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Biological Control 20, 57–64 (2001)doi:10.1006/bcon.2000.0875, available online at http://www.idealibrary.com on

Host Range, Temperature Response, Survival, and Overwinteringof Alternaria cirsinoxia

S. Green,1 K. Mortensen, and K. L. BaileyAgriculture and Agri-Food Canada (Saskatoon Research Centre), 107 Science Place, Saskatoon, Saskatchewan S7N 0X2, Canada

Received March 20, 2000; accepted August 13, 2000; published online December 13, 2000

Alternaria cirsinoxia was evaluated for its host range,he influence of temperature on mycelial growth, andurvival and overwintering on Canada thistle (Cirsium

arvense) in Saskatchewan. With the exception of leafyspurge, the host range of A. cirsinoxia was limited tospecies within the Asteraceae. Canada thistle, safflower,and sunflower were most susceptible to A. cirsinoxia, thelatter two being crop species of lesser importance inSaskatchewan. Mycelium of A. cirsinoxia grew best at aconstant temperature of 25°C and in temperature cycleswhich alternated around a mean of 20–25°C. Myceliumdid not grow when exposed to constant temperatures of0, 40, or 45°C for 7 days. However, at 0°C, myceliumsurvived and was able to resume growth, whereas at 40or 45°C, mycelium was killed. In the field, A. cirsinoxiaproduced viable conidia on senescent, basal Canadathistle leaves for at least 3–4 months after inoculation in1998 and 1999. Sporulation tended to be higher in 1998than in 1999, possibly favored by the warmer, drier, andsunnier conditions prevailing during July to mid-Sep-tember in 1998. A. cirsinoxia also overwintered and pro-duced viable conidia on infected Canada thistle leavesin the field, and at constant 4°C, when sampled fromNovember 1998 until April 1999. Sporulation of leavesoverwintering in the field was lowest in April 1999, prob-ably due to inoculum degradation as a result of surfaceflooding in the plots. Clusters and chains of chlamydos-pores were abundant on overwintering leaf and stemdebris of Canada thistle in field plots inoculated 10months previously. A. cirsinoxia subsequently sporu-lated on this infected debris. Based on these host-rangetests, the risks to major nontarget crop species inSaskatchewan should be minimal after the inundativeapplication of A. cirsinoxia as a bioherbicide for Canadathistle. However, this pathogen appears able to persistand remain potentially infectious in the field for a pro-longed period of time after inoculation. Hence, longevityand spread of A. cirsinoxia should be evaluated furtherto minimize the potential risks to susceptible minor cropspecies. © 2000 Academic Press

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

57

Key Words: Alternaria cirsinoxia; host range; sur-vival; overwintering; Canada thistle; Cirsium arvense;bioherbicide.

INTRODUCTION

Alternaria cirsinoxia Simmons & Mortensen is a re-cently described species originally isolated from dis-eased Canada thistle (Cirsium arvense [L.] Scop.) inSaskatchewan, Canada, in 1993 (Simmons andMortensen, 1997). A. cirsinoxia can cause severe foliarnecrosis of Canada thistle, an important perennialweed of crops in regions of North America (Moore,1975), including Saskatchewan (Thomas et al., 1996).The inundative application of fungal pathogens as bio-herbicides is an alternative approach to weed manage-ment (Charudattan, 1991) with potential for Canadathistle. Due to its pathogenicity on this weed, A. cirsi-noxia is being evaluated as a bioherbicide for Canadathistle in the cropping systems of the Canadian prai-ries (Green and Bailey, 2000a,b; Green et al., 2001).

To date, A. cirsinoxia has been reported only onCanada thistle and sow thistle (Sonchus arvensis L.) inSaskatchewan (K. Mortensen, unpublished), but itspathogenicity to other species within the Asteraceaeand to important unrelated weed and crop species ofthe Canadian prairies has not been elucidated. Agri-cultural crop safety is an important issue which needsto be addressed early in the developmental phase ofcandidate bioherbicides. As a potential bioherbicideagainst Canada thistle in Saskatchewan, A. cirsinoxiashould be sufficiently host specific so as to avoid dam-age to the cereal and brassica crops grown most widelyin this province.

