roots were exposed to DCPA, Tha greater aaouat ef translocatlon frou tha hypoeotyls of susceptible specie# sight also explain tha toxicity of DCPA to cuarglag grass when only the coleoptlles are exposed. Other DCPA trenslocation cxperiueats should be sade using cotyledon stags plants and special emphasis pieced on the role ef hypocotyl uptake. Studies should also be uade of the proposed block of DCPA translocatlon frou root to hypocotyl. future experimental design ef DCPA trenslocation experiments should Include plants which ere treated for longer periods of time, e.g. 7 days instead of 24 aad 36 hours, aad plants that are traatad ia tha dark. In addition, temperature, humidity and light conditions should he kept mere constant and the treatments should be applied more accurately with the eld of e ulcroeyringe. 39 IH VITRO METABOLISM STUDIES The results of two experiments Indicated that DCPA-C1* waa not natabolixad (in vitro) in either resistant or susceptible weed species. All the thin layer chromatography stripe which vere spotted with the plant extract DCPA-C*^ treatments and those spotted vlth the DCPA-C** standard had radioactive peaks at the Ef value of one* The solvent- treated strips had 00 radioactive peek. The results of the netabellsn study are in agreement with Skinner et al (1964) who reported that DCPA was metabolised only slightly in plants* The major portion of the DCPA metabolites found in the plant* he postulated* was absorbed froa the soil* The experiment reported herein* should be repeated using living plants instead of water extracts In order to verify the preliminary obeervatlons found with water extracts* The Rf values for the non-radioactive DCPA and its metabolites monoaethyl 2,3,5*b-tetrachloroterephthalat* (**~acid fora) and 2,3,5,6- totrachloroterephthallc acid (add fora), were determined in e preliminary experiment te be 1* *71, end *071* respectively. SUMMARY The phytotoxicity, treeslocation end fete of DCPA were studied ia both realateat ead susceptible weed species ia order to gain an insight into the selective Mechanise of this herbicide. The meet important findings ef the report ero summarised below. 1. The hypoeotyls of two susceptible weed species, purslane ead splay eearanth, were inhibited by 50 percent et DCPA concentrations of 45 and 2 ppn, respectively; whereas, tha hypoeotyls ef tho resistant species, flora's paintbrush, required 7,000 ppa for aa equivalent Mount of inhibition. Spanish needle hypoeotyls were not significantly inhibited at 10,000 ppa. 2. loots of all tha species traatad were aore tolerant of DCPA then the hypoeotyls. The roota of susceptible purslane ead spiny aaaraath were inhibited by 50 percent at 101 aad 40 ppa, respectively. The roots ef flora's paintbrush aad Spanish needle were not inhibited at 10,000 ppa. 3. Tha order of increasing tonicity of DCPA to tho speclas tested waa spanlah needle, flora's paintbrush, purslane aad spiny aaaraath. 4. DCPA inhibited tha elongation of hypocotyl epidermal cells aad tha root hairs of spiny amaranth. Tha root hairs of spiny aaaraath produced ia tho presence of DCPA wore highly distorted and wore certainly functionally impaired. The toxicity of DCPA aay bo due ia 41 part te the Inhibition end distortion of root hairs. This aechanlsa is probably aost Important whan seeds garalnate on the surface of tba sell and have Halted hypocotyl contact vlth DCFA. 5. Selective absorption and translocatlon of DCFA did not appear to account for tha wide range of tolerance observed with tha susceptible aad resistant weed species. 