Biological Control 19, 167–174 (2000)doi:10.1006/bcon.2000.0860, available online at http://www.idealibrary.com on

Effects of Leaf Maturity, Infection Site, and Application Rateof Alternaria cirsinoxia Conidia on Infection

of Canada Thistle (Cirsium arvense)S. Green1 and K. L. Bailey

Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan S7N 0X2, Canada

Received January 4, 2000; accepted June 29, 2000

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The effects of leaf maturity, infection site, and ap-plication rate of Alternaria cirsinoxia conidia on thepre- and postpenetration phases of infection of Can-ada thistle were studied. Leaf maturity had no effecton the germination of conidia, but appressoria for-mation was significantly higher on the oldest leafthan on the youngest leaf. There were no differencesin the frequency of leaf penetration in the youngestand the oldest leaves, but consistently larger lesionsformed on the oldest leaf at 10 days after inoculationthan on the youngest leaf. Penetration of the oldestleaf was greatest at the leaf tip, compared with themidleaf area or leaf base. Increasing the applicationrate of A. cirsinoxia conidia (105/ml) from 200 to 800L/ha, with either continuous leaf wetness or inter-mittent drying during application, resulted in in-creasing conidial densities on the phylloplane from3–4 to 9–13 conidia per 1.67-mm2 field of view. Inter-mittent drying during application reduced germina-tion of conidia, but leaf penetration did not differgreatly among treatments. Disease severity on wholeplants at 14 days after inoculation was not affectedby application rate or leaf-wetness treatment. Allplants scored 5–6 on the Horsfall–Barratt ratingscale, with severe infection occurring only on theolder, basal leaves. Thus, the plants recovered bydeveloping new leaves at the apical portion. Theseresults show that A. cirsinoxia is primarily patho-genic on older, senescing leaves of Canada thistle,irrespective of the application rates tested on wholeplants. This characteristic limits its potential as abioherbicide for Canada thistle. © 2000 Academic Press

Key Words: Alternaria cirsinoxia; Cirsium arvense;infection process; leaf maturity; application rate; bio-herbicide.

1 To whom correspondence should be addressed. Fax: 1 306 9567247. E-mail: greens@em.agr.ca.

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INTRODUCTION

Canada thistle (Cirsium arvense (L.) Scop.) is aneconomically important perennial weed of arable andpasture lands in Europe, North America, and NewZealand (Moore, 1975; Bourdot and Harvey, 1996).Chemical, cultural, and mechanical management toolsare available, but control of this weed is often poor dueto its vigor and persistence (Moore, 1975). Alternativecontrol methods for Canada thistle are being sought,including biological control using fungal pathogens(Frantzen and Van Der Zweerde, 1994; Bourdot andHarvey, 1996).

Alternaria cirsinoxia Simmons & Mortensen is a re-cently described species originally isolated from dis-eased Canada thistle in Saskatchewan, Canada (Sim-mons and Mortensen, 1997). A. cirsinoxia can causeevere foliar necrosis of Canada thistle and was se-ected as a candidate bioherbicide for this weed.

An important part of the development of A. cirsi-oxia as a bioherbicide is examination of aspects of itsiology on Canada thistle. Histopathological studiesound that conidia of A. cirsinoxia could infect Canadahistle rapidly, but appressoria formation and leaf pen-tration were highly variable among leaves sampledGreen et al., 2000). One source of variability may beeaf maturity, since preliminary observations indicatedhat older leaves of Canada thistle were most likely toarbor infection by A. cirsinoxia. For diseases causedy many Alternaria spp., susceptibility increases aslant tissues mature (Rotem, 1994). For the Canadahistle/A. cirsinoxia interaction, definitive informations lacking on the relationship between leaf maturitynd infection.Field applications of A. cirsinoxia have been carried

ut using 105 conidia/ml in water at a rate of 300liters/ha with inconsistent levels of infection (K. L.Bailey, unpublished). It may be possible to increasedisease levels on whole plants more consistently byincreasing the rates of conidial deposition, since a sin-

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gle conidium of A. cirsinoxia has the potential to incitea lesion on Canada thistle (Green et al., 2000). How-ever, conidia of A. cirsinoxia are large, approximately55–80 3 19–23 mm with beaks up to 160 mm in length(Simmons and Mortensen, 1997), and these tended toblock the standard Teejet 8002 and 8004 sprayer noz-zles when applied at inoculum densities higher than105 conidia/ml (K. L. Bailey, unpublished). More thanone sprayer pass at 105 conidia/ml may be required toincrease conidial deposition on the leaf surfaces and,therefore, increase disease levels. However, multipleapplications could cause conidia to run off the leavesdue to excess application volume, resulting in reducedconidial deposition. Allowing leaves to dry betweenpasses could prevent this from occurring. No informa-tion exists on how the application rate of A. cirsinoxiatranslates to inoculum dose, as measured by conidialdeposition on the phylloplane, nor whether conidialdensity or distribution influence infection and diseasedevelopment on Canada thistle.