Alternaria spp. develop under a range of climaticconditions, have long infectious periods, and can sur-vive environmental extremes (Rotem, 1994). Thegrowth response of A. cirsinoxia to temperatures pre-vailing during the cropping season, from May untilSeptember in Saskatchewan, is not known. In addition,the potential for A. cirsinoxia to survive underSaskatchewan conditions (subhumid to semiarid, with

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58 GREEN, MORTENSEN, AND BAILEY

short, hot summers and long, cold winters) and retainthe ability to sporulate within a cropping season aswell as from one season to the next has yet to bedetermined.

Typically, the bioherbicide tactic involves annual ap-plications of the agent sufficient to control the weedwithin each cropping season (Charudattan, 1991).Therefore, the prolonged survival of the agent in thefield, from one season to the next, is unnecessary forcontrol and could be regarded as a potential environ-mental risk if susceptible crop species are grown sub-sequently. On the other hand, residual activity of ex-tremely host-specific bioherbicides may be advanta-geous for the control of weeds emerging in the followingspring. Therefore, the survival and overwintering ca-pacity of A. cirsinoxia on Canada thistle needs to beaddressed, and this can be evaluated over time bysampling infected tissues from the field and determin-ing their potential to sporulate under standardizedconditions in the laboratory. Sporulation of A. cirsi-noxia can be induced by exposure of moist, infectedplant material to a near ultraviolet light/dark cycle,followed by drying (Walker, 1980). Large conidia withlong single or branched beaks are produced singly onconidiophores (Simmons and Mortensen, 1997), whichenables easy distinction of A. cirsinoxia from otherAlternaria spp.

The objective of this study was to determine the hostrange of A. cirsinoxia, the influence of temperature onmycelial growth, and the survival and overwintering ofinoculum on Canada thistle in Saskatchewan to assessthe biological control potential of this agent.

MATERIALS AND METHODS

Inoculum Preparation

A single isolate of A. cirsinoxia (93-109 B1), origi-ally collected from a Canada thistle plant at Watrous,askatchewan in 1993, was used throughout the study.tock cultures were maintained as conidia frozen in 5%kim milk and 20% glycerol at 273°C. For studies onost range, survival, and overwintering, conidia wereroduced using the methodology described by Walker1980) and stored at 4°C. Inoculum concentration wasdjusted to approximately 105 conidia/ml in distilled

water with the aid of a hemocytometer.

Host Range

Canada thistle and sow-thistle plants were grownfrom rootstocks taken from naturally occurring thistlepopulations near Saskatoon, Saskatchewan. For allother test species (Table 1), seeds were obtained fromcommercial seed sources or from the Saskatoon Re-search Centre seed collection. Plants were grown in15-cm-diameter pots in a mixture of field soil/peat/

vermiculite (3:2:1, v/v) in a controlled-environmentchamber (Model E-15, Conviron, Winnipeg, Canada)with a 16-h photoperiod (280 mE/m2/s) at 24°C. Plantswere watered daily directly into the pot soil and fertil-ized weekly with a 20:20:20 (N:P:K) fertilizer. Plantswere thinned to four to six plants per pot. For each testspecies, five replicate pots were inoculated at the four-to six-leaf stage, and five uninoculated pots served ascontrols. The conidial suspension was sprayed ontoplants until runoff using an airbrush sprayer (ModelH-5; Paasche Airbrush Ltd., Chicago, IL) at 250 kPaconstant air pressure. All plants were placed in a dewchamber (Model E-54U-DL; Percival, Boone, IA) at20 6 2°C for 24 h in the dark and then moved to acontrolled-environment chamber as above. Infectionwas assessed on leaves and stems every 7 days using arating scale of 0–9, where 0 5 no symptoms and 9 5greater than 90% of plant tissue necrotic (Mortensen,1988). At 21 days after inoculation, all above-groundplant tissue was harvested, weighed, dried at 100°C for24 h, and reweighed. The experiment was conductedtwice, and disease rating, fresh weight (FW), and dryweight (DW) data from each trial were pooled if nosignificant interaction was observed between trials andtreatments and analyzed using analysis of variance(GLM procedure; SAS, 1985). Separation of means wasconducted using the least significant difference (LSD) ttest at P # 0.05. Standard errors of the means werealso calculated. For the presentation of biomass data,the mean DW was subtracted from the mean FW(FW minus DW) of inoculated plants and expressed asa percentage of the FW-DW of the respective controlplants (Table 1). This calculation of water content pro-vided an indication of the degree of healthy, nonne-crotic tissue present at 21 days in inoculated plantscompared with the control plants.

