Importance of Plant Spacing and Sclerotial Position to Developmentof Sclerotinia Wilt of Sunflower

H. C. HUANG and J. A. HOES, Research Scientists, Plant Pathology, Agriculture Canada, Research Station, Morden,

Manitoba, ROG lJO

ABSTRACTHUANG, H. C., and J. A. HOES. 1980. Importance of plant spacing and sclerotial position to

development of Sclerotinia wilt of sunflower. Plant Disease 64:81-84.

Wilt of sunflower in Manitoba was due to infection of underground tissue by Sclerotiniasclerotiorum. The taproot-hypocotyl axis was the primary site of infection; lesion development on

this tissue resulted in wilting of leaves and sudden death of the plant. The penetration site on the

taproot lesion was within the zone of lateral roots. Studies in artificially infested field plots showed

that sclerotia were the primary source of inoculum and that their spatial distribution affected the

incidence of wilt. Wilt incidence was highest when sclerotia were buried next to seed and decreased

with increasing distance between sclerotia and seed. The plant at the site of infestation developed

wilt symptoms first and became a primary infection locus (PIL) from which the pathogen spread by

root contact from plant to plant in sequential order. Plant spacing affected efficiency and time of

spread to adjacent plants and the number of new infections developing from each PIL.

Additional key words: basal stem rot, Helianthus annuus, plant density, root rot, Whetzelinia

sclerotiorum

Head rot and wilt or basal stem rot orroot rot caused by Sclerotinia sclero-tiorum (Lib.) de Bary [(=S. libertianaFuckel (20); = Whetzelinia sclerotiorum(Lib.) Korf and Dumont (13)] areimportant diseases of sunflower (Helian-thus annuus L.) in Manitoba (8) and arelimiting factors in production (10).Isolates from sunflower with Sclerotiniawilt in Manitoba (5), Montana (22), andWisconsin (12) all belonged to the samespecies. Sunflower production in Mani-toba dropped from about 77,000 hectaresin 1972 to 8,500 hectares in 1974 (3). Thisdecline was due in part to severe yieldlosses caused by Sclerotinia head rot andwilt.

Other diseases caused by S. sclero-tiorum include white mold of bean (7),stalk rot of cruciferous seed crops (15),stalk rot of potato (17), and drop oflettuce (16). All these diseases are due toinfection of aboveground parts byairborne ascospores produced fromapothecia arising from sclerotia at or nearthe soil surface. Ascospore infection isconditioned by a long period of wetnessand by senescent flower tissue that servesas a food base for the germinatingascospore (1). Ascospores likewise infectaboveground parts of sunflower andcause head rot. This disease is prominentwhen high moisture conditions existduring late July, August, and September,months when sunflowers are flowering,

Accepted for publication 4 March 1979.

00191-2917/80/000017$03.00/001980 American Phytopathological Society

seed is being produced, and senescingflorets are abundant.

In contrast to head rot, Sclerotinia wiltof sunflower may occur at the seedlingstage. Most wilt symptoms developduring the flowering and seed develop-ment stages, but conditions are dry. Wiltsymptoms are first observed in individualplants scattered throughout the field. Asthe disease progresses, plants are affectedin series of two or more. Plants withincipient wilt are characterized bychlorosis and wilting of leaves, often onone side only. Water-soaked lesionsoccur on the taproot and on some fibrousroots. A severely diseased plant shows"drop" in which all leaves wilt suddenly; alesion at the stem base is characteristic.The lesion is continuous and extendsfrom the taproot along the hypocotyl toas much as 50 cm along the basal stem.Most fibrous roots are rotted and thecortex has sloughed off. White myceliuminterspersed with sclerotia occur on thebrown, wet lesion, underground as well asaboveground. Mycelium and sclerotiaalso occur on the outside of the taprootand the stem base and on the outside ofthe laterally extending fibrous roots (9).Wilting of leaves, the nature of the lesion,and the presence of large sclerotia andwhite mycelium are symptoms and signscharacteristic of wilt disease (22).

