Diversity of Fusarium species associated with discolored ... · Diversity of Fusarium species...

Epidemiology / Épidémiologie

Diversity of Fusarium species associated withdiscolored ginseng roots in British Columbia1

Z.K. Punja, A. Wan, R.S. Goswami, N. Verma, M. Rahman, T. Barasubiye,K.A. Seifert, and C.A. Lévesque

Abstract: Crown discoloration on roots of American ginseng (Panax quinquefolius) is a major problem that causes areduction in quality and can be a limiting factor to ginseng production in some regions of Canada. The symptomsresult from an accumulation of phenolic compounds within disorganized and disrupted cells, accompanied by thepresence of fungal hyphae in affected cells. Twelve Fusarium species were recovered on isolation media during June–September of 2004 and 2005 from ginseng roots displaying root-surface discoloration in British Columbia. Many of thesame species were also recovered from soil from ginseng fields and cereal-straw mulch used during ginseng production.Hybridization of labeled PCR amplicons from DNA samples from affected root tissue to a DNA array designed usingoligonucleotides specific to 26 Fusarium species was used to identify the Fusarium species present in ginseng roots.Over 90% of symptomatic root samples contained F. equiseti and several other Fusarium species, includingF. sporotrichioides and F. avenaceum, that were absent in nonsymptomatic tissues. An in vitro pathogenicity test wasused to screen 180 Fusarium isolates originating from affected roots. Reddish brown lesions developed 10 daysfollowing inoculation with 58 isolates of F. equiseti and F. sporotrichioides and to a lesser extent with 7 isolates ofF. avenaceum and F. culmorum. Greenhouse and field inoculations confirmed the pathogenicity of F. equiseti andF. sporotrichioides isolates to ginseng roots. Pathogenicity tests conducted with 340 additional isolates representing 10Fusarium species originating from wheat- and barley-straw mulch and soil from ginseng fields, as well as from cerealhosts worldwide, identified a proportion of isolates (52.5%) of F. equiseti, F. sporotrichioides, F. avenaceum, andF. culmorum that produced reddish brown lesioned areas. These cereal-infecting Fusarium species can induce a tissueresponse following infection that results in root-surface discoloration.

Key words: epidemiology, molecular detection, Panax quinquefolius, rusty root.Punja et al.: ginseng / Fusarium

species / root discoloration / DNA array / F. equiseti 353Résumé : La coloration anormale des racines de ginseng (Panax quinquefolius) est un problème important qui diminuela qualité et peut être un facteur limitant pour la production de ginseng dans certaines régions du Canada. Lessymptômes sont le résultat d’une accumulation de composés phénoliques à l’intérieur de cellules désorganisées etdésintégrées, accompagnée de la présence d’hyphes fongiques dans les cellules affectées. Entre juin et septembre de2004 et de 2005, en Colombie-Britannique, douze espèces de Fusarium furent obtenues sur milieu de culture à partirde racines de ginseng montrant, en surface, des symptômes de coloration anormale. Les mêmes espèces furent aussiisolées du sol de culture de ginseng et de paillis de céréales utilizé pour la production du ginseng. Pour identifier lesespèces de Fusarium présentes dans les racines de ginseng, l’hybridation d’amplicons marqués obtenus par PCR àpartir de l’ADN d’échantillons de tissu racinaire affecté fut utilizée sur puces à ADN avec oligonucléotides spécifiquesaux 26 espèces de Fusarium. Plus de 90 % des racines avec symptômes contenaient le F. equiseti et plusieurs autresespèces de Fusarium, y compris le F. sporotrichioides et le F. avenaceum, qui étaient absents des tissus sanssymptômes. Un test de pathogénicité in vitro fut fait pour trier 180 isolats de Fusarium provenant de racines affectées.Des lésions brun rougeâtre se sont développées 10 jours après l’inoculation de 58 isolats de F. equiseti et deF. sporotrichioides, et, à un moindre degré, de 7 isolats de F. avenaceum et de F. culmorum. Des inoculations en serreet au champ ont confirmé le pouvoir pathogène des isolats de F. equiseti et de F. sporotrichioides sur des racinesginseng. Les tests de pathogénicité, menés avec 340 isolats additionnels représentant 10 espèces de Fusarium provenant

Can. J. Plant Pathol. 29: 340–353 (2007)

340

Accepted 22 August 2007.

Z.K. Punja,2 A. Wan, R.S. Goswami, N. Verma, and M. Rahman. Department of Biological Sciences, Simon Fraser University,8888 University Drive, Burnaby, BC V5A 1S6, Canada.T. Barasubiye, K.A. Seifert, and C.A. Lévesque. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada,960 Carling Avenue, Ottawa, ON K1A 0C6, Canada.

1All editorial decisions for this paper were made by J.G. Menzies.2Corresponding author (e-mail: [email protected]).

de paillis de blé et d’orge et de sol de champs de ginseng, ainsi que de céréales à travers le monde, ont permis dedéterminer le pourcentage d’isolats (52,5 %) de F. equiseti, de F. sporotrichioides, de F. avenaceum et deF. culmorum qui produisent des plages de lésions brun rougeâtre. Ces espèces de Fusarium des céréales peuventinduire une réponse du tissu après infection suivie par l’apparition de coloration anormale de la surface des racines.

Mots-clés : épidémiologie, détection moléculaire, Panax quinquefolius, racine rouille.

Introduction

American ginseng (Panax quinquefolius L.; hereinafterginseng), a member of the family Araliaceae, is cultivatedin British Columbia (B.C.) and Ontario, Canada, for exportto Asian markets, where the roots are widely used in tradi-tional medicine. The plants are initiated from seed andgrown on raised beds under shade cloth, and a thick layer ofcereal straw is placed on the soil surface to simulate a forest-canopy environment. The plants are harvested after 3–4 years of growth and the roots are dried and trimmed priorto sale. Ginseng root quality is determined by size, shape,and overall appearance. Roots without any blemishes arehighly valued and surface discoloration can significantly re-duce their marketability. Superficial reddish brown toorange–brown discolored areas that develop near the crownof ginseng roots, followed by blistering and darkening ofthe affected tissue (Rahman and Punja 2005a), is a problemdescribed as rusty root (rusted root) (Hildebrand 1935;Zinssmeister 1918) and greatly reduces root marketability.

