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disease of rutabagas (turnips). Ont. Minist.Agric. Food Factsheet 258/635. 3 pp.

4. Evertsen, J. A. 1974. Biological aspects of turnipmosaic virus in relation to the rapeseed crop insouthern Ontario. M.Sc. thesis. University ofGuelph, Ontario, Canada. 46 pp.

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7. Provvidenti, R. 1980. Evaluation of Chinesecabbage cultivars from Japan and the People'sRepublic of China for resistance to turnipmosaic virus and cauliflower mosaic virus. J.

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Immunodiffusion tests with sodium dodecylsulfate (SDS)-treated plant viruses and plantviral inclusions. Univ. Fla. Agric. Exp. Stn. Inst.Food Agric. Sci. Bull. 788. 39 pp.

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10. Stobbs, L. W. 1988. Screening for resistance toturnip mosaic virus in canola germplasm. Phyto-protection. In press.

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Phosphonate Levels in Avocado (Persea americana) Seedlings and SoilFollowing Treatment with Fosetyl-Al or Potassium Phosphonate

D. G. OUIMETTE, Graduate Research Assistant, and M. D. COFFEY, Professor, Department of Plant Pathology,University of California, Riverside 92521-0122

ABSTRACTOuimette, D. G., and Coffey, M. D. 1989. Phosphonate levels in avocado (Persea americana)seedlings and soil following treatment with fosetyl-Al or potassium phosphonate. Plant Disease73:212-215.

The levels of ethyl phosphonate and phosphonate in avocado seedlings and soil were determinedusing high-performance ion chromatography at 1, 2, 4, 6, and 8 wk following foliar or soilapplications of either 3 mg/ ml of fosetyl-Al or 2.1 mg/ ml of potassium phosphonate. After soiltreatment with either potassium phosphonate or fosetyl-Al, phosphonate persisted in soil for 2 and4 wk, respectively. With fosetyl-Al, low levels of ethyl phosphonate were present in soil, roots, andstems 1 wk after application, but none was detected thereafter. In contrast, no ethyl phosphonateresidues were detected in either soil or avocado tissue 1 wk following foliar application offosetyl-Al. Soil treatment with both potassium phosphonate and fosetyl-Al resulted in muchhigher phosphonate levels being present in all tissues compared with foliar treatment (up to 78 and94 times more in the root samples following potassium phosphonate and fosetyl-Al treatment,respectively). Following both soil and foliar applications of the two fungicides, high phosphonatelevels were maintained in avocado tissues for the 8-wk period of the experiments, suggesting thatphosphonate is stable in plants. The phosphonate levels found in roots after either soil or foliarapplications were sufficiently high to account for a direct antifungal effect in controlling avocadoroot rot caused by Phytophthora cinnamomi.

Fosetyl-Al is an ethyl phosphonatefungicide shown to be efficacious againstsome important diseases caused bysoilborne Phytophthora spp. as well assome downy mildew diseases (3,6-13,19,21). Fosetyl-Al is unique amongfungicides in that it is translocated inboth the xylem and phloem (9). Thisproperty permits its use as a foliar sprayor, as in the case of some tree crops, atrunk injection for control of root rotscaused by Phytophthora sp. (5,10,21).

Fosetyl-Al is hydrolyzed to phos-phorous acid in plants and soil (9,20). Inaqueous systems, phosphorous acid(H3 PO3) is in equilibrium with phosphonicacid (H2PHO3) such that essentially all ofthe molecules are in the phosphonic acidstate. Two phosphonate anions (HPHO 3and PH03-2 ) can result from the

Accepted for publication 5 October 1988.

ionization of phosphonic acid, which haspKa values of 1.3 and 6.7 (9). Mostprevious literature has used the termsphosphorous acid and phosphite(1,4-7,9,14,15,17-19,21-24) to denotethe phosphonate moiety. According tothe International Union of Pure andApplied Chemistry (16), the correct termfor the anionic form of phosphonic acidis phosphonate. This term will be usedhere. Unbuffered phosphonic acid isphytotoxic because of its low pH, and sois commonly used as a salt, such assodium or potassium phosphonate, in thepH range of 6.2-6.7 (9,20).