The objectives of this study were to evaluate theeffects of leaf maturity and infection site on germina-tion, appressoria formation, and infection of Canadathistle by A. cirsinoxia conidia and to determine howpplication rate of A. cirsinoxia, with continuous leaf

wetness or intermittent drying, determines the densityof conidia deposited on the phylloplane, conidial viabil-ity, and infection.

MATERIALS AND METHODS

Plant Preparation

Canada thistle plants were grown from rootstockstaken from field populations near Saskatoon,Saskatchewan. Roots were rinsed in water, cut into10-cm sections, and planted in a soilless mix (one partsand to four parts sphagnum peat moss, vermiculite,and calcium carbonate with a 16-8-12 N-P-K con-trolled-release fertilizer) in 10-cm-diameter plasticpots. Plants were grown in a greenhouse at 20 6 3°Cand supplementary lighting was provided with 430-Whigh-pressure sodium lamps (230 mE/m2/s) (PhilipsSon-Agro, Scarborough, ON) for about 6 weeks afterplanting until the 8- to 10-leaf stage.

Inoculum Preparation and Plant Inoculation

The isolate of A. cirsinoxia used in this study wasriginally collected from a Canada thistle plant atatrous, Saskatchewan in 1993. Stock cultures wereaintained as conidia frozen in 5% skim milk and 20%

lycerol at 273°C. Conidia were produced using theethodology described by Walker (1980) and were

dded to distilled water with no surfactant to give auspension of 105 conidia/ml as determined by using a

hemocytometer. For the leaf maturity and infection

site experiments, the conidial suspension was sprayedonto plants until runoff using an airbrush sprayer(Model H-5; Paasche Airbrush Ltd., Chicago, IL) at 250kPa constant air pressure. For the conidial densityexperiment, plants were sprayed using a cabinetsprayer (Model RP1; R & D Sprayers, Opelousas, LA)with a single 8802 TeeJet nozzle and 50-mesh screen at250 kPa constant air pressure. Inoculated plants wereimmediately placed in a dew chamber (Model E-54U-DL; Percival, Boone, IA) at 20 6 2°C and continuous

arkness for 24 h. Plants were then placed in a con-rolled-environment chamber (Model GR48; Conviron,

innipeg, Canada) with a 16-h photoperiod (500 mE/m2/s) and 24/15°C day/night temperatures.

Effect of Leaf Maturity on Germination of Conidia,Appressoria Formation, Leaf Penetration, andPercent Leaf Area Infected

Eight replicate plants were inoculated. At 24 h afterinoculation, 10-mm-diameter leaf disks were sampledfrom the mid-leaf area of the first fully expanded,nonsenescent leaf at the base of each plant (referred toas the oldest leaf), the fourth leaf from the base of eachplant, and the uppermost fully expanded leaf on eachplant (referred to as the youngest leaf). To assess ger-mination, leaf disks were stained with a 10-ml dropletf lactophenol cotton blue (Fisher Scientific, Fairawn, NJ) and examined using a compound micro-cope (Nikon Optiphot compound microscope; Nipponogaku, K.K., Tokyo, Japan). Approximately 100

onidia per disk were counted within random fields ofiew on eight disks, and conidia were considered toave germinated when the germ tube was at least halfhe width of the conidium. Values were expressed asercentages of germination of the total number ofonidia counted. To assess appressoria formation andeaf penetration, leaf disks were cleared and stainedor 24 h in a solution of Chlorazole Black E (Sigmahemical Co., St. Louis, MO) and destained for approx-

mately 24 h in a saturated solution of chloral hydrateKeane et al., 1988). Disks were mounted in 50% glyc-rine and examined using a compound microscope. Ap-ressoria formation and successful leaf penetration (in-icated by the presence of an infection hypha in thepidermal cell below an appressorium) were assessedn eight disks for approximately 100 germinatedonidia per disk within random fields of view. Leafenetration values were based on the sum of all ap-ressoria forming an infection hypha for every germi-ated conidium counted. Appressoria formation and

eaf penetration values were expressed as a percentagef the total number of germinated conidia counted.ercentage of leaf area infected was assessed visually

or the oldest remaining leaf at the base of each plant,he third and fifth leaves from the base of each plant,