Previous studies have implicated both abiotic (physiolog-ical) and biotic factors as the causes of discoloration of theginseng root surface. The abiotic factors include anaerobicsoil conditions during root growth (Lee et al. 2004), ethyl-ene production under stress conditions (Campeau et al.2003b), and soil conditions in which iron accumulates totoxic levels in the tissues (Lee et al. 2004; Yinping et al.1997). Recovery of Cylindrocarpon destructans (Zinssm.)Scholten and Fusarium species from affected roots (Parkeand Shotwell 1989), as well as Rhexocercosporidium sp.(Reeleder et al. 2006), have also led investigators to postu-late that these fungi may play a role in symptom induction.In some ginseng-growing regions in Canada, rusty root andassociated economic losses have been severe enough to pre-clude further cultivation of the crop.

The superficial reddish brown discolored areas on af-fected roots contain phenolic compounds (Campeau et al.2003a; Lee et al. 2004; Rahman and Punja 2005a). This dis-coloration, however, is distinct from the symptoms of rootrot, caused by species of Rhizoctonia, Cylindrocarpon,Pythium, and Phytophthora (Brammall 1994; Parke andShotwell 1989; Punja 1997; Rahman and Punja 2005b),which penetrate deeper into the cortex of the root and inmany cases destroy the entire root. In a preliminary study(Punja et al. 2006) we observed that Fusarium species werethe most frequently isolated fungi from affected tissues. Theobjectives of this study were to examine the cellularchanges taking place and to investigate the role thatFusarium species may play in root discoloration on gin-seng. Using light microscopy, isolation methods, and DNA-based detection assays, coupled with pathogenicity tests, weshow that a complex of Fusarium species that have their or-

igins in the straw mulch that is used extensively in ginsenggardens appear to be involved in the development of rustyroot on ginseng in B.C.

Materials and methods

Source of ginseng rootsGinseng fields from which root samples were obtained

were located near Kamloops, B.C., a region characterizedby hot and dry summer growing conditions. Four farms lo-cated within a 50 km radius of each other were sampled atapproximately monthly intervals during June–September of2004 and 2005. Ginseng roots were randomly dug up andthose displaying reddish brown discolored areas (Fig. 1)were collected. In all farms, samples were taken from 1- to4-year-old plants and the incidence of discoloration withinfields ranged from 1% to 30%. Roots were transported tothe laboratory and stored at 4 °C until fungal isolationswere conducted, generally within 1–3 weeks.

Light microscopy of discolored tissuesTissue pieces (2 mm2) containing discolored areas were

cut out from 2-year-old symptomatic field-grown roots. Thetissue blocks were fixed in formalin – acetic acid – alcoholfor 72 h, transferred to 70% ethanol, and then passedthrough an ethanol series (70%, 85%, 95%, and 100%, 2 heach). Tissues were preinfiltrated with a 100% ethanol–2-hydroxyethylmethacrylate mixture (Technovit® 7100;Marivac Ltd., Halifax, N.S.) (1:1 v/v) for 2 h. Samples wereinfiltrated with Technovit (100 mL of Technovit 7100 and1 g of hardener) for 24 h at 4 °C and embedded inTechnovit 7100 according to the manufacturer’s instructions(Marivac Ltd.). Serial sections (4 µm thick) were cutthrough the tissues, both in transverse and in cross section,with an LKB pyramitome (Diversified Equipment Company,Lorton, Va.) and stained with an aqueous solution ofToluidine Blue O (0.05%) for 30 s followed by rinsing indistilled water for 30 s. The sections were dried at 50 °C for10 min, sealed under cover slips using Permount™ (FisherScientific Canada, Ottawa, Ont.), and examined under a lightmicroscope at 100× and 400× magnification. Observationsmade included the extent of cellular integrity, depth and celltype of the affected area, presence of fungal mycelia, andaccumulation of phenolic compounds in the tissues. A mini-mum of 30 sections of each tissue sample were examinedand photographed using a Pentax *ist camera (Pentax, Tokyo,Japan). The process was repeated using tissues obtained atdifferent sampling times from roots collected during 2004and 2005 that displayed a range of symptoms. To detect thepresence of lignin, tissue samples from symptomatic rootswere fixed in formalin – acetic acid – alcohol for 48 h.

Punja et al.: ginseng / Fusarium species / root discoloration / DNA array / F. equiseti 341

They were transferred to 70% EtOH, dehydrated through atertiary butyl alcohol series (70%, 80%, 90%, and 100%,1 h each), and transferred into a mixture of paraffin oil : ter-tiary butyl alcohol (1:1 v/v). The tissues were infiltratedwith Paraplast™ (Fisher Scientific Canada, Ottawa, Ont.) /paraffin oil / tertiary butyl alcohol under vacuum withfrequent changes to fresh Paraplast over 4 days, and thenembedded in fresh Paraplast. Sections (10 µm thick) weremade with a LKB pyramitome, stained with a saturatedaqueous solution of phloroglucinol in 18% HCl, andmounted in glycerol. Tissues were examined under blue light(wavelength 450–490 nm) to detect fluorescence and photo-graphed.

Isolation of microbes from affected rootsRoots were washed under running tap water and gently

scrubbed with a cheesecloth pad to remove adhering soil.Small tissue pieces (1–2 mm2), consisting mostly of epider-mal tissues, were excised to a depth of 1 mm from reddishbrown discolored areas. Initially, the tissue segments wererinsed in water for 1 min and plated onto potato dextroseagar (PDA), malt extract agar, nutrient agar, and water agar,all without antibiotics, to assess which groups of fungi werepresent. Subsequently, Komada’s medium (Komada 1975)was included for isolations from roots collected duringAugust–September 2004. At each sampling time, around15 roots were included from each farm. In 2005, PDA with150–200 mg/L streptomycin sulphate and Komada’s me-dium were used for isolation. In addition, a brief surface-sterilization procedure was included, consisting of a 30 sdip in 70% EtOH followed by 30 s in 5% bleach (Javex™,containing 5.25% NaOCl). Tissue pieces were rinsed in wa-ter and plated onto the agar media, which were incubatedon the laboratory bench at 20–23 °C under ambient fluores-cent lights. After 7–14 days, developing colonies weretransferred to PDA and retained for identification to genusor species level using morphological criteria and compari-son with identified cultures of known fungi.

Recovery of Fusarium species from straw mulch and soilTo determine the extent of colonization of straw mulch

and soil in ginseng gardens by Fusarium species and the di-versity of species present, isolations were made ontoKomada’s medium. Samples of wheat- and barley-strawmulch and soil were obtained at monthly intervals duringJuly–September in 2004 and 2005 from the same fourginseng farms from which discolored root samples were

previously collected. Straw samples were taken from thesoil–straw interface and soil was collected near the crownof ginseng roots directly beneath the straw mulch. Leaf andstem pieces, including internodal regions, of straw samples(about 0.5 cm long) were plated onto Komada’s medium(five segments per dish) with 15 replicate Petri dishes persample. Percent recovery of Fusarium species was recordedafter 7–10 days of incubation at 20–23 °C. Fusarium colo-nies were characterized by pink, carmine-red, blue, or light-purple pigmentation on Komada’s medium. A subset ofrepresentative morphologically distinct colonies was trans-ferred to PDA for subsequent identification to species leveland for inclusion in pathogenicity tests.