Although the mode of action offosetyl-Al still remains controversial, it islikely that it exerts a direct antifungaleffect on disease control mediated by itsbreakdown product, the phosphonateanion, which is capable of inhibitinggrowth and sporulation of a pathogensuch as Phytophthora (6,7,9,14,15).Phosphonate itself has been usedsuccessfully in controlling root and heart

rot of pineapple caused by P. cinnamomiRands and P. parasitica Dastur (24), rootrot of avocado caused by P. cinnamomi(14,21), and Phytophthora gummosiscaused by P. parasitica and P.citrophthora (R. & E. Sm.) Leonian (19).

Registration of fosetyl-Al on non-bearing avocados to control avocadoroot rot has been granted recently inCalifornia, with full registration antic-ipated once toxicological studies havebeen completed. At present, little isknown concerning the fate of ethylphosphonate or the more fungitoxicmetabolite phosphonate in avocadotissues and soil (9). To fully evaluate theefficacy of fosetyl-Al and potassiumphosphonate against avocado root rot, itis desirable to determine the nature andlevels of fungicide residues and theirdistribution within the plant, especiallythe roots. In addition, some knowledgeof the persistence of ethyl phosphonateand phosphonate in both plants and soilwould be useful, because this has a directbearing on the number of applications offungicide that will be required.

In this paper we employ high-performance ion chromatography (HPIC)to determine both the persistence anddistribution of ethyl phosphonate andphosphonate in avocado seedlingsfollowing either a foliar or soil applicationof fosetyl-Al or potassium phosphonate.

MATERIALS AND METHODSFungicide treatments. Avocado seed-

lings (Persea americana Mill. 'TopaTopa') were grown in 4-L pots containingU.C. mix no. 5 (50% peat moss, 50% finesand, plus 2.2 kg of dolomite, 1.5 kg ofsuperphosphate, 148 g of KNO3, and 148g of K2SO4 per cubic meter) for 12 wk.The plants then were treated withequivalent rates (25.5 meq phosphonate)o 1989 The American Phytopathological Society

212 Plant Disease/Vol. 73 No. 3

of either 2.1 mg/ml of potassiumphosphonate or 3 mg/ ml of fosetyl-Al aseither a foliar or soil application.Phosphorous acid (99.6%) was obtainedfrom Fisher Scientific, Tustin, CA.Fosetyl-Al (96.4% technical grade) waskindly supplied by Rhone-Poulenc Ag.Co., Research Triangle Park, NC.Titration of phosphorous acid with 10 MKOH to 6.2 yielded a mixture ofmonobasic and dibasic potassiumphosphonate. Both chemicals werebuffered with 25.5 mM MES-hydrate (4-morpholine ethane sulphonic acid).

The soil application method consistedof 500 ml per pot applied as a drenchapproximately 4 hr after irrigation. Themethod for foliar application involvedimmersion of the foliage in the fungicidesolution and then placement of the pot onits side while the foliage dried, so as toprevent any fungicide runoff fromcontaminating the soil. The same foliarapplication was repeated 24 hr later.There were six plants per fungicidetreatment per experiment and the plantswere arranged randomly on a greenhousebench. Additionally, 0.1% (v/v) TritonB-1956 (Rohm and Haas, Philadelphia,PA) was used as a surfactant with thefoliar applications of both fosetyl-Al andpotassium phosphonate.

At 1, 2, 4, 6, and 8 wk after fungicideapplication a soil sample was removedfrom six pots of each treatment, to adepth of 10 cm from four equidistantpoints around the base of the plants,using a 15-mm-diameter cork borer. Theplants then were removed from the potsand washed thoroughly with runningwater to remove soil from the roots plusany removable fungicide residue remain-ing on the foliage surface. After theexcess water on the plants had dried, theywere separated into roots, stems, leaves,and, beginning 2 wk after initialapplication, newly emerged leaves. Thetissue was chopped finely with a razorblade, mixed well, and a 2-g fresh weightsample was removed for fungicideanalysis. Five additional 2-g samples ofeach tissue were dried thoroughly in aforced-air oven at 65 C for 3 days todetermine their dry weight. Six replicateswere made of each tissue analysis for eachtreatment.