Influence of Temperature on Mycelial Growth

Mycelial plugs, 5 mm in diameter, were cut from themargin of 7-day-old A. cirsinoxia cultures activelygrowing on potato dextrose agar (PDA) and placed inthe center of fresh PDA plates (10 cm diameter). Theplates were then placed in a thermogradient apparatus(McLaughlin et al., 1985) consisting of 100 randomlyarranged, individual, temperature-controlled cells pro-viding constant and alternating temperature combina-tions between 0 and 45°C at 5°C intervals. For thealternating temperatures, the system had a 12-h ther-moperiod moving from the low to high temperature insix steps (cycle 1) and from the high to low temperaturein six steps (cycle 2). There were three replicate platesper cell and the plates were subjected to a 16-h photo-period (225 mE/m2/s) from fluorescent tubes placed 75cm above the thermogradient apparatus. After 7 days,radial mycelial growth was measured in millimeters,

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59HOST RANGE AND SURVIVAL OF Alternaria cirsinoxia

including the original 5-mm plug. Therefore, a finalmeasurement of 5 mm indicates no growth. Plates inwhich no growth occurred were placed in an incubatorfor 7 days with a 12-h photoperiod (28 mE/m2/s) at2/24°C day/night temperatures, and the presence orbsence of growth was recorded. The experiment wasonducted twice and the data from each trial pooled fornalysis. The data presented are the means and stan-ard errors of the means for each temperature regime.

TAB

Effect of Alternaria cirsinoxia on Selec

Plant species

ApiaceaeCarrot (Daucus carota L. subsp. sativus Hoffm.)Asteraceae

Aster (Aster sp.)Canada thistle (Cirsium arvense [L] Scop.)Dandelion (Taraxacum officinale Weber in Wiggers)Diffuse knapweed (Centaurea diffusa Lam.)Jerusalem artichoke (Helianthus tuberosus L.)Russian knapweed (Acroptilon repens [L.] DC.)Safflower (Carthamus tinctorius L.)Scentless chamomile (Matricaria maritima L.)Sow thistle (Sonchus arvensis L.)Sunflower (Helianthus annuus L.)rassicacaeMustard (Brassica juncea L. cv. ‘Cutlass’)Mustard (Sinapis alba L. cv. ‘Gisilba’)Oilseed rape (Brassica campestris L. cv. ‘Tobin’)Oilseed rape (Brassica napus L. cv. ‘Westar’)aryophyllaceaeChickweed (Stellaria media [L.] Vill.)

ChenopodiaceaeSugar beet (Beta vulgaris L.)

CucurbitaceaeCucumber (Cucumis sativus L.)

EuphorbiaceaeLeafy spurge (Euphorbia esula L.)

HypericaceaeSt. John’s wort (Hypericum perforatum L.)

LeguminosaeAlfalfa (Medicago sativa L.)Soybean (Glycine max [L.] Merr.)

MalvaceaeRound-leaf mallow (Malva pusilla Sm.)Velvetleaf (Abutilon theophrasti Medic.)