To recover Fusarium species from soil, 1 cm3 sampleswere diluted to 10–2 and 0.5 mL was plated onto each offour replicate Petri dishes containing Komada’s mediumand incubated at 20–23 °C for 7–10 days. To establish a re-lationship between the extent of colonization of strawmulch and populations of F. equiseti (Corda) Sacc.[Gibberella intricans] in the underlying soil, a total of18 different ginseng farms in B.C. were sampled during Au-gust 2004 as described previously. Straw and soil sampleswere collected at random locations in the gardens and as-sayed as described above. The number of F. equiseti colo-nies was converted to CFU/g soil after the moisture contentin each sample was determined. A regression analysis wasconducted using Jmp IN 4 (SAS Software Inc., Cary, N.C.).In 2005, a comparison of three different Fusarium-selectivemedia was made for ease of identification and frequency ofrecovery of Fusarium colonies from selected samples ofsoil from ginseng fields. The media included were Kerr’smedium (Kerr 1963), Nash and Synder’s medium (Nash andSynder 1962), and Czapek–Dox agar medium (Abildgren etal. 1987).

Pathogenicity tests

LaboratoryTen genera of fungi, including Fusarium, were recovered

from root tissues in 2004 and more than 200 cultures weretested for pathogenicity on whole ginseng roots in vitro.Roots 2–4 years of age were dug from various gardens asdescribed previously and washed, and roots with blemisheswere discarded. Roots were stored at 4 °C for periods notexceeding 3 months prior to use and visibly healthy whiteroots with no discolored areas were placed on moistenedpaper towels inside a plastic container. Mycelial plugs(5 mm diameter) taken from the margin of 7- to 10-day-old

342 Can. J. Plant Pathol. Vol. 29, 2007

Fig. 1. Symptoms of root discoloration on field-grown American ginseng (Panax quinquefolius) roots and light microscopy of affected tissues.(A) One-year-old seedlings emerging through the layer of straw mulch in June. Reddish brown discoloration can be seen near the crown ofsome plants. (B) Early symptoms of root discoloration, showing flecking and coalescing of spots. (C) Typical crown discoloration on theupper part of the root. Scale bars in B and C = 1 cm. (D) Cluster of epidermal cells in a small spot, showing intense staining with ToluidineBlue O at early stages of symptom development. Scale bar = 15 µm. (E) Hyphae ramifying through affected epidermal cells. Scale bar = 10 µm.(F) Transverse section through an affected area showing necrotic cells in the centre surrounded by cells with disorganized cytoplasm inadvanced symptom development. Scale bar = 20 µm. (G) Transverse section through a large affected area showing intense accumulation ofphenolic compounds. The arrows depict the margin of the affected area. Scale is the same as in (F). (H) Developing lesion contained by azone of phellogen or cork cambium (arrows), which has restricted further advancement of the affected area. Note the raised appearance of theaffected area, stained greenish blue. Scale bar = 50 µm. (I) Fully developed zone of cork cambium (arrows) and sloughing off of affectedtissues in very advanced symptomatic roots. Scale bar = 50 µm. (J) Fluorescence of the cells in the zone of active division (indicated bybox D in (I)) stained with phloroglucinol and examined under blue light, reflecting an accumulation of lignin. Scale bar = 20 µm.

colonies on PDA were placed mycelial side down on theroots, about 10–12 plugs per root, and the containers weresealed. Two replicate roots were used for each culture andmost of the isolates were tested a minimum of two times.During 2005 an additional 100 isolates of Fusarium species

that originated from roots were tested for pathogenicityusing this in vitro procedure.

The pathogenicity of a subset of Fusarium species recov-ered from straw mulch and soil over the 2 years of study(around 300 isolates) was also assessed using the in vitro


inoculation method on ginseng roots and they were as-signed to pathogenicity groups. In addition, 37 isolates offour species of Fusarium originating from cereal speciesand soil from different geographic regions were tested forpathogenicity. Each isolate was tested on two replicateroots and the experiment was performed at least twice. Thepresence of superficial reddish brown lesions ranging in di-ameter from 1 to 5 mm on inoculated roots was assessed af-ter 7–10 days of incubation under laboratory conditions.The isolates were assigned to groups using the followingscale: highly aggressive, more than 12 lesions per root;moderately aggressive, 5–10 lesions per root; weakly ag-gressive, 1–4 lesions per root; and nonpathogenic, no le-sions. Inoculated roots were compared with control roots,which received PDA plugs, as well as with the underside ofthe inoculated roots not in contact with the inoculum.

Greenhouse and fieldA ginseng farm located in Kamloops, B.C., in which dis-

coloration symptoms were not observed on roots in 2005was selected. Groups of two or three 3-year-old plants withsurrounding rhizosphere soil were gently dug up and trans-planted into 15 cm diameter pots, covered with polyethyl-ene bags, and transferred to the greenhouse. The soil in thisfield was a silt–loam, pH 7.1, organic-matter content 1.6%,and NO3 (16 ppm), P (44 ppm), K (330 ppm), Ca(3660 ppm), Mg (419 ppm), and S (20 ppm). Inoculum of20 Fusarium isolates, moderately to highly aggressivebased on the laboratory assay, was produced on autoclavedoat kernels (1200 cm3 in a 2 L flask). The kernels were in-oculated with mycelial plugs from PDA colonies and incu-bated at room temperature for 2 weeks with occasionalshaking. Infested kernels were mixed with soil (about100 cm3 of inoculum in each 15 cm diameter pot) and strawmulch was placed over the soil surface. There were fourreplicate pots for each isolate. The plants were maintainedunder ambient greenhouse conditions (temperature range19–26 °C) and watered twice weekly over a period of2 months (June–July). Control plants were uninoculated orreceived autoclaved oat kernels only. The roots were then re-moved from the pots and washed, and the extent of discolor-ation was rated as described above for each isolate. A secondset of inoculations was made during October–Decemberusing field-grown roots inoculated as above and placed in thegreenhouse (temperature range 17–24 °C).