6. In all of the species studied with the exception of spiny aaaranth, the DCPA which wee absorbed by the hypocotyl was translocated to a greater extant than that absorbed by tha cotyledons aad roots, loots translocated tha least DCPA of tho three plant parte traatad; however, roota appeared to aceumalata DCPA. 7. DCPA vaa translocated wore readily In an aeropetal direction than In tha baslpltal direction whan applied to tha hypocotyl. 8. A United study shoved that DCPA vaa not Metabolised la water extracts of either susceptible or resistant weed species. APPENDIX 43 APPEMDtX TABLE SUHBES I - The Tolerance to DCFA of So m Coaoealy Found Woods la Kawaii. • Scientific Km n Cosuson Maas family _ Katina b Aoaraathua splnoaus spiny aaaraath Aoaranthacaaa M Bldeus pilose epanish noodle Coaposltas T Cenchrus_ achlwtus sand bur Graninas S Coreaopos didjaus swlaaerass Crucifaraa T S3aSJOS. purple aut sedge Cyparacsae T l^tura strMwaiuo jInsea wood Solaaacaaa X fJPE* crabgrass Graniaaa S & £ * * & £ • wire grass Craninaa X M U * MbsbAsa flora'a paintbrush Coaposltaa T H ixta cardan spurgs Ruphorblacaaa S M U l S M L £arvlflore gallnaoga Conpoaltae T & £ t * U s a olgracca purslane Portulacaceaa S UftM&U SSfMt richerdie Kubiaceae X-I bristly foxtail Graniaaa 8 Solanua nodiflorun popolo Solaaacaaa 1>I IffiM&iSS jnfSfcttfttS ckcklsbur Conpoaltae X a - Adapted f t w Ronaoovahi (1963) Hava 11 Pans Science, vol. 34, no, 1 and unpublished data of Dapartaeat of Horticulture, University of Eavall. b - T - Tolarant X - Internedlat* (lnflstanced by sail typo) 9 - Susceptible 4 4 Appendix 2 - The Effect of DCTA on tho Growth of Purslane Hypocotyl* Average hypocotyl Length (sen) Replication ...... -...... Treatnent (ppn)...... Sun 0 ...1 5 10 . M 1 10.64 • 8.34 6.92 4.83 5.68 38.65 2 12.16 8.31 6.51 6.22 6.00 39.20 3 10.64 7.83 6.88 6.32 3.50 37.17 Sun 33.46 24.43 20.31 19.39 17.38 113.02 Mean 11.13 8.16 6.77 6.46 5.79 Analyaia of Variance Table Source i l H a* Ireatnent 4 54.48 13.62 ** Keplicatlon 2 .44 .22 no Error 8 .... 1*7?...... 213 &.&L ...... M ... Cogporleono of Hooa Dlfferencee b kisssl M b s ! ______Ifitiasl 5 0 ( p p o ) 3.36 ** 2.37 ** ,98 * .67 1 0 (ppn) 4,69 ** 1.76 ** .31 — 5 (ppn) 4.38 ** 1.39 ** «• ■S M N M M l(ppn) 2,99 ** wmm m m m m a - Average of 16 individual hypocotyl* b - Duncan'* Multiple Range Test ** - Significant at 1 parcant level * - Significant at 3 parcant level aa - Sot significant 45 Appendix 3 • 9 m Effect ef DCPA m 9 m Growth ef Purslane Boots Average Root length (taa) Replication ..... X m a a n a e B CecMil 0 10 50 1 ^ 4 5 « ^ 4 3.83 3.53 21.87 2 6.41 5.40 4,58 4.37 3,28 24.04 3 4.84 4.38 3.38 4.08 3.16 20.64 Sen 16.30 14.82 12.78 12.28 9.97 66.55 Mean 5.56 4.94 4.28 4.09 3.32 Analysis. of .Variant* Table.. Source Oft la s I freemens 4 8.257 2.189 ** 18.09 Replication 2 1.186 .593 * 4.90 1£f snar — . »______* 9 6 9 .... .121... Jotal mmm mmm — J I t — J f a l R ... S & m a l ------U m s l ______M m ? ______Igjtel 50(pj#s) 2.24 ** 1.62 ** .94 * .77 * KKppn) 1,47 *• .85 * .17 5 « - Average based on 10 Individual roots h * Duncan1* Multiple Range feet A* * Significant at 1 percent level * * stealMeaar «t 5 percent level Appendix 4 • 1be Effect ef DCPA m Che Growth ef Spiny Amaranth B y y a w t y U Average Hypocotyl length (a©) 1epllcatton .. -. .... -... treatment lean)...... Su b 0 10 1 1 0 5 * i s h o *i ?k 4ti3 52.00 2 21,59 11.S5 7.20 S.88 5,03 51.56 3 19,12 11.54 7.13 7.70 4.09 51.58 Sum 59.24 34.40 22.07 20.41 14.80 155.14 Mean 19.73 12.20 7.34 4.80 5.40 £&B£8______ S & K 8 & i £ 88. & Treatment 4 407.839 100.940*4 Replicetion 2 .03 .915 3 9.255 1.157 14 *HM» Caatrol Itojfa) 5(p®®y 10fee*3 50(pp«) 13.95 •* 6 . 