PoaceaeBarley (Hordeum vulgare L. cv. ‘Manley’)Green foxtail (Setaria viridis [L.] Beauv.)Oats (Avena sativa L. cv. ‘Derby’)Wheat (Triticum aestivum L. cv. ‘Hy 320’)

SolanaceaeTomato (Lycopersicon esculentum Mill.)

a Disease rating on a scale of 0–9, where 0 5 no symptoms and 9ata are the mean 6 SE of five replicates in each of two trials. Allb Biomass data are the mean FW minus the mean DW (FW minus

of the FW 2 DW of the respective control plants. Data are the meac All data followed by * are significantly different from the contro

Survival and Overwintering

Survival during the cropping season. Field plotswere 1.8 3 3 m2 consisting of dense natural stands ofCanada thistle in bare soil at the Agriculture and Agri-Food Canada Research Farm, Saskatoon, Saskatche-wan. This experiment was conducted twice, with 10replicate plots inoculated on July 9, 1998, and 4 repli-cate plots inoculated on June 24, 1999, approximately

1

Species under Controlled Conditions

Disease ratinga

Biomassb

(% of control)Leaves Stems

0 0 92.9

.1 6 0.2* 0.5 6 0.3 90.1

.6 6 0.1*c 4.3 6 0.1*c 55.7*c

.2 6 0.6* — 93.4

.8 6 0.3* 0 90.5

.0 6 0.5* 1.0 6 0.3* 97.7

.9 6 0.6* 3.1 6 0.8* 55.0

.6 6 0.8* 7.5 6 0.4* 33.8*

.2 6 0.2* — 88.3

.4 6 0.4* 0.2 6 0.2 87.1

.2 6 0.6* 6.0 6 0.4* 75.2*

0 0 92.50 0 94.90 0 110.20 0 100.9

0 0 100.3

0 0 110.9

0 0 107.8

.8 6 0.5* 4.2 6 0.4* 76.7

0 0 93.4

0 0 117.70 0 107.3

0 0 96.60 0 94.6

0 0 101.40 0 95.90 0 118.60 0 105.8

0 0 97.1

reater than 90% of plant tissue necrotic, 14 days after inoculation.trols (not shown) were scored zero disease.) of inoculated plants at 21 days after inoculation expressed as a %five replicates in each of two trials.P 5 0.05.

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60 GREEN, MORTENSEN, AND BAILEY

4–6 weeks after thistle emergence. The conidial sus-pension was sprayed in the evening onto the plantsuntil runoff using a backpack sprayer (Model GS; R&DSprayers, Inc., LA) with four 8804 TeeJet nozzles at275 kPa constant pressure supplied from a CO2 tank.Temperature, precipitation, and hours of bright sun-shine were monitored throughout the study using aweather station located approximately 1.5 km from thefield site. Every 1–3 weeks until the end of October, fivenecrotic, basal leaves were sampled at random frominfected Canada thistle plants in each plot. The leaveswere washed in running tap water and dried overnightin a growth chamber (described above). The leaveswere then placed on moist blotting paper in trays,misted with distilled water, and covered with clearplastic bags premoistened on the inside. The moistenedleaves were moved to a 20°C incubator for 24 h with12 h near-ultra violet (NUV) light provided by 40-Wblack-light bulbs, followed by 12 h dark to stimulatesporulation. The leaves were dried for 24 h in a growthchamber and then examined using a dissecting micro-scope (Leica Wild M28, Leica AG, Heerbrugg, Switzer-land) at 53 magnification. Survival of A. cirsinoxia wasassessed by recording the density of sporulation withinnine fields of view on both the upper and the lower leafsurfaces using the following sporulation density index:0 5 no conidia; 1 5 1–10 conidia; 2 5 approx. 10–50onidia; 3 5 approx. 50–100 conidia; 4 5 approx. 100–00 conidia; and 5 5 over 200 conidia. Sporulationensities from the nine fields of view were pooled toive a mean value for each leaf surface at each sam-ling date. To assess viability at each sampling,onidia were washed from a few leaves into distilledater, plated onto water agar in petri plates, placed indark incubator at 22°C for 24 h, and examined withcompound microscope (Nikon Optiphot compound mi-

roscope, Nippon Kogaku, K.K., Tokyo, Japan), andercentage of germination was recorded. Sporulationensity data were analyzed using analysis of varianceGLM procedure). Means were separated by the leastignificant difference (LSD) test at P # 0.05. Standardrrors of the means were also calculated.Overwintering. In early October 1998, infectedanada thistle leaves from the field plots inoculated onuly 9, 1998 were placed inside nylon bags consisting ofne-mesh window screen material, five leaves per bag.wenty bags were placed on the soil surface in the fieldlots and secured with wide-meshed snow fencing.wenty bags were also placed in a cold room at con-tant 4°C. In mid-November 1998, and in early Janu-ry, mid-February, and early April 1999, four bagsere removed from each location and sporulation of A.