Field inoculations were conducted during the growingseason of 2005 in the same ginseng field in which no root-discoloration symptoms were detected. Plots were demar-cated, each measuring 1 m × 5 m and containing 40–60four-year-old ginseng plants. Inoculum of five isolates ofF. equiseti found to be highly aggressive in the laboratoryassay was produced on shredded-wheat cereal (Nabisco™brand). Moisture was added to 10% (v/v) and the substratewas autoclaved, inoculated with mycelial plugs, and incu-bated under laboratory conditions for 2 weeks with occa-sional shaking. The inoculum was placed on the soil surfaceadjacent to the crown of the root, which was exposed by re-moving the soil and straw mulch, and this was thenreplaced. Control plots received either no inoculum orautoclaved substrate only. The plants were watered immedi-ately after inoculation and included in the normal grower’s

management practices. There were two replicate plots foreach isolate, randomized within a block, and the trial wasconducted twice. In the first trial, inoculations were per-formed on 10 May 2005 and the roots were harvested on26 July 2005. In the second trial, inoculations were per-formed on 26 July 2005 and roots were harvested on27 September 2005. All of the roots in each plot were dugup and washed, and the percentage of roots with one ormore reddish brown discolored areas was rated. The datafrom each trial were kept separate and the mean incidenceof rusty root was determined.

Identification of Fusarium speciesOne hundred cultures were selected to represent all the

different morphological types of Fusarium colonies recov-ered from ginseng roots, straw mulch, and soil. The cultureswere single-spored and were compared with previouslyidentified cultures of Fusarium species from culture collec-tions and research laboratories for preliminary identificationto species level. A molecular analysis using sequence com-parisons of the elongation factor 1 (EF-1) alpha gene fromall isolates was also conducted. DNA was extracted fromlyophilized mycelium of these cultures as described byRosewich et al. (1999). Specific primer sets were used toamplify a 700 base pair (bp) fragment from the EF-1 alpharegion (Barasubiye et al. 2005), which was then sequenced inboth directions using the BigDye™ Terminator V3.1 cyclesequencing kit (Applied Biosystems, Foster City, Calif.). TheEF-1 alpha chromatograms were assembled using Seqman(DNAStar Inc., Madison, Wis.) and the isolates were identi-fied using BLAST on FUSARIUM-ID version 1.0 (Geiser etal. 2004) and GenBank.

Molecular detection of Fusarium species in ginsengtissues

DNA samples used for molecular detection were obtainedfrom 55 ginseng roots with root-discoloration symptoms aswell as from healthy roots from four ginseng farms (PR,RD, AS, HE) in B.C. collected during July–September 2005,and from field-inoculated ginseng roots that developed dis-coloration symptoms. The DNeasy™ Plant Mini Kit


Fig. 2. Dried ginseng roots showing the progression of discoloredareas near the crown. The arrows point to darkened shriveled areasin which the epidermal cells accumulate phenolic compounds. Theepidermis of these roots has not yet sloughed off. Scale bar = 1 cm.

(Qiagen, Valencia, Calif.) or the UltraClean™ Soil DNAKit (Mo Bio Laboratory Inc., Carlsbad, Calif.) was used forDNA extraction. The presence of Fusarium species in theDNA samples was first confirmed using polymerase chainreaction (PCR) primers designed to amplify a portion of the

ITS region specific to the genus Fusarium. The primersITS-Fu-f and ITS-Fu-r and PCR conditions as described byAbd-Elsalam et al. (2003) were used for this purpose. In ad-dition, hybridization of digoxigenin-labeled PCR-derivedEF-1 amplicons to an array of DNA oligonucleotides de-


Fig. 3. Recovery of Fusarium species from ginseng roots, straw mulch, and soil and artificial inoculation of roots. (A) Recovery ofF. equiseti from four out of five affected root-tissue segments on Komada’s medium. The bottom, slower growing colony was identified asFusarium tabacinum. (B) Recovery of F. equiseti (pink colonies) and carmine-red colonies of several other Fusarium species from wheat-straw segments collected from ginseng fields. (C) As in (B), showing Fusarium colonies following soil dilution onto Komada’s medium.Petri dishes are 9 cm in diameter. (D) In vitro root-inoculation method used to assess the pathogenicity of Fusarium isolates. Mycelialplugs were placed along the length of root and incubated for 7–10 days at 20–23 °C. The plugs shown are those of F. equiseti (left-handroot) and F. solani (two right-hand roots). (E–G) Reddish brown lesions developing 7 days following inoculation with Fusarium species invitro. (E) Fusarium equiseti. (F) Fusarium sporotrichiodes. (G) Fusarium avenaceum. (H) Development of root-discoloration symptoms onginseng following greenhouse inoculation of roots with F. equiseti. (I) As in (H) but inoculated with F. sporotrichioides. A control root ison the right. (J) Development of root-discoloration symptoms on ginseng following field inoculation of roots with F. equiseti. (K) Close-up of field-grown root inoculated with F. equiseti. Note initial blistering near the crown.

signed from sequences of a portion of the EF-1 alpha genefrom 26 Fusarium species was used to directly detect andidentify the complex of Fusarium species on ginseng rootsusing procedures described previously (Barasubiye et al.2005; Fessehaie et al. 2003; Goswami et al. 2006).

Results

Root-discoloration symptomsThe ginseng-production environment at the farms from

which roots were sampled included placement of a layer ofwheat- or barley-straw mulch, which ranged from 4 to10 cm in thickness, on the soil surface (Fig. 1A). Earlysymptoms of root discoloration were observed near thecrown region and consisted of reddish areas on the root sur-face (Figs. 1B, 1C). When the roots were washed, the dis-colored regions were found to consist of small flecks and

coalesced spots representing the early stages of diseasedevelopment (Fig. 1B). At advanced stages of symptom de-velopment, seen later in the growing season, the upper thirdof the root was orange–brown (Fig. 1C). When the rootswere dried, the areas near the crown affected by root discol-oration appeared shriveled and dark brown (Fig. 2).