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169BIOLOGY OF Alternaria cirsinoxia ON CANADA THISTLE

and the youngest remaining leaf on each plant 10 daysafter inoculation. The experiment was conducted twice.

Effect of Infection Site on Germination of Conidia,Appressoria Formation, and Leaf Penetration

Eight replicate plants were inoculated. At 24 h afterinoculation, 10-mm-diameter leaf disks were sampledfrom the tip, middle, and base of the oldest leaf of eachplant. The midleaf vein was avoided during sampling.Germination, appressoria formation, and leaf penetra-tion were assessed as above. The experiment was con-ducted twice.

Effect of Application Rate, with Continuous LeafWetness or Intermittent Drying during Application,on Conidial Density on the Leaf Surface andInfection

Five replicate plants per treatment were inoculated.Treatments consisted of spraying plants with the samestock suspension of 105 conidia/ml, at rates equivalentto field application rates of 200 liters/ha (one sprayerpass), 400 liters/ha (two sprayer passes), 600 liters/ha(three sprayer passes), and 800 liters/ha (four sprayerpasses). For 600 and 800 liters/ha, each sprayer passwas carried out either in quick succession before theconidial suspension could dry on leaves (continuousleaf wetness) or with 10-min delays between eachsprayer pass to allow the conidial suspension to dry onthe leaves (intermittent drying). To assess the densityof conidial deposition, 10-mm-diameter leaf disks werecut from the tip, middle, and base of the fourth leaffrom the base of each plant at 24 h after inoculationand the number of conidia were counted within fiverandomly selected fields of view per leaf disk using a103 objective. The leaf area within each field of view

as 1.67 mm2 and values were expressed as the meannumber of conidia per field of view. At 24 h afterinoculation, 10-mm-diameter leaf disks were sampledfrom the middle of the oldest leaf of each plant andassessed for germination and leaf penetration asabove, except that the number of penetration sites wascounted within 10 random fields of view per leaf disk,summed, and expressed as the total number of pene-tration sites per leaf disk. Disease development onwhole plants was assessed at 7 and 14 days after inoc-ulation using the Horsfall–Barratt visual grading sys-tem with a rating scale of 0–11 (Horsfall and Barratt,1945), whereby 0 5 0% diseased and 11 5 100% dis-eased. The experiment was conducted three times.

Data Analysis

A randomized, complete block design was used in allexperiments. Since the variances were not indepen-dent of the means, percentage data were arcsine trans-formed and actual counts square-root transformed be-

fore an analysis of variance (GLM procedure; SAS,1985). Separation of means was conducted using theleast significant difference (LSD) test (P # 0.05). Stan-dard errors of untransformed means were also calcu-lated. For each experiment, data from each trial werepooled for analysis if no significant interaction wasobserved between trials and treatments. In the exper-iment on the effect of application rate, conidial densitydata were not normal, and therefore untransformeddata were ranked using a Kruskal–Wallis ranking sys-tem before analysis of variance and means separationusing the LSD test. For all experiments, data pre-sented are the untransformed means, standard errorsof these means, and the results of means separationusing the LSD test.

RESULTS

Effect of Leaf Maturity on Germination of Conidia,Appressoria Formation, Leaf Penetration, andPercent Leaf Area Infected