irsinoxia was induced, assessed, and analyzed asbove. In mid-May 1999, four leaves and stem piecesere sampled from infected Canada thistle plants inach of four field plots inoculated on July 9, 1998,

porulation was induced as above, and the materialas given a general rating for no sporulation, sparse

porulation, moderate sporulation, or dense sporula-ion. Conidial viability on water agar was determineds described above. Leaf and stem material were alsoleared and stained in a solution of Chlorazole Black ESigma Chemical Co., St. Louis, MO) for 24 h andestained for approximately 24 h in a saturated solu-ion of chloral hydrate (Keane et al., 1988). The mate-ial was mounted in 50% glycerine and examined forverwintering structures using a compound micro-cope. This experiment was conducted once.

RESULTS

Host Range

A. cirsinoxia was pathogenic on all Asteraceae spe-cies tested (Table 1). The highest disease ratings wererecorded on leaves (4.8–6.6) and stems (4.3–7.5) ofsafflower, sunflower, and Canada thistle and on leavesof asters (6.1) and diffuse knapweed (4.8). All other testspecies, except leafy spurge, were immune (Table 1).Infection caused a significant reduction (P # 0.05) inthe biomass of safflower, Canada thistle, and sunfloweronly compared with their respective controls (Table 1).

Influence of Temperature on Mycelial Growth

The optimum constant temperature for radial myce-lial growth of A. cirsinoxia was 25°C (Fig. 1A), withoptimum temperature ranges of 20–25°C within 5°Ccycles (Fig. 1B), 20–30°C within 10°C cycles (Fig. 1C),15–30°C within 15°C cycles (Fig. 1D), and 10–30°Cwithin 20°C cycles (Fig. 1E). High temperatures (40–45°C) were more detrimental to mycelial growth thanlow temperatures (0–5°C) (Figs. 1B, 1C, and 1D), al-though mycelium grew during a 25–45°C temperaturecycle (Fig. 1E). No mycelial growth occurred at 0, 40, or45°C constant temperatures (Fig. 1A), but when plateswith no growth were subsequently incubated for 7 daysat 22/24°C, the 0°C cultures resumed growth, whereasthe 40 and 45°C cultures did not.

Survival and Overwintering

Survival during the cropping season. A. cirsinoxiamaintained the potential to sporulate on infected, se-nescing, basal leaves of Canada thistle from late June/early July until sampling ended in late October, in boththe 1998 and the 1999 seasons (Figs. 2 and 3). Conidialviability was 90% or above at each sampling in bothseasons (data not shown). In 1998, there was a signif-icant effect of leaf surface (P 5 0.001) and a signifi-cant interaction (P 5 0.001) between leaf surface andsampling date, with greater sporulation on the upperleaf surface than on the lower leaf surface until the endof September (Fig. 2A). Sporulation peaked at the end

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61HOST RANGE AND SURVIVAL OF Alternaria cirsinoxia

of August and mid-September samplings in 1998, withmean sporulation density indexes of 2.8 and 2.7, re-spectively, on the upper leaf surface. Sporulation de-clined after mid-September, with a slight increase be-tween early and late-October (Fig. 2A). Temperatureswere warm from the time of inoculation until earlySeptember 1998, with maximum temperatures consis-tently above 20°C and minimum temperatures largelywithin the 10–15°C range (Fig. 2B). Rainfall occurredaround the time of inoculation, but conditions were dryfrom mid-July until mid-August 1998 (Fig. 2B) with adaily mean rainfall of 0.5 mm. Bright sunshine duringthis time was consistent (Fig. 2C) with a mean of 11.5 hbright sunshine per day. From mid-September onward,frequency of rainfall increased and both temperatureand hours of bright sunshine decreased (Figs. 2B and2C).