Light microscopy of discolored rootsSmall reddish brown flecks and spots on field-grown

roots (Fig. 1B) and advanced discoloration (Fig. 1C) wereexamined under the light microscope. The former consistedof a small cluster of epidermal cells within the spots, whichstained a greenish blue with Toluidine Blue O (Fig. 1D),and hyphae could be seen ramifying through some of thecells. Hyphae in the epidermis grew into cortical cells(Fig. 1E). Affected cells with hyphae stained greenish blue,while healthy cells did not. A transverse section through a


Source of isolatesFusarium speciesrecovereda

Percent recoverybNo. of isolatestestedc

Pathogenicity ratingd

Mean Range High Moderate Low None

Ginseng tissues with rusty roote F. equiseti 31 3–42 76 10 11 21 34(n = 360) F. acuminatum 11 0–19 8 — — — 8

F. avenaceum 8 0–14 8 — 3 1 4F. sporotrichioides 8 0–11 7 2 3 1 1F. culmorum 5 0–7 4 — 2 1 1F. solani 4 0–15 6 — — — 6F. oxysporum 3 0–7 8 — — — 8F. tricinctum 2 0–4 2 — — — 2P. tabacinum 28 6–51 12 — — — 12

Soil from ginseng fields f F. equiseti 57 14–93 40 17 5 8 10(n = 32) F. avenaceum 11 0–14 6 — 1 3 2

F. acuminatum 8 0–15 4 — — — 4F. solani 8 0–16 7 — — — 7F. oxysporum 4 0–8 4 — — — 4F. culmorum 2 0–5 2 — 2 — —F. sporotrichioides 2 0–4 2 — 2 — —P. tabacinum 8 0–21 — — — — —

Wheat- and barley-straw mulchg F. equiseti 42 10–85 194 25 30 43 96(n = 32) F. acuminatum 25 6–34 12 — — — 12

F. culmorum 14 0–18 14 — 3 6 5F. sporotrichioides 8 0–12 7 — 3 2 2F. avenaceum 5 0–9 5 — 2 2 1F. tricinctum 3 0–5 2 — — — 2F. graminearum 1 0–3 1 — — — 1F. pseudograminearum 1 0–3 1 — — — 1F. crookwellense 0.5 0–1 1 — — — 1F. torulosum 0.5 0–1 1 — — — 1

aFusarium species were identified by morphological comparison with known Fusarium cultures grown on potato dextrose agar (PDA) and with EF-1alpha gene sequence homology.

bValues are from all replicates and sample sources, including four sampling times (June–September), two years (2004 and 2005), and four farms. The frequencyof recovery of each species is expressed relative to the total Fusarium colonies present in all samples. Colonies of Fusarium species were rated after 7–10 days.

cIsolates were selected at random from the total available.dPathogenicity was determined by placing 10–12 mycelial plugs of each isolate from 7- to 10-day-old PDA cultures on a ginseng root and rating the

number of visible lesions that developed after 7–10 days of incubation at 20–23 °C. For each isolate, two replicate roots were used and the experimentwas conducted a minimum of two times. Data represent the number of isolates in each pathogenicity category.

eTissues from 15 ginseng roots at each sampling time were plated onto PDA and Komada’s medium and incubated at 20–23 °C.fOne cubic centimetre of soil was diluted to 10–2 and 0.5 mL was plated onto each of four replicate dishes of Komada’s medium.gFive straw segments from each sample were placed onto each of 15 dishes of Komada’s medium and incubated at 20–23 °C.

Table 1. Frequency of recovery of 12 Fusarium species and Plectosporium tabacinum from American ginseng (Panax quinquefolius)farms in British Columbia during 2004 and 2005 and pathogenicity to ginseng roots in vitro.

larger affected area showed a central cluster of necroticcells surrounded by cells with disorganized cytoplasm(Fig. 1F). The affected area included the uppermost 8–12cell layers of the epidermis. At more advanced stages ofsymptom development, the number of affected cells in-creased and the area widened (Fig. 1G), and there was evi-dence of cell disruption and intense staining with ToluidineBlue O in the larger coalesced area (Fig. 1G). A transversesection through a large cluster of cells revealed a narrowzone of dividing cells that restricted further expansion ofthe affected area into the cortex (Fig. 1H). At more ad-vanced stages of discoloration, the actively dividing zone ofcells, presumed to be phellogen or cork cambium, gave theaffected area a raised appearance and the epidermis wasruptured (Fig. 1I). The underlying actively dividing zoneconsisted of lignified cells that fluoresced under blue lightwhen stained with phloroglucinol (Fig. 1J).

Recovery of Fusarium species from roots, straw mulch,and soil

PDA followed by malt extract agar yielded the greatestdiversity of fungi recovered from discolored root tissues.The genera identified, in order of decreasing frequency,were Fusarium, Penicillium, Trichoderma, Pythium,Rhexocercosporidium, Rhizopus, Cylindrocarpon, Alternaria,Aspergillus, and Rhizoctonia. Some unidentified species ofbacteria were also recovered. Fusarium species representedup to 65% of the total fungal colonies recovered on PDA.The use of Komada’s medium facilitated preliminary identi-fication of Fusarium species from affected tissues, strawmulch, and soil from ginseng fields. Colonies of F. equisetideveloped pinkish white aerial mycelium (Fig. 3A) and theunderside was pink–brown (Fig. 3B). Colonies of a largenumber of other Fusarium species developed a carmine-redcolor (Figs. 3B, 3C). Colonies of F. solani (Mart.) Sacc.[Nectria haematococca] were pale blue, while those of F.oxysporum Schlechtend.:Fr. developed a light-purple tingeon this medium after 2–3 weeks (data not shown). By com-parison, Fusarium colonies on Kerr’s, Nash and Snyder’s,and Czapek–Dox media were mostly white, making tenta-tive identification of the species difficult (data not shown).

Pathogenicity testsFusarium species induced the development of superficial

reddish brown lesions following in vitro inoculation(Fig. 3D). The most aggressive isolates recovered from af-fected roots were identified using morphological criteria andsequence comparisons of the EF-1 alpha gene as F. equiseti,followed by F. sporotrichioides Sherb., F. avenaceum (Fr.)Sacc., and F. culmorum (W.G. Smith) Sacc. (Table 1,Figs. 3E–3G). Several other species, including F. acumi-natum Ellis & Everh, F. solani, F. oxysporum, and F. tri-cinctum (Corda) Sacc. were also identified but they did notinduce any lesions on the root. Control roots also lacked le-sions after 10 days of incubation.

The mean frequency and range of frequencies of recoveryof Fusarium species from B.C. farms are shown in Table 1,together with pathogenicity ratings on ginseng roots. Thespecies recovered at the highest frequency from strawmulch, discolored root tissues, and soil from ginseng fieldswas F. equiseti. A total of 11 other Fusarium species were

recovered at varying frequencies (Table 1). Pathogenicitytests conducted on over 300 isolates confirmed thatF. equiseti, F. sporotrichioides, F. avenaceum, and F. culmo-rum produced reddish brown lesions on ginseng roots.However, only certain isolates of these species (around35%–50% of the total) were pathogenic, with the remaindercausing no lesions (Table 1). The highest proportion ofpathogenic isolates of F. equiseti was recovered from soilfrom ginseng fields and discolored root tissues, followed bystraw mulch. The relationship between population levels ofF. equiseti and recovery from straw mulch sampled from 18ginseng farms is shown in Fig. 4. As the degree of coloni-zation of straw increased, population levels in the underly-ing soil increased (R2 = 0.79, P ≤ 0.0001). The highestpopulation level among the 18 farms sampled in B.C. in2004 was 2.5 × 104 CFU/g soil.