Plant and soil analysis. Plant tissuesamples were ground to a fine powder inliquid nitrogen using a mortar and pestle.The powder was transferred to a 15-mlplastic vial and 10 ml of deionized waterwas added. For soil analysis, moistsamples weighing 5 g were placed in 15-ml plastic vials and 10 ml of water wasadded. The vials were shaken for 1 hr on areciprocal shaker at 180 strokes/ min andthen allowed to stand for 30 min to allowthe debris to settle. Next, 1.5 ml of thesupernatant was removed, placed in anEppendorf microfuge tube, and centri-fuged for 10 min. Finally, the supernatantwas transferred to a clean microfuge tube

and either analyzed immediately orstored at -20 C. Before injection into thechromatography apparatus, the extractwas diluted if necessary, passed through aSep-Pak C18 cartridge (Waters Associates,Milford, MA) on the end of a 3-mldisposable syringe, and into another 3-ml

syringe with a Swinney holder (Fisher

Scientific) containing a 13-mm GS-type

filter, pore size 0.22 gim. Five duplicate

soil samples were allowed to dry in a

forced-air oven at 65 C for 3 days to

determine soil dry weight.

Ion chromatography system. The

HPIC apparatus was a Dionex 2000i/P

with a model AMMS-1 anion micro-

membrane suppressor coupled withconductivity detection (Dionex Corp.,

Sunnyvale, CA). For phosphonateanalysis, an AS4A separator column was

used along with two types of guard

columns, an MPIC-NGl and an HPIC

AG4A. The eluent for the AS4A column

consisted of 0.53 mM NaCO3 and 1.54

mM Na2HCO3 used at a flow rate of 2.2ml/ min. For ethyl phosphonate analysis,

an AS6 separator column was used along

with an MPIC-NGl and an HPIC AG6guard column. The eluent consisted of 20

mM NaOH run at a flow rate of 2.2

ml/min. The suppressor regenerants for

phosphonate and ethyl phosphonate

analysis were 15 mM H2 SO 4 run at 2.0

ml/min and 30 mM H2SO 4 run at 3.5

ml/ min, respectively. The detector

sensitivity was set at 3 ,S and the

chromatograms were compared with

standards of ethyl phosphonate or

phosphonate as well as spiked and

untreated samples. The data were

recorded on a Spectra-Physics 4270integrator, and fungicide levels were

determined by comparing peak height

with a standard curve (20).

RESULTSFungicide residues in avocado tissues

following soil application. One weekafter soil application of fosetyl-Al,avocado roots and stems contained 2 and3 ,ug of ethyl phosphonate per gram freshweight, respectively, while none wasdetected in leaves. Ethyl phosphonatewas not detectable in any tissue after 2 wk.

Phosphonate levels in avocado rootsduring the 8-wk experiment were notdifferent after treatment with eitherfungicide, with the exception of week 4,in which the potassium phosphonatetreatment yielded higher levels. With thefosetyl-Al treatment, the phosphonatelevels in the roots ranged from 208 to 751,ug/ g fresh weight, respectively (Table 1).After potassium phosphonate treatment,the phosphonate levels were 356 ,g/gfresh weight at 1 wk, increasing to 1,399,ug/g fresh weight after 4 wk and thendecreasing steadily to 213 ,ug/g freshweight by 8 wk.

The phosphonate levels in stemsduring the 8-wk experiment were similarafter treatment with either fungicide,with the exception of week 4, in which thepotassium phosphonate treatment yieldedhigher levels (Table 1). The phosphonatelevels at 8 wk after treatment with eitherfungicide were not significantly differentfrom those found after 1 wk.

After 1 wk, plants treated withpotassium phosphonate contained almostfour times as much phosphonate in theirleaves compared with the fosetyl-Al treat-ment (Table 1). Thereafter, the levels ofphosphonate in the leaves were not dif-ferent with the two fungicide treatments.

Fungicide residue in avocado tissuesfollowing foliar treatment. One weekafter foliar applications of fosetyl-Al, noethyl phosphonate was detected in any

Table 1. Levels of phosphonate in seedlings of Persea americana 'Topa Topa' up to 8 wk after soiltreatment with 500 ml of either 3 mg/ ml of fosetyl-Al or 2.1 mg/ml of potassium phosphonatew

Phosphonate (jug per gram fresh weight)Y

Fungicidex Week Roots Stems Leaves New leaves

Potassium phosphonate 1 356 c' 221 d 221 a ...2 510 bc 534 cd 105 bcd 175 c4 1,399 a 1,561 a 132 bc 298 ab6 512 bc 784 bc 72 bcd 116 c8 213c 382d 47d 131 c