In 1999 there was no interaction between leaf sur-face and sampling date, with similar sporulation den-sities on the upper and lower leaf surfaces during theseason (Fig. 3A). Sporulation densities on the upper

FIG. 1. Radial mycelial growth of Alternaria cirsinoxia on PDAafter a 7-day exposure to constant and alternating temperaturecycles; constant temperatures (A), 5°C cycles (B), 10°C cycles (C),15°C cycles (D), and 20°C cycles (E). Data are the means withstandard error bars.

leaf surface in 1999 were generally lower than thoserecorded in 1998 (Figs. 2A and 3A). Overall meansporulation increased (P # 0.05) to a peak of 1.7 on thedensity index in early August 1999, which was anequivalent (P # 0.05) level of sporulation to thatecorded in October (Fig. 3A). The 1999 summeronths were generally cool, wet, and cloudy. Maxi-um and minimum temperatures commonly fell below

0 and 10°C, respectively (Fig. 3B), and rainfall wasrequent, particularly from late June until late JulyFig. 3B) with a daily mean rainfall of 3.8 mm duringhis time. Hours of bright sunshine were also variablerom late June to late August (mean of 8.8 h/day), witheveral periods of zero sunshine (Fig. 3C).Overwintering. A. cirsinoxia overwintered and

porulated on infected Canada thistle leaves in theeld, and at constant 4°C, from November 1998 untilpril 1999. There was a significant effect of leaf surface

P 5 0.001), with greater sporulation on the uppereaf surface than on the lower leaf surface at each

FIG. 2. Mean sporulation density (using an index of 0–5, where0 5 no conidia and 5 5 .200 conidia) of Alternaria cirsinoxia on theupper and lower leaf surfaces of Canada thistle (A), total dailyrainfall (mm) and daily maximum and minimum temperatures (°C)(B), and total bright sunshine h/day (C) during the 1998 field seasonat Saskatoon, Saskatchewan. Data in (A) are the means with stan-dard error bars.

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62 GREEN, MORTENSEN, AND BAILEY

sampling date (Figs. 4A and 4B). Conidial viability wasabove 88% at each sampling (data not shown). Whenaveraged over both leaf surfaces, sporulation on leavesin the field was similar when sampled in November,January, or February, scoring approximately 3 on thedensity index, but declined significantly (P # 0.05) inApril to approximately 1.5. From November untilMarch, there was generally good snow cover, with tem-peratures in December and January frequently drop-ping to 220°C and below (Fig. 4C). By the end ofMarch, temperatures increased to above zero (Fig. 4C),causing a thaw and some surface flooding in the plots.When averaged over both leaf surfaces, sporulation oninfected leaves at constant 4°C was greatest (P #0.05) in November, with a density index of 2.5, andlowest (P # 0.05) in April, with a density index of 1.8.Clusters and chains of chlamydospores were abundanton overwintering leaf and stem debris of Canada this-tle collected from four field plots sampled in May 1999,10 months after inoculation (data not shown). A. cirsi-noxia subsequently sporulated in May on 14 of 16

FIG. 3. Mean sporulation density (using an index of 0–5, where0 5 no conidia and 5 5 .200 conidia) of Alternaria cirsinoxia on theupper and lower leaf surfaces of Canada thistle (A), total dailyrainfall (mm) and daily maximum and minimum temperatures (°C)(B), and total bright sunshine h/day (C) during the 1999 field seasonat Saskatoon, Saskatchewan. Data in (A) are the means with stan-dard error bars.

overwintering leaves collected and on 12 of 16 overwin-tering stem pieces collected. Sporulation was generallymoderate to dense on the leaves and sparse to moder-ate on the stem pieces.