Spread of the pathogen and develop-ment of new infections are aspects of wiltdisease that are not fully understood.Bisby (4) believed that plants are "usuallyattacked at or near the soil surface" andnoted that the disease occurred mainly inpatches, a feature also observed by Jones(12). Transplanted sunflower plants

artificially inoculated with mycelium (4)or already wilted and carrying mycelium(22) caused wilt of neighboring plants ofsunflower (4) and of hemp (22). Youngand Morris (22) concluded that thepathogen penetrated the sunflowermainly through roots and spread rapidlyalong the row unless the stalks were 30 cmapart. Direct evidence of spread of thepathogen by root contact was notpresented, however, and the primarysource of inoculum was not emphasized.

This paper describes the pattern of wiltdistribution in the field, the site ofprimary infection, the mode of spread ofthe pathogen, and the effects of inoculumposition and plant spacing on theepidemiology of Sclerotinia wilt ofsunflower. The studies were conductedon naturally and artificially infested soils.

MATERIALS AND METHODSStudies on naturally infested soils.

Pattern of wilt distribution in commercialfields in Manitoba. In 1972, a field of thecultivar Peredovik at the seed develop-ment stage was severely diseased withSclerotinia wilt. The rows were 90 cmapart, and variable distances separatedthe within-row sunflowers. A total of3,231 plants were inspected for wilt.Whether diseased plants occurred singlyor within clumps was noted. Single plantswere taken as those separated fromadjacent ones in the same row by morethan 20 cm. A plant was consideredbelonging to a clump if it was separatedfrom adjacent ones by less than 20 cm.The number of diseased and healthyplants in the same clump was counted,but the sequential order of diseased andhealthy plants was not recorded.

In 1977, 2,791 plants of the cultivarKrasnodarets were inspected in a field inwhich wilt was severe. Row spacing was90 cm and within-row spacing of plantsvaried. Series of one or more diseasedplants bounded by one or more healthyplants were recorded, and the kind andsize of series were noted. The data wereanalyzed by the "doublet" method of VanDer Plank (19), in which two adjacentdiseased plants comprise a doublet, threediseased plants comprise two doublets,four diseased plants comprise threedoublets, etc. The expected number N ofdoublets in a population of n plants of

which m are diseased is - (m-l), with a

standard error of x Presence of

Plant Disease/January 1980 81

significantly more than the theoreticalnumber of doublets was taken to suggesttransmission of the pathogen betweenadjacent plants.

Primary site of infection. The studywas conducted on clay loam soil at theResearch Station. Samples were taken atthe beginning of flowering when only afew scattered sunflower plants showedadvanced symptoms of Sclerotinia wilt.The plants were grown in rows 90 cmapart, and row-to-row spread of thepathogen was precluded (22). Twenty-one plants that had incipient wilt andwere bordered by healthy plants weredug. Leaves on one side of the selectedplants were wilting, and the lesions wereunderground and typical of thoseassociated with Sclerotinia wilt. Thelength of the lesion and the distancebetween its center and the soil surfacewere measured. The center of the lesionwas assumed to be the site of penetrationby the pathogen. The depth of the zonealong which lateral roots occurred wasalso recorded. For identification of thepathogen, the taproot along with thehypocotyl of each plant was washedunder tap water and incubated in a plasticbag for 7 days at room temperature.

Transmission of the wilt pathogen. Ata later date, 64 pairs of wilted plants wereselected from the same Research Stationfield plot. One plant of each pair wasseverely wilted, while the other showedincipient wilt symptoms only. After thesoil was carefully removed to a depth of20 cm or more, the root systems of thepair were examined in situ for signs oftransmission of the pathogen. Thedistance between the centers of the stemsof the two plants was recorded. Five pairsof plants were dug, taken to the labora-tory, and processed for identification of

the pathogen.Studies on artificially infested soil.

Three experiments using the cultivarKrasnodarets were conducted in a field atthe Research Station. The field wasconsidered to be free of S. sclerotiorumbecause a cereal-fallow rotation had beenfollowed for more than 12 yr. Plots wereinfested with sclerotia harvested fromdiseased sunflower heads the previous falland stored in a dry condition at roomtemperature until use. Seed was plantedin freshly dug furrows 5 cm deep and 600cm long. Rows were 90 cm apart. Afterinoculation and planting, the furrowswere filled and packed.