Fosetyl-Al 1 208 c 191 d 57 cd ...2 751 b 566cd 136b 211 bc4 522bc 1,140b 131 bc 337a6 706 b 1,084 b 86 bcd 317 a8 488 bc 543 cd 105 bcd 116 c

wSoil application consisted of a 500-ml drench to each 4-L pot approximately 4 hr after irrigation.'Fungicides were adjusted to pH 6.2 with KOH and buffered with 25.5 mM MES-hydrate.Samples of avocado tissue (2 g) were ground to a fine powder in liquid nitrogen and extracted with10 ml of water for 1 hr in a 15-ml plastic vial on a reciprocal shaker at 180 strokes/ min. The extractwas passed through a Sep-Pak C18 cartridge and a 0.22-gm filter before injection into thechromatography apparatus. Data is the mean of six replicates. To obtain ,ug phosphonate pergram dry weight, multiply the fresh weight values for roots, stems, and leaves by 5.4, 4.2, and 3.2,respectively. The limit of detection using ion chromatography is 0.5 ,g phosphonate/g freshweight.Values within columns for each tissue sample followed by the same letter are not significantlydifferent (P = 0.05) according to Duncan's multiple range test.

Plant Disease/March 1989 213

tissue. During the 8-wk duration of theexperiment, there was no difference inthe phosphonate levels in the rootstreated with either fungicide (Table 2).After 1 wk, fosetyl-Al treatment resultedin a threefold difference in phosphonatelevels of leaves compared with thepotassium phosphonate treatment, withno difference in the levels thereafter(Table 2). The patterns of distribution ofphosphonate in avocado tissues werealmost identical after treatment witheither compound for the 8-wk durationof the experiment (Table 2).

Levels of ethyl phosphonate andphosphonate in soil following soilapplication of fungicides. One week aftersoil application of fosetyl-Al, the soilcontained 7 ,ug of ethyl phosphonate pergram of soil dry weight; none wasdetected at 2 wk and thereafter. At 1, 2,and 4 wk after fosetyl-Al soil treatment,the soil contained 88, 58, and 13 ,ug ofphosphonate per gram dry weight,respectively. One and two weeks aftertreatment with potassium phosphonate,the soil contained 146 and 44 gg ofphosphonate per gram dry weight,respectively; none was detected after 4 wk.

DISCUSSIONIn these studies it was determined that

ethyl phosphonate was short-lived inboth soil and avocado tissues, with nodetectable residues found in avocado orsoil 2 wk after soil treatment, or inavocado 1 wk after foliar application. Incontrast, high levels of phosphonate weredetected in both soil and avocado tissuesand these residues persisted for 4 wk insoil and for the 8-wk duration of theexperiments in avocado tissue.

Compared with the soil treatment,considerably lower levels of phosphonatewere detected in avocado tissue afterfoliar treatment with either fosetyl-Al orpotassium phosphonate, and these levelsalso persisted for the duration of the 8-wkexperiment. The phloem mobility ofphosphonate was confirmed by itsdetection in the roots following carefulfoliar application of either phosphonateor fosetyl-Al. No ethyl phosphonate wasdetected in root tissues following foliarapplication, even at 1 wk after foliarapplication of fosetyl-Al.

The persistence of phosphonate inavocado tissue 8 wk after chemicaltreatment provides circumstantial evi-dence that it is not readily oxidized tophosphate by the plant. This is inagreement with previous findings that nogrowth response was detected in plantsgrowing in soil where phosphonate wasused as the sole source of phosphorusfertilizer (17). Robertson and Boyer (23)concluded that phosphonate and phos-phate are separate biological entities,apparently due to a difference indistribution of electrical charges on thephosphonate molecule relative to that ofthe phosphate molecule, despite the factthat the molecules have a very similaratomic spatial arrangement (23). Theyalso determined that phosphonatesolutions were resistant to oxidation bymolecular oxygen at temperatures up to60 C and over a pH range of 1.5-7.6, andsuggested that phosphonate might be auseful buffer for biological studiesbecause of its chemical stability andrelative biological inactivity (22). Suchphysiochemical and biological propertiesmay account for the persistence of

Table 2. Levels of phophonate in seedlings of Persea americana 'Topa Topa' up to 8 wk after foliartreatment with either 3 mg/ ml of fosetyl-Al or 2.1 mg/ ml of potassium phosphonatew