DISCUSSION

There are a number of important ecological consid-erations related to the inundative application of A.cirsinoxia as a bioherbicide. Two key considerationsare the host range of A. cirsinoxia and its survival inhe field after inoculation. These issues were addressedn this study to predict the risk of infection of econom-cally important nontarget plant species and to gainnformation on the environmental fate of the pathogen.

With the exception of leafy spurge, itself a commoneed of the Canadian Prairie Provinces (Best et al.,980), pathogenicity of A. cirsinoxia was limited topecies within the same family as Canada thistle, thesteraceae. Weed species in the Asteraceae that were

FIG. 4. Mean sporulation density (using an index of 0–5, where0 5 no conidia and 5 5 .200 conidia) of Alternaria cirsinoxia on theupper and lower surfaces of Canada thistle leaves placed in the field(A), in a constant 4°C room (B), and total daily snowfall (cm) anddaily maximum and minimum temperatures (°C) (C) from October1998 until April 1999 at Saskatoon, Saskatchewan. Data in (A) and(B) are the means with standard error bars.

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63HOST RANGE AND SURVIVAL OF Alternaria cirsinoxia

fairly susceptible to A. cirsinoxia included both of thenapweeds, suggesting that A. cirsinoxia could have aroader spectrum of activity than on Canada thistlelone. Of the crop species tested within the Asteraceae,. cirsinoxia was most pathogenic to sunflower andafflower, both minor crops in Saskatchewan. The ma-or crops in Saskatchewan, such as wheat, oilseed rape,nd barley (Fung et al., 1999), were immune to A.

cirsinoxia, indicating that if it were developed as abioherbicide, this pathogen could be used safelyagainst Canada thistle in these production systems.However, young plants were inoculated in this studyand harvested 3 weeks after inoculation without beingexamined for latent infection. Alternaria spp. havebeen reported causing latent or symptomless infectionsin seeds, healthy leaves, and young fruits of varioushosts (Norse and Wheeler, 1971; Mortensen et al.,1983; Bashan, 1994; Rotem, 1994), with symptoms notappearing until leaf or fruit maturity (Norse andWheeler, 1971; Mortensen et al., 1983). Therefore, theissue of latent infections needs to be addressed in host-range tests for bioherbicides (Cerkauskas, 1988; Ma-kowski and Mortensen, 1998, 1999), and should beincorporated as a part of further field testing for a morecomplete assessment of the impact of A. cirsinoxia onnontarget species. The most important crop speciescould also be reinoculated at a later growth stage toconfirm their immunity, since susceptibility to manyAlternaria spp. increases as plant tissues mature(Rotem, 1994).

Mycelial growth of A. cirsinoxia was optimal at con-stant 25°C or when exposed to temperature cycles fluc-tuating around a mean of 20–25°C. Testing the growthresponse of A. cirsinoxia to fluctuating temperaturesprovided conditions closer to the field situation thangrowth tests using constant temperatures. For exam-ple, average minimum and maximum temperaturesduring May–September, the growing season inSaskatchewan, range from 5–12 to 18–25°C, respec-tively, with occasional extremes of up to 40°C anddown to 213°C (Environment Canada, 1993). Althoughexposure to continuous 40 and 45°C for 7 days killedmycelium, short periods of exposure to these highertemperatures did not. This study indicates that tem-peratures fluctuating within the normal range forSaskatchewan during the growing season should notlimit the short-term survival of A. cirsinoxia.

A. cirsinoxia remained potentially infectious, pro-ducing viable conidia on senescent Canada thistleleaves in the field for at least 2–3 months after inocu-lation in the 1998 and 1999 cropping seasons. Longinfectious periods are a well-established characteristicof Alternaria spp. since they start sporulating in livingtissues and can continue to sporulate abundantly onnecrotic and dead tissues (Rotem, 1994). In anotherstudy, Alternaria brassicae (Berk.) Sacc. and A. bras-icicola (Schwein.) Wiltsh. produced viable conidia on

nfected leaves of brassica crops in the United Kingdomor as long as the leaf tissues remained intact (2–3

onths) (Humpherson-Jones, 1989). The sporulationotential of A. cirsinoxia on Canada thistle appeared toe higher during the summer of 1998, than during theame period in 1999. This may be a response to theifferent weather patterns experienced in each season.or Alternaria spp., survival is better in dry than inoist conditions (Rotem, 1994). Conditions in the sum-er of 1998 were warmer, drier, and sunnier than in

ummer 1999, with temperatures closer to the opti-um for mycelial growth of A. cirsinoxia than in 1999.