Effect ofplant spacing on wilt develop-ment in neighboring plants. Ten sclerotiawere placed at each of three sites in eachfurrow. The sites were centrally locatedand 150-160 cm apart, depending on thespacings between seeds subsequentlyplanted. Seed was planted just above theinoculum, and planting of a row wascompleted by spacing seeds at 10, 20, 30,or 40 cm. Each treatment consisted of sixrows. Wilt development was monitoredweekly. Each wilted plant was markedwith a stake and the date of wilt wasrecorded.

Effect of vertical and horizontal seed-inoculum distance on wilt development.For vertical seed-inoculum distance, seedwas sown within the row at 30-cm spacingto preclude most of the plant-to-plantspread of the pathogen (22). Ten sclerotiawere buried at each plant site at varyingvertical distances from the seed. Treat-ment consisted of placing sclerotia 4 cmabove the seed, at seed level, or 5, 15, or25 cm below the seed. For horizontalseed-inoculum distance, seed was sown at60-cm spacing to preclude infection fromsclerotia belonging to different plants in

the same row. Sclerotia were buried atseed level at varying horizontal distancesfrom each seed site. Treatment consistedof placing one dose of inoculum (fivesclerotia) adjacent to the seed or fourdoses crosswise in four directions 10, 20,or 30 cm from the seed. The study wasconducted as a randomized block (single-row plots) with six replicates.

RESULTSWilt distribution in commercial fields

in Manitoba. Of the 3,231 plantsinspected in 1972, 912 occurred singlyand 2,319 occurred in clumps of two toseven or more plants. The number ofplants belonging to clumps of two ormore and the number of clumps variedfrom 132 in 22 clumps of six plants to 780in 390 clumps of two. The incidence ofwilt increased from 66% in single plantsto 83% in clumps of seven to 10 plants(Fig. 1).

Of the 2,791 plants inspected in 1977,997 were wilted. Of the diseased plants,129 occurred singly and 194, 174, 164,130, 60, 14, 40, 18, and 10 occurred inseries of two to 10 plants, respectively; 64plants occurred in series of 12 to 21plants. The estimated total number ofdoublets was 356, with a standard error of19. However, 622 doublets were actuallyobserved; the 266 more doublets thanexpected was 14 times the standard errorof 19. Because the plant population waslarge, the difference was considered to behighly significant (19) and suggested thatthe wilt pathogen had been transmittedfrom plant to plant.

Primary site of infection. Lesions ontaproots of the 21 plants with incipientwilt measured 2-7 cm in length andaveraged 4.2 cm. All lesions yieldedmycelium and sclerotia characteristic ofS. sclerotiorum when incubated in thelaboratory. The lesion centers or thesites of penetration occurred at soildepths of 1.5-8.5 cm, with the majorityoccurring at 4-6 cm. The depth of the soilprofile containing lateral roots rangedfrom 5-12 cm and averaged 8 cm. Thesites of penetration were thus alwayswithin the zone in which lateral rootsoccurred.

Transmission of the wilt pathogen.Transmission of S. sclerotiorum by rootcontact was evident from the diseased-pair study. In 54 pairs of plants separatedby 30 cm or less, wilting of the "receptor"plant with incipient wilt always occurredon the leaves facing the severely infected"donor" plant. The taproots of the 54receptor plants were characterized,without exception, by a lesion on thesame side as the wilting leaves. Lateralroots originating from lesion areas ondonor and receptor plants intermingled

and showed signs of the wilt pathogen.When plant distance ranged from 31 to 40cm, however, transmission by rootcontact of the pathogen was evident inonly five of the eight pairs examined. Itwas not evident in the two pairs with

(1)4W

CL

0m-W

1 2 3 4 5 6 7-10

No. Plants /ClumpFig. 1. Effect of plant density on incidence of sunflower wilt caused by Sclerotinia scerotiorum.Data are based on 912 plants occurring singly; the number of plants belonging to clumps variedfrom 132 in clumps of six plants to 780 in clumps of two.

82 Plant DiseaseNol. 64 No. 1

plant distance of more than 40 cm,indicating that infection arose indepen-dently in these pairs. Mycelium andsclerotia characteristic of S. sclerotiorumwere produced from the lesions ontaproots of all 10 plants that had beenremoved from the field and incubated inplastic bags at room temperature.