When 37 reference isolates representing four species ofFusarium originating from cereal hosts from different geo-graphic regions were inoculated onto ginseng roots in vitro,the pathogenicity of F. equiseti, F. avenaceum, F. sporo-trichioides, and F. culmorum was confirmed (Table 2). Theisolates ranged from highly aggressive to nonpathogenicwithin each species.

Inoculations conducted in the greenhouse with severalFusarium species confirmed the ability of F. equiseti(Fig. 3H) and F. sporotrichioides (Fig. 3I) to induce superfi-cial reddish brown lesions on the root surface. The diseaseincidence (frequency of roots with discolored areas) overthe two experiments for these two species was 37 ± 9%(mean ± SD). Control roots developed no discoloration. In-oculations conducted in the field during 2005 with five iso-lates of F. equiseti showed that four isolates inducedsymptoms of discoloration (Figs. 3J, 3K) in both trials atfrequencies ranging from 10% to 40% (Fig. 5). Controlroots developed no discoloration. There were differences infinal disease incidence among isolates in the two trials butthe general trends were similar except for isolate Fus18ZP2,which did not cause any symptoms in the first trial. Themean maximum daytime temperatures during the field inoc-ulation experiments were 34 °C from May–July and 32 °Cfrom July–September. Soil samples taken from inoculatedplots adjacent to roots revealed a F. equiseti population of


Fig. 4. Relationship between recovery of Fusarium equisetifrom straw mulch and inoculum levels in the underlying soil.Samples were collected from 18 ginseng farms during August2004 and plated onto Komada’s medium.

3.2 × 104 CFU/g following soil dilution and plating ontoKomada’s medium.

Molecular detection of Fusarium species in ginsengtissues

Using Fusarium primers ITS-Fu-f and ITS-Fu-r, a bandapproximately 400 bp in size was detected in discolored tis-sues originating from four ginseng farms, as well as in fieldinoculated discolored roots showing discoloration symp-toms (Fig. 6). Healthy ginseng root samples did not showthis band. Using a EF-1 Fusarium DNA array, a moredetailed analysis of the specific Fusarium species wasobtained. In 1-year-old plants from B.C. with root-discoloration symptoms, six Fusarium species were de-

tected, including F. equiseti (Fig. 7A), the most aggressivespecies on ginseng roots. From 4-year-old roots, five spe-cies were detected (Fig. 7B). When roots inoculated withF. equiseti in the field experiment were included in theDNA hybridization analysis, strong detection of this specieswas observed, as expected. Five other Fusarium specieswere also present (Fig. 7C). A summary of the results fromthe DNA hybridization analyses conducted on over 50 dis-colored roots from four ginseng farms in B.C., in whichyoung lesions were distinguished from older lesions, isshown in Table 3. The total number of species detectedranged from five to eight, and in all farms, older lesionsyielded a greater number of Fusarium species than youngerlesions. Fusarium equiseti was detected in seven out of


Fusarium species Isolate No. or codeHost or substrateof origin Geographic origin Source

Pathogenicityrating onginseng rootsa

F. avenaceum Fav/Avwmb12 Wheat Oak Lake, Manitoba A. Xue ModerateF. avenaceum Fav Wheat Grenfell, Saskatchewan M. Fernandez LowF. avenaceum F.av.1 Wheat Booth Siding, Saskatchewan R. Clear LowF. avenaceum F.av.2 Wheat Dawson Creek, British Columbia R. Clear NoneF. culmorum 3288 Unknown Unknown H.C. Kistler NoneF. culmorum F.07.03.62 Wheat Whitman, Washington T. Paulitz LowF. culmorum F.07.03.66 Wheat Whitman, Washington T. Paulitz NoneF. culmorum F.07.03.51 Wheat Whitman, Washington T. Paulitz NoneF. culmorum F.cul Wheat Scott, Saskatchewan M. Fernandez LowF. culmorum F.cul.1 Wheat Dawson Creek, British Columbia R. Clear NoneF. culmorum F.cul.2 Wheat Hague, Manitoba R. Clear NoneF. culmorum Fcu/Cuwab24 Wheat Lethbridge, Alberta A. Xue LowF. equiseti 25796(CBS406.86) Soil Unknown K. O’Donnell NoneF. equiseti 96-M11 Unknown Alaska H.C. Kistler LowF. equiseti 96-M12 Unknown Alaska H.C. Kistler LowF. equiseti 96-M14 Unknown Alaska H.C. Kistler LowF. equiseti Feq/Eqwon30 Wheat Ottawa, Ontario A. Xue HighF. equiseti F.e Wheat Scott, Saskatchewan M. Fernandez ModerateF. equiseti F.e Wheat Unknown R. Clear NoneF. equiseti F.e#85 Cereal seed Quebec, 2003 R. Clear ModerateF. equiseti F.e#86 Cereal seed Quebec, 2003 R. Clear LowF. equiseti F.e#87 Cereal seed Quebec, 2003 R. Clear HighF. equiseti F.e#88 Cereal seed Quebec, 2003 R. Clear NoneF. equiseti F.e#89 Barley seed Stonewall, Manitoba, 2001 R. Clear NoneF. equiseti F.e#90 Barley seed Stonewall, Manitoba, 2001 R. Clear LowF. equiseti F.e#91 Barley seed Stonewall, Manitoba, 2001 R. Clear ModerateF. equiseti F.e#92 Barley seed Stonewall, Manitoba, 2001 R. Clear LowF. equiseti FR311–4 Wheat crowns Fort Vermilion, Alberta K. Turkington NoneF. equiseti FR310 Wheat crowns Fort Vermilion, Alberta K. Turkington HighF. equiseti 2004WRS Barley Boisevain, Manitoba A. Tekauz NoneF. equiseti 2005WRS Barley Rosebank, Manitoba A. Tekauz ModerateF. equiseti 2006WRS Barley Westbourne, Manitoba A. Tekauz LowF. equiseti F.e Wheat Glenlea, Manitoba J. Gilbert NoneF. sporotrichioides 96-M32 Unknown Unknown H.C. Kistler ModerateF. sporotrichioides Fsp/Spwmb53 Wheat Oak Lake, Manitoba A. Xue ModerateF. sporotrichioides F.sporo.1 Wheat Unknown R. Clear NoneF. sporotrichioides F.sporo.2 Wheat Unknown R. Clear Moderate

aPathogenicity was determined by placing 10–12 mycelial plugs from 7- to 10-day-old potato dextrose agar cultures on a ginseng root and rating thenumber of visible lesions that developed after 7–10 days of incubation at 20–23 °C. For each isolate, two replicate roots were used and the experimentwas conducted a minimum of two times.