Consistently high numbers of conidia of A. cirsinoxiagerminated at 24 h after inoculation on all leaves ofCanada thistle (87–93%) (Fig. 1A). Appressoria forma-tion at 24 h was most numerous (P # 0.05) on theldest leaf, with 285 appressoria formed per 100onidia, and least (P # 0.05) on the youngest leaf, with6 appressoria formed per 100 conidia (Fig. 1B). Therequency of appressoria formation varied on both theoungest leaf and the fourth leaf compared with theldest leaf (Fig. 1B). There was no significant differ-nce in leaf penetration at 24 h on the youngest leafnd the oldest leaf but penetration was lower (P #.05) on the fourth leaf than on the oldest leaf (Fig. 1C).eaf penetration was also more variable on the young-st leaf and the fourth leaf than on the oldest leaf (Fig.C). As infection progressed, lesion expansion and co-lescence was greater on the older, basal leaves thann the younger, uppermost leaves (data not shown).nfection and necrosis of young leaves was often lim-ted to small, pin-prick-sized lesions. There was a sig-ificant interaction between trials and treatments forercentage of leaf area infected 10 days after inocula-ion, but in both trials the youngest leaf had the lowestP # 0.05) percentage of leaf area infected comparedith all older leaves (Figs. 1D and 1E). The oldest leafas consistently 100% infected (Figs. 1D and 1E).

ffect of Infection Site on Germination of Conidia,Appressoria Formation, and Leaf Penetration

No clear pattern emerged for the effect of infectionite (leaf base, midleaf, or leaf tip) on the oldest leaf ofanada thistle on infection by A. cirsinoxia at 24 h.onidial germination at the three sites ranged between4 and 89% (Fig. 2A). Although appressoria formation

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170 GREEN AND BAILEY

at 24 h was most numerous (P # 0.05) at the leaf base(Fig. 2B), most penetration at 24 h occurred (P # 0.05)at the leaf tip (Fig. 2C). At all three sites, infectionprogressed well, with over 200 appressoria formed per100 germinated conidia (Fig. 2B) and with 25% orgreater leaf penetration by conidia (Fig. 2C).

Effect of Application Rate, with Continuous LeafWetness or Intermittent Drying during Application,on Conidial Density on the Leaf Surface andInfection

There was a significant interaction between trialsand treatments for the number of conidia of A. cirsi-noxia deposited on the surface of the fourth leaf ofCanada thistle. However, in all three trials, conidialdensity per 1.67-mm2 field of view increased threefoldfrom 3–4 conidia at 200 liters/ha to 9–13 conidia at 800liters/ha, with similar patterns in all three trials (Figs.

FIG. 1. Effect of leaf maturity on percentages of germination offter inoculation with Alternaria cirsinoxia (trials 1 and 2 combined),(D) and trial 2 (E). Columns are the means with standard error ba

ccording to LSD test.

3A, 3B, and 3C). Generally, conidial densities at 600and 800 liters/ha were similar for both continuous leafwetness and intermittent drying during application(Figs. 3A, 3B, and 3C). The majority of conidia weredeposited on the midleaf area in two of the three trialswhen averaged over all application rates (Fig. 3D). Inall trials and treatments, conidia were generally evenlydistributed without clumping.

There was a significant interaction between trialsand treatments for conidial germination at 24 h, withsimilar trends (Figs. 4A, 4B, and 4C). When appliedwith continuous leaf wetness, germination was above90% in all three trials, and there were no differences ingermination among application rates (Figs. 4A, 4B,and 4C). However, germination of conidia applied withintermittent drying was consistently lower (P # 0.05)han at any application rate with continuous leaf wet-ess (Figs. 4A, 4B, and 4C). In trials 1 and 2, applica-

idia (A), appressoria formation (B), and leaf penetration (C) at 24 hd on percentage of leaf area infected 10 days after inoculation in trialColumns with different letters are significantly different (P # 0.05)

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171BIOLOGY OF Alternaria cirsinoxia ON CANADA THISTLE

tion with three intermittent drying periods at 800 li-ters/ha resulted in lower (P # 0.05) conidial germina-ion than application with two intermittent dryingeriods at 600 liters/ha (Figs. 4A and 4B).There were few statistical differences among treat-ents for the number of leaf penetration sites at 24 h

ince variability from disk to disk was high (Fig. 4D).he only statistically different treatments were contin-ous leaf wetness at 600 liters/ha with 41 penetrationites per disk (P # 0.05) compared to continuous leafetness at 200 liters/ha and 800 liters/ha with inter-ittent drying; the latter resulted in 19–21 penetra-

ion sites per disk (Fig. 4D). At 14 days after inocula-ion, there were no differences among the treatmentsor disease rating on whole plants. All treatmentscored 5–6 (25–75% diseased) on the Horsfall–Barratt

FIG. 2. Effect of infection site on the oldest leaf on the percent-ages of germination of conidia (A), appressoria formation (B), andleaf penetration (C) at 24 h after inoculation with Alternaria cirsi-noxia. Columns are the means for trials 1 and 2 combined withstandard error bars. Columns with different letters are significantlydifferent (P # 0.05) according to LSD test.

scale (Fig. 4E) and all plants regrew, producing new,healthy shoots with severe necrosis confined to theolder, basal leaves only.