Phosphonate (gg per gram fresh weight)Y

Fungicidex Week Roots Stems Leaves New leaves

Potassium phosphonate 1 15 aZ 21 c 42 de ...2 13 a 70 bc 118 abc 128 ab4 18 a 209 a 114 abc 103 bc6 11 a 130 ab 74 cde 37 d8 14a 75bc 19e 24d

Fosetyl-Al 1 16 a 43 bc 140 ab ...2 8a 126ab 166a 157a4 14a 59bc 85bcd 73c6 15a 121 ab 73cde 30d8 12 a 95 bc 49 de 13 d

'Foliar treatment consisted of completely immersing the foliage in fungicide solution then layingthe pot on its side while the foliage dried, preventing any fungicide from reaching the soil. Thesame treatment was repeated 24 hr later.

'Fungicides were adjusted to pH 6.2 with KOH and buffered with 25.5 mM MES-hydrate. TritonB-1956 was added at 0.1% to act as a surfactant.Samples of avocado tissue (2 g) were ground to a fine powder in liquid nitrogen and extracted with10 ml of water for I hr in a 15-ml plastic vial on a reciprocal shaker at 180 strokes/ min. The extractwas passed through a Sep-Pak C18 cartridge and a 0.22-,um filter before injection into thechromatography apparatus. Data is the mean of six replicates. To obtain ,ug phosphonate pergram dry weight, multiply the fresh weight values for roots, stems, and leaves by 5.4, 4.2, and 3.2,respectively. The limit of detection using ion chromatography is 0.5 gg phosphonate/g freshweight.Values within columns for each tissue sample followed by the same letter are not significantlydifferent (P = 0.05) according to Duncan's multiple range test.

214 Plant Disease/Vol. 73 No. 3

phosphonate in the avocado plant andexplain the long-term disease controlachieved with some target pathogens(9,10,24).

Persistence of phosphonate in soil wasless than that found in plants. After 4-6wk, no phosphonate was detectable insoil following either potassium phos-phonate or fosetyl-Al treatment. However,the behavior of phosphonate in anartificial potting mix, such as the oneused in this study, may differ from thebehavior in field soils. Adams andConrad (1) investigated the transition ofphosphonate to phosphate in soil andfound that its oxidation proceeded onlywhen microbial activity was not restrictedby the presence of toluene. It has alsobeen found that soil microorganisms cantake up phosphonate from a simplesynthetic media, oxidize it to phosphatein the cell, and release it upon autolysis ofthe cells (1,4,18). A similar mechanismmay have occurred in these studies,resulting in the disappearance ofphosphonate from the soil over arelatively short time period. In addition,the high water solubility of phosphonate(>50%) may have facilitated leachingfrom the soil container.

With few exceptions, there were nosustained differences found in the level ofphosphonate in avocado tissue followingfoliar application of potassium phos-phonate or fosetyl-Al. Once inside theplant, phosphonate appears to be quitestable, although the ultimate fate of themolecule is unknown. Whereas the exactmechanisms involved in disease controlby fosetyl-Al are still not fully understood,critical determinants must be theconcentration and persistence of theactive metabolite phosphonate in theplant tissues targeted by the pathogen. Bycomparison with previous in vitro studieson the inhibitory effects of potassiumphosphonate towards P. cinnamomi, thelowest levels of phosphonate (8-18 ,pg/gfresh weight) found in the roots afterfoliar application of potassium phos-phonate or fosetyl-Al in the present studyare sufficient to account for a directinhibition of P. cinnamomi. For example,Fenn and Coffey (14) and Coffey andBower (6) reported that EC50 values ofpotassium phosphonate for inhibition ofradial growth of mycelium of P.cinnamomi on solid media ranged from4.2 ,ug/ml to 9.0 ,ug/ml. In addition,Coffey and Joseph (7) found thatpotassium phosphonate was highlyinhibitory to critical stages in the lifecycle of P. cinnamomi; 1 ,ug/ ml ofpotassium phosphonate inhibited oosporeproduction by 60-78%, while the EC 5ovalues for inhibition of sporangiumproduction and zoospore release were 1.8and 6 Mg/ml, respectively. Because therapid increase in secondary inoculum byP. cinnamomi under conducive environ-mental conditions is primarily responsiblefor the highly destructive nature of