Table 2. Reference isolates of four Fusarium species originating from cereal hosts used to inoculate ginseng roots.

eight samples and F. sporotrichioides was present in fiveout of eight samples, including the sample in whichF. equiseti was absent (Table 3).

Discussion

The light-microscopic observations of discolored areason ginseng roots showed that the affected cells were mostlylocalized to the uppermost 8–14 cell layers, or 100–200 µmdepth, and they stained greenish blue with ToluidineBlue O, indicating an accumulation of phenolic compounds(Gutmann 1995; Krishnamurthy 1999; Punja 2004). Furtherexpansion of the affected area appeared to be restricted bythe development of a narrow layer of dividing cells in theregion adjacent to the cortex, which resembled a phellogenor cork cambium layer (Esau 1965; Jones 1931). The cellwalls in this zone were found to contain lignin. These ob-servations suggest that the ginseng roots had initiated a re-sponse by attempting to restrict the continued expansion ofthe affected areas, resembling the responses of some plantspecies to wounding or pathogenic infection (Esau 1965;Hammerschmidt 1984; Jones 1931). Production of fluores-cent phenolic compounds in and around discolored areas or“rust spots” was also reported by Campeau et al. (2003b).

DNA of F. equiseti was detected in over 90% of discol-ored root samples (comprising 50 roots) from four farms inB.C., although mean percent pathogen recovery from theseroot tissues on Komada’s medium over the 2 years of studywas around 31%. This low recovery rate could have beendue to the rapid accumulation of fungitoxic phenolic com-pounds in affected tissues, which could have rendered thepathogen inviable. Biochemical analysis of discolored gin-seng tissues has revealed a marked elevation of specificphenolic compounds, including several flavonoids (quercetin,catechin, tannic acid) (Rahman and Punja 2005a), which areintermediates or end-products of the phenylpropanoid path-way leading to the production of lignin and other defense-

related compounds in plants (Boerjan et al. 2003; Friend1981). Phenolic compounds play a role in limiting patho-genic invasion of plant tissues during defense responses(Matern and Kneusel 1988; Nicholson and Hammerschmidt1998). Fungitoxicity of a number of flavonoid compoundsto Fusarium species infecting barley has been reported(Skadhauge et al. 1997).

Once the epidermal cell layers are disrupted, the underly-ing tissues can be further colonized by secondary fungi, aswas observed in this study. Many fungal genera and a num-ber of nonpathogenic Fusarium species were also isolatedon PDA from discolored tissues. These organisms may pro-mote further tissue decay and thereby reduce the recoveryof primary Fusarium species, confounding the interpreta-tion of isolation data. Nonpathogenic isolates includedPlectosporium tabacinum (J.F.H. Beyma) M.E. Palm,W. Gams & Nirenberg (anamorph Fusarium tabacinum),which is prevalent in soil and has been implicated as thecause of some root diseases (Palm et al. 1995), as well asF. solani, which can cause root decay on ginseng seedlings(Punja 1997). The in vitro root inoculation test was a usefulapproach to rapidly evaluate fungal isolates for their abilityto induce reddish brown lesions. The test was shown to bereliable when pathogenic isolates were tested consecutivelyon different roots over a period of 2 years. Highly aggressiveisolates identified in the laboratory test also induced symp-toms in roots in the greenhouse and field inoculation trials.

Previous reports on the development of root-discolorationsymptoms (also referred to as rusty root) on ginseng grownin other parts of the world have implicated such factors asoxidative stress, anaerobic conditions, and high Fe levels insoil as the cause (Lee et al. 2000; Lee et al. 2004; Yang etal. 1997; Yinping et al. 1997). Environmental-stress factorsthat increase root exudation or oxidative-stress responses in


Fig. 5. Incidence of disease resulting from inoculation of ginsengroots under field conditions with five isolates of F. equiseti.Trial 1 was initiated on 10 May (inoculation date) and roots wereharvested on 26 July 2005. Trial 2 was initiated on 26 July 2005and the roots were harvested on 27 September 2005. Data arefrom two replicate plots in each trial, consisting of 80–120 rootsin total. Control roots developed no discoloration.

Fig. 6. Detection of Fusarium species in discolored ginseng roottissues using the polymerase chain reaction (PCR) with specificprimers ITS-Fu-f and ITS-Fu-r. M, 100 bp ladder; lane 1, positivecontrol (DNA from strain NRRL 31084 of F. graminearum);lanes 2–5, discolored ginseng tissues from field-grown roots;lane 6, roots inoculated in vitro with isolate ZP22–2 ofF. equiseti; lane 7, roots inoculated in the field with isolateZP22–2 of F. equiseti; lane 8, healthy ginseng root.

roots (Okubara and Paulitz 2005), as well as ethylene pro-duction (Campeau et al. 2003b; Okubara and Paulitz 2005),may predispose ginseng roots to infection by Fusarium spe-cies, since these stresses can reduce the resistance of rootsto necrotrophic fungal pathogens (Okubara and Paulitz2005). Our observations do not rule out the possibility thatfungi other than Fusarium species that elicit a response inroots while attempting to penetrate the upper cell layers can

induce root-discoloration symptoms. For example, a recentreport implicates species of Rhexocercosporidium as thecausal agent of root-discoloration symptoms (rusted root)on ginseng (Reeleder et al. 2006). The role of this fungus inthe development of symptoms on ginseng roots grown inB.C. remains to be confirmed.

The DNA hybridization assay was more sensitive thanthe recovery of Fusarium species from affected tissues on


Fig. 7. DNA array hybridization assay to a panel of 82 specific oligonucleotides representing 26 different Fusarium species and severalclades above or below the species level. The specific oligonucleotides designed from EF-1 alpha gene and intron sequences wereamino-modified and spotted in duplicate on nylon membranes. They were arranged in 6 rows × 14 columns in two replicates. Positivehybridizations can be seen as chemiluminescence-labeled spots at specific locations along the array representing each species. (A and B)DNA samples originating from ginseng roots of different ages with discolored root symptoms from British Columbia. (C) DNAsamples from ginseng roots inoculated with F. equiseti under field conditions (trial 2). C, control, reverse complement oligo of forwardPCR primer; F. eq, F. equiseti; F. pseu, F. pseudograminearum; F. tri, F. tricinctum; F. acu, F. acuminatum; F. aven complex,F. avenaceum complex; F. meri, F. merismoides; F. sam, F. sambucinum.