4 0 * * 1.74 na 1.29 am 10(ppe) 12.94 ** 5.40 ** .56 aa mmm 5(PP«) 12.39 ** 4.84 ** *•*«» M# l(pp*a) 7.55 mm* #*• mmm a * Average# ef 10 individual hypocotyl 4 - Duncan'• Multiple Rente Teefc ** * Significant •« I percent level m * Met cigaitieaat at 5 percent level 47 Appendix $ * B m Effect of fiOPA so Che Growth af Spiny Aseraatfc Boots Average a»ot Laogefc (an) application ...... ftNMBMMft .£mm1 . ...3m..... 1 10,so *"35^StT A.§7 - & s 57.80 2 10.36 8.78 7.06 3.91 4.69 56.80 3 9,67 8.44 7.20 6.39 3.40 37.10 Sum N J l 23.86 20.03 17.97 16.U 111.70 Mean 10.28 8.62 6.98 5.99 5.37 *** m a t 9JL m Treatment 4 48.222 11.81 4* Replication 2 .103 •053 8 1,803 •226 m J L m m J M & m m iiiTiiMSICo&ustirloooe i inwi»i¥iiiijBiiirTSiisf Mean mTbwmi BrT^iiSSwSr^TSgSSw^^5r7JSiTjflwowww^ii l f i i m c M ^ Coeteal liwmti ...... lOCppn) 50(ppe) 4.91 4 5.23 44 1 .6 1 4 6 .62 m 10(ppct) 4.29 4* 2.63 46 •99 66 Hvm) 3.30 46 1.64 66 1(PP®) 1.66 46 • * Avsrags* of 10 JadferjUUisl roots h * OwBcsn1* Mult; Ip is Stst 44 * Sigaiiicaet st 1 psresofc level 4 * Significant at 5 percent level aa • Sot ijp tftm i 48 Apjxm dix 8 • Ktfmz o f 8C7A tb* Growth of Haira*s Fainebvuoh SypocoCyU ttaMAJwalnat I^Wfcg? fcis 7.000 10.000 1 3 0 ^ » w 15.6 n . T 106.9 2 26.9 20.5 23.0 17.9 15.5 113.2 3 29.0 20.8 20.8 17.0 13.0 100.6 Sues 90.4 95.1 §4.1 50.5 38.4 221.3 Wmm 36.2 25.2 22.0 16.3 12.8 S oareo i i M m Zroateaent 4 562.05 140.514# Replication 2 17.44 8.72 a# t r r o f .. I . - ____ ML 5.000— ...... 7.0O0 10*000 17.4 12.4 9.2 4.0 7,000 13.4 8.4 5.2 5,000 8.2 3.2 m 3,006 3.0 • - Avatasga# of 10 iaAitrlfcal hypoeotyls b « Puncoa'a Multiple iUaga foot ** - Significant ot 1 p t a m t level m - Hot significant 49 Appandix 7 * Ulitt »£ DCPA on th* (tewfh of flora** f a t t t M Roota Average Boot La^gth (on) ..Sue Q 3.000 5.000 7.000 10.000 1STT* i r r r 2 0 * loTo 5 0 151.3 2 26.3 31.0 30.6 27.2 29.4 144.5 3 23.2 34.6 30.8 32.5 24.7 147.8 Sue 81.0 97.0 89.7 89.7 86.2 443.6 Miwt 27.0 32*3 29.9 29.9 28.7 Aaaiyaia of Yaria&aa Xai>U S o i a r c a II t t f i t T r a a t e a a t 4 45.48 11.37 ** p l i c a t i o n 2 4 . 6 2 2 . 3 1 a * ...... 8 ...... 5 9 , . 1 1 ...... 7 . 3 8 1 4 1 0 9 . 2 1 8 ** Avuta^ao of I# laflvtduil roota &l * Hot so Appendix t • Xffoot of DCPA on tho Crowd* of Spanish Koodlo Bypocotyl* Avorago Hypocotyl Longtb (cm) S m lim te a ______sgAJfeMttMaiigad ______»« 0 1.000 3.000 7.000 10.000 1 s f t r aJTs l o r t s x s f r r 243.2 2 54,0 50.2 $4.2 42.8 41.8 243.8 3 50.9 31.3 41.8 48.5 43.5 236.0 StK 156.7 148.4 1 5 0 . 0 134.9 133.0 723.0 52.2 49.3 50.0 43.0 44.3 iftHnit, ffii , w Sewycp M i t % Trentrasnt 4 139.53 33.88 b o ftoplleatiea 2 7.54 3.7 ao 9 131.21 18.9 • • Mwca|«« of 10 individual hyjxwiotyio no • Eofc significant it 5 peseta t lcrn 51 Appendix f • EfiOct of DC9k on the Growth of Spools!* Hoodie Boots Average Boot Length (»>) Benliestlext ...... -.. DCBA Xrestnsnt t m Y . .. i » . l.QQQ 3.000 7.000 10.000 1 rfSr* 22V3 2?T3 26v6 29.2 131.9 2 2 4 . 7 25.8 29.2 22.0 25.8 127.3 3 21.8 23.7 22.9 28.4 25.0 U 1.8 S o n 73.2 71*1 29.4 76.8 8 0 . 0 381.2 U s m 2 4 . 4 23.9 26.5 25.6 26.7 i i ML ML 4 17.80 4 . 4 5 no Replication 2 10.26 5 . 1 3 os f i g a s - ...... 8 50 . 3 6 6.29 o * Averages of 10 individual roots os • Hot significant at 5 percent lovol Abugi Smart. <1964) Susceptibility* Ibctora to toe plant modifying toe response »i • given species to treatment. a t i k u l m a m z s a m s M s M r n . * Academic Appleby, A. P. <1962) A « influence ef toil temperature end toll placement on to* phytoioKtc properties ef EPTC end etoer carbamate herbicides. Ph.D. Thesis, Oregon State University. Appleby, A* P., W. &. A s t t o aad S. C. Ung. (1965) Soil placement, studies with S P R ead etoer carbamate berbleidee to Arena aatlva. Weeds Keeeeeto, eel. 5, pp. 115-122. Ashton, p. 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