DISCUSSION

This study provides information on the biology of therecently described host–pathogen combination of Can-ada thistle and A. cirsinoxia and identifies some im-

ortant ecological characteristics of A. cirsinoxia thatnfluence its potential as a bioherbicide. For A. cirsi-oxia to be a successful bioherbicide for Canada thistle,

t must incite levels of infection sufficient to inhibit orreatly retard growth and reproduction of this weed.or a foliar bioherbicide, the youngest tissues at thepical meristem of Canada thistle should be the mainarget.

Numerous studies with Alternaria spp. on variousost plants have shown that mature leaves at the plantase are more susceptible to infection than theounger, uppermost leaves (Allen et al., 1983; Miller,983; Barna and Gyorgyi, 1992; Mridha and Wheeler,993; Hong and Fitt, 1995). Von Ramm (1962) andorse (1973) found that germ tubes of Alternaria lon-ipes (Ell. & Ev.) Mason were significantly longer onoung leaves of tobacco (Nicotiana tabacum L.) than onature leaves and that longer germ tubes resulted in

ewer penetrations (Von Ramm, 1962). In the Canadahistle–A. cirsinoxia interaction, the effect of leaf ma-urity of Canada thistle manifested itself at differenttages during infection by A. cirsinoxia. Conidial ger-ination was consistently high irrespective of leaf ma-

urity, but an accurate assessment of germ-tube lengthould not be made because of frequent curling andoubling-back during growth. However, appressorialounts reflected the extent of the prepenetration phasend these were highest and least variable on the oldesteaf. Stimulation of infection by plant pathogenic fungi

ay be affected by the structure and thickness of leafpicuticular waxes, the cuticle, or the presence of cer-ain fungistatic leachates (Agrios, 1978). Such leaforphological factors could also alter as leaves mature

nd may exert an influence on appressoria formationy A. cirsinoxia on Canada thistle.Despite fewer appressoria on the youngest leaf ofanada thistle than on the oldest leaf, the levels ofenetration were not significantly different, indicatinghat young and old leaves were equally susceptible tohis stage of infection. However, 10 days after inocula-ion, the oldest leaf was consistently 100% infected,hereas lesion development on the youngest, upper-ost leaf was always more limited, with plant recovery

rom the shoot tip. These results suggest that leafaturity exerted a large effect in the postpenetration

hase of infection, during growth of A. cirsinoxiaithin leaf tissues. In another study, tobacco leaves ofll ages were susceptible to penetration by Alternaria

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172 GREEN AND BAILEY

alternata (Fr.) Kreissler, but young, expanding leaveswere able to halt the advance of the fungus within thehost tissues by initiating a zone of rapid cell divisionthat surrounded the infection site. This zone stainedmore intensely than the normal tissue (Stavely andSlana, 1971). Older, fully expanded tobacco leaveswere unable to undergo this cell division response,resulting in considerably larger lesions than onyounger leaves. Barna and Gyorgyi (1992) suggestedthat the greater susceptibility of old tobacco leaves toA. alternata, compared with young leaves, was due to ahigher sensitivity to the enzymes and toxins producedby the pathogen during infection. Canada thistle candevelop a range of structural defence responses againstA. cirsinoxia infection, including formation and mobi-lization of lignin, callose, and silicon at infection sites(Green et al., 2000). These defence responses may in-

FIG. 3. Effect of application rate (liters/ha) of Alternaria cirsinoumber of conidia deposited on the leaf surface within a 1.67-mm2 fieite on the fourth leaf (leaf base, midleaf, or leaf tip) on the number ore the means with standard error bars.

hibit the activity of pathogen-produced enzymes andtoxins and slow the rate of infection. Young leaves ofCanada thistle are likely to be more metabolically ac-tive than older leaves, and the activity of enzymesinvolved in these biochemical defence responses maybe higher. In future studies, a comparison of the rateand extent of these resistance responses in young andold leaves of Canada thistle may help to identify whythey differ in their susceptibility to A. cirsinoxia.