This may have favored the development of A. cirsinoxiaon Canada thistle. Although long periods of brightsunshine, as experienced in 1998, could be considereddetrimental to A. cirsinoxia, melanization of conidia ofAlternaria spp. and protection of mycelium within leaftissues will limit degradation by UV radiation (Rotem,1994). In addition, exposure to long periods of light inthe NUV wavelength at relatively high temperaturespromotes induction of conidiophores of some Alternariaspp. (Leach, 1967). Since A. cirsinoxia also requiresNUV to induce sporulation, prolonged bright sunshineand warm conditions may have contributed to the con-tinuation of A. cirsinoxia in the field. There was amarked decline in the sporulation potential of A. cirsi-noxia after mid-September in 1998, which was notobserved in 1999. This decline, to levels similar for thesame time of year in 1999, was associated with theonset of cooler temperatures, more frequent rainfall,and shorter periods of bright sunshine. This furtherstrengthens the possibility that warm, dry weathermay promote survival of A. cirsinoxia.

A. cirsinoxia survived the Saskatchewan winter of1998–1999 on leaf and stem debris of Canada thistleon the surface of field plots and continued to produceviable conidia when debris was sampled in the spring.During the winter, air temperatures remained belowfreezing for about 2 months and occasionally droppedto 230°C. However, subzero conditions are generallynot detrimental to the survival of fungal propagules(Rotem, 1994), and good snow cover will have providedinsulation. A decline in the sporulation potential ofleaves sampled in April may have been due to thespring thaw and flooding in the plots, which reducedthe viability of overwintering inoculum. High moisturelevels and microbial activity tend to reduce the longev-ity of fungal propagules, and for this reason, myceliumof other Alternaria spp. degrade faster when buried inthe soil than on the soil surface (Rotem, 1994). It ispossible that cultivation and burial of infected Canadathistle debris will also reduce the survival of A. cirsi-noxia.

A. cirsinoxia formed chlamydospores, which are spe-cialized resting bodies (Rotem, 1994), on leaf and stemdebris of Canada thistle overwintering in the field.These chlamydospores were similar in appearance to

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64 GREEN, MORTENSEN, AND BAILEY

those reported for other Alternaria spp. on their hosts(Basu, 1971; Tseunda and Skoropad, 1977; Patterson,1991). Since overwintering debris containing chlamy-dospores subsequently supported sporulation underideal conditions, chlamydospores may be an importantsurvival mechanism of A. cirsinoxia and a potentialsource of infection of Canada thistle shoots emerging inthe year following inoculation.

These studies have shown that the risks to majornontarget crop species should be minimal if A. cirsi-noxia were to be applied inundatively as a bioherbicidefor Canada thistle—an encouraging result for the con-tinued development of this pathogen as a bioherbicide.However, A. cirsinoxia has the potential to persist inthe field for a prolonged period of time after inocula-tion. Therefore, more studies are needed to determinethe long-term survival of buried and nonburied myce-lium and chlamydospores of A. cirsinoxia, the presenceof latent infections, and the potential for disseminationof this pathogen. Such studies could assist the devel-opment of appropriate use recommendations for A. cir-sinoxia to minimize the risk of infection of closely re-lated minor crop species.

ACKNOWLEDGMENTS

The authors thank Ms. Margaret Molloy, Mr. Tim Nelson, and Ms.Anita Lemke for technical assistance and Mr. Jon Geissler for pro-duction of conidia. Financial support for this study was provided byMicroBio RhizoGen Corp. and an Agriculture and Agri-Food CanadaMatching Investment Initiative.

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