Komada’s medium. For example, F. torulosum andF. tricinctum were detected in the DNA hybridization assaybut their recovery in culture was very low. Therefore, thepathogenicity of only a few isolates of these species couldbe tested on ginseng. The detection of DNA of Fusariumspecies in discolored roots was not used as the sole indica-tor of their role in the causation of symptoms, since a largenumber of nonpathogenic species and considerable varia-tion in pathogenicity among isolates within a species wereobserved. This limitation was recognized by Lievens andThomma (2005) in their discussion on the use of pathogen-detection arrays. Consequently, pathogenicity tests on re-covered isolates were necessary to establish their role in thecausation of root-discoloration symptoms.

All of the Fusarium species recovered from straw mulchand ginseng tissues in this study have also been isolatedfrom cereal-straw residues, root tissues, and wheat and bar-ley seed (Gale 2003; Parry et al. 1995; Pereyra et al. 2004;Shaner 2003). However, F. equiseti has been recovered atonly low levels (0.2%–4.6%) from diseased cereal speciesand is not considered to be one of the primary pathogenscausing head blight (Gale 2003; Parry et al. 1995; Pereyraet al. 2004; Shaner 2003). This species can be isolated fromsoil and its ability to colonize wheat-straw residue is exten-sive. Together with F. sporotrichioides, it was found at highlevels colonizing buried wheat residue (Pereyra et al. 2004).

Pathogenic isolates of F. equiseti can cause crown infec-tion, root rot, and fruit and seed decay on several plant spe-cies, including muskmelon (Adams et al. 1987; Sumbaliand Mehrotra 1982), cotton (Chimbekujwo 2000), cumin(Reuveni 1982), and cowpea (Aigbe and Fawole 1999). Thepathogen has also been isolated from the roots of diseasedasparagus (Vujanovic et al. 2006) and wheat plants(Demirci and Dane 2003) and can cause minor root andculm rot on wheat (Fedel-Moen and Harris 1987; Nash andSynder 1965). While F. equiseti was considered a minorpathogen on wheat and barley seedlings, it caused a signifi-cant reduction in root length (Fedel-Moen and Harris 1987;

Strausbaugh et al. 2005). On potato, F. equiseti inducedsmall superficial lesions, particularly around the lenticels(Rai 1979). These lesions coalesced to produce surface dis-coloration not unlike that seen on ginseng, which later pro-gressed into the tuber to cause a dry rot (Rai 1979).Fusarium equiseti can also cause root decay on ginsengseedlings (Punja 1997), and we occasionally observed thissymptom on inoculated roots under high soil moisture con-ditions in the greenhouse experiments.

The initial symptoms of root discoloration at the crownof ginseng roots are consistent with the mode of infectionby F. equiseti on other host plants, as well as by otherFusarium species recovered in this study. All of the remain-ing pathogenic Fusarium species recovered from affectedginseng tissues, except F. sporotrichioides, are able to causecrown infection on other crop species, especially cereals(Paulitz et al. 2002; Smiley and Patterson 1996;Strausbaugh et al. 2005). Fusarium equiseti, as well asthese other Fusarium species, can colonize seed of numer-ous plant species, making seed transmission a potentialsource of inoculum for disease development (Gordon 1952;Shaner 2003). The potential for transmission of Fusariumspecies on ginseng seed has not yet been established.

Recent advances in the development of methods for con-trolling fusarium head blight on wheat, notably fungicideapplications (Ioos et al. 2005; Mesterházy 2003), may proveto be useful in reducing Fusarium infection on ginseng.Colonization of straw mulch and crop residues on the soilsurface significantly increases the inoculum potential ofFusarium species and the severity of fusarium head blight(Dill-Macky and Jones 2000; Parry et al. 1995; Pereyra etal. 2004; Shaner 2003). The survey of 18 ginseng farmssuggested that higher frequencies of straw-mulch coloniza-tion enhanced the populations of F. equiseti in soil. The re-sults presented in this study demonstrate the uniqueinteractions of Fusarium species commonly found on cerealspecies with ginseng roots, causing root-discolorationsymptoms that resemble those described as rusty root


PR Farm RD Farm AS Farm HE Farm

Fusarium species New Old New Old New Old New Old

F. equiseti + + + – + + + +F. tricinctum + + – + – + + +F. sporotrichioides – + + + + + – –F. acuminatum – + – + – + + +F. avenaceum + + – – – – + +F. torulosum + – – – – + + +F. solani – – – + – + – +F. cf. merismoides – – + + – + – –F. graminearum + – + – – – – +F. redolens – – – – – – – +F. pseudograminearum – – – – – – + –

Total species 5 5 4 5 2 7 6 8

Note: Fusarium species were detected using a EF-1 DNA array containing specific oligonucleotides.Lesions were characterized as new or old based on their visual appearance on roots (see Fig. 1). Newlesions were small, with no raised corky areas characteristic of older, spreading lesions. Over 50 rootscollected during July–September 2005 were assayed.

Table 3. Molecular detection of Fusarium species within discolored ginseng root tissues(rusty root lesions) originating from four ginseng farms in British Columbia.

(Hildebrand 1935; Zinssmeister 1918). These fungi elicit atissue response that is manifested by the appearance of red-dish brown surface discoloration due to accumulation ofphenolic compounds. The surface discoloration is likely anoutcome of oxidation and polymerization of these phenoliccompounds.

Acknowledgments

Funding for this research was provided by Chai-Na-TaGinseng Corp., The Natural Sciences and Engineering Re-search Council of Canada Collaborative Research and De-velopment Program, and the Associated Ginseng Growersof British Columbia. We are indebted to numerous ginsenggrowers, particularly Doug Murdoch, Ray Dunsdon, andMenno Schellenberg, for providing ginseng root and strawsamples for this study. We thank Terry Holmes, Pacific For-estry Centre Microtechnique Laboratory, Victoria, B.C., forinvaluable assistance with all aspects of the histopatho-logical work. The assistance provided by Ken Ng, JayaJayaraj, and Geoff Bradley with field sampling and JorgeLussio and Lisa Leippi with laboratory work is gratefullyacknowledged. We thank Kerry O’Donnell, USDA Agricul-tural Research Service, Peoria, Illinois, for confirming theidentification of F. equiseti isolates, and all of the individu-als listed in Table 2 for generously providing cultures ofFusarium species.

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