To a certain extent, inconsistencies in infection ofweeds by bioherbicide pathogens may be overcome byincreasing the inoculum load to an optimum level. A.cirsinoxia has large conidia (Simmons and Mortensen,1997) and inoculum concentrations of more than 105

conidia/ml have tended to clog the standard sprayernozzles used. This study has shown that multiple ap-plications of 105 conidia/ml can be used to increase

conidia with continuous leaf wetness or intermittent drying on thef view in trial 1 (A), trial 2 (B), and trial 3 (C), and effect of infection

nidia per field of view for trials 1, 2, and 3 combined (D). Data points

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173BIOLOGY OF Alternaria cirsinoxia ON CANADA THISTLE

inoculum deposition on the phylloplane of Canada this-tle, resulting in good retention of conidia and uniformdistribution without the problems of inoculum clump-ing or runoff. The large conidia of A. cirsinoxia appearo persist well at the point of deposition on the leafurface. The use of multiple applications to increaseonidial deposition was a satisfactory approach only forhe purpose of this study. This is not a practicalethod of increasing the inoculum dose in the field.he use of larger nozzles, with a greater flow rate,ould enable a single application of A. cirsinoxia at areater concentration without clogging the nozzles. De-ermining the optimal nozzle size for application ofnoculum is an important area in the development ofathogens as bioherbicides. However, pathogens thatequire very high rates of inoculum for weed controlre not the most efficacious bioherbicides.

FIG. 4. Effect of application (liters/ha) rate of Alternaria cirsinoercentage of germination on Canada thistle in trial 1 (A), trial 2 (B)fter inoculation, and on disease rating (E) on whole plants at 14 daith standard error bars. Columns with different letters are signific

Increasing conidial density had no effect on the ger-mination of A. cirsinoxia, unlike the bioherbicide Col-letotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp.malvae, which showed reduced germination at higherconidial densities (Morin et al., 1996). Although inter-mittent drying of leaves during application reducedconidial germination of A. cirsinoxia in all three trials,it did not reduce overall leaf penetration substantially.A sufficient number of viable conidia remained to es-tablish infection levels comparable with all other treat-ments. The multicellular and highly melanized conidiaof Alternaria spp. often well survive intermittent leafwetness periods (Rotem, 1994), and this characteristiccould be studied further with A. cirsinoxia to improvethe ecological understanding of this recently describedspecies. However, with regard to its potential as abioherbicide for Canada thistle, disease levels on whole

conidia with continuous leaf wetness or intermittent drying on thed trial 3 (C) at 24 h after inoculation, on leaf penetration (D) at 24 hfter inoculation in trials 1 and 2 combined. Columns are the means

tly different (P # 0.05) according to LSD test.

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plants appeared to be influenced more by differences inleaf maturity than by application rate or leaf-wetnesstreatment. Increasing the average conidial densities ofA. cirsinoxia from 3–4 to 9–13 conidia per 1.67-mm2

leaf area did not cause significant enhancement of dis-ease at any stage of the infection process. In fact, thetrend for disease development on whole plants of Can-ada thistle was similar to that described for the leaf-maturity experiment: the most severe disease levelsremained limited to the basal leaves, enabling theplant to recover despite incremental inoculation ofwhole plants.

This study strengthens previous observations sug-gesting that A. cirsinoxia is ecologically adapted toCanada thistle as a pathogen mainly of senescing,basal leaves, irrespective of the application ratestested. This characteristic limits the bioherbicidal po-tential of A. cirsinoxia, since Canada thistle plants willusually recover from infection, and may also explainthe low degree of efficacy reported in preliminary fieldtrials. In addition to providing valuable information onthe biology of this host–pathogen interaction, datafrom this study can be used to standardize inoculationand host tissue sampling procedures for future bioher-bicide studies with Canada thistle.

ACKNOWLEDGMENTS

The authors thank Mr. Jon Geissler for production of conidia.Financial support for this study was provided by MicroBio RhizoGenCorp. and an Agriculture and Agri-Food Canada Matching Invest-ment Initiative.

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tavely, J. R., and Slana, L. J. 1971. Relation of leaf age to thereaction of tobacco to Alternaria alternata. Phytopathology 61,73–78.on Ramm, C. 1962. Histological studies of infection by Alternarialongipes on tobacco. Phytopathol. Z. 45, 391–398.alker, L. 1980. Production of spores for field studies. Adv. Agric.Technol. 12, 1–5.