Effect of plant spacing on wilt develop-ment in neighboring plants. Wilt devel-oped at 37 of the 72 sites where inoculumhad been deposited. In each of the 37cases, the plant at the inoculum sitedeveloped wilt first and became a primaryinfection locus (PIL). In turn, plantsadjacent to the PIL developed wilt asa result of the spread of the pathogen.Continued plant-to-plant spread of S.sclerotiorum ultimately produced seriesof wilted plants. The longest seriesconsisted of nine plants and occurred in arow with 10-cm plant spacing. The serieswas represented as (-8-7-5-4-3-1-2-4-5-),each figure indicating the number of theweek during which a plant wilted. Week 1began on 22 June 1976. The differencebetween two successive figures in theseries indicated the time required by thepathogen to spread from plant to plantand to cause new infections. The exampleshows that the pathogen spread in twodirections and caused wilt of plants insequential order. The symptom severityduring development of a series wasalways greater in the PIL than in theextreme plants of the series.

Plant spacing affected efficiency andtime of spread of the pathogen and thusthe number of new infections. Thepathogen spread from nine of 12 PIL atthe 10-cm spacing but from only one ofnine PIL at the 40-cm spacing. The timeneeded for pathogen spread averaged 1.5wk at the 10-cm spacing, 2.6 wk at the 20-cm spacing, and 4.3 wk at the 30-cmspacing. Spread at the 40-cm spacingoccurred in pnly one instance and to oneplant only. As a result of pathogenspread, about four to five times as manyplants were killed at the 10-cm spacingthan at the 30-cm spacing. Pathogenspread was negligible at the 40-cm

spacing (Table 1).Effect of vertical and horizontal seed-

inoculum distance on wilt development.Proximity of seed and sclerotia markedlyaffected the incidence of wilt. More than50% of the plants developed wilt symp-toms when sclerotia were buried at seedlevel near the seed (Tables 2 and 3). Wiltincidence decreased significantly whensclerotia were buried 4 cm above or 5 or15 cm below the seed (Table 2). Similarly,wilt incidence decreased significantlywhen seed and sclerotia were separatedhorizontally by 10 cm or more (Table 3).

DISCUSSIONData on the distribution of Sclerotinia

wilt of sunflower in commercial fieldsindicated that when more plants weregrouped together, more wilt developed.Once a plant was infected, the possibilityof other plants in the same clumpdeveloping wilt increased, and thenumber of doublets that developedexceeded expectation significantly. Thesedata suggested that wilt developed as aresult of spread of the pathogen and thatplant spacing was an important factor inwilt development. The hypothesis wasconfirmed in studies in artificiallyinfested plots. Wilt developed fastestwhen the inoculum was closest to theseed. The pathogen then spread fromthese primary infection loci to adjacentand other neighboring plants in sequen-tial order. The closer the plant spacing,the more efficient the spread of thepathogen, the less time needed forinfection, and the more plants thatdeveloped wilt. One primary infectionlocus caused the death of as many as eightneighboring plants spaced at 10 cm in therow. Transmission of the pathogen wasminimal at spacings of 30 and 40 cm,confirming the observation of Young andMorris (22). Wide spacing promoteddisease escape because of fewer potentialinfection loci and because of decreasedincidence of transmission. Burdon andChilvers (6) found that varying the plantdensity of Lepidium sativum had aneffect on the frequency of primary

infection loci of Pythium irregularesimilar to that produced by varying thedensity of the inoculum. An analogoussituation likely exists in the Helianthus-Sclerotinia system.

Sclerotia of S. sclerotiorum are theprimary source of inoculum, and infec-tion of underground parts results indevelopment of wilt symptoms in sun-flower. This was clearly evident from astudy in an artificially infested soil.Hyperparasitism (11) by a soilbornemicroorganism may have been the causeof failure at the 35 sites where wilt did notdevelop. However, infections wereestablished in plants at the remaining 37sites where sclerotial inoculum wasintroduced. The infections were due tomycelium originating from the sclerotia,because conditions were not conducive tocarpogenic germination and apotheciawere not observed. Sclerotial germina-tion by production of mycelium wasapparent in studies in which S. sclero-tiorum produced secondary sclerotia insoil (7,2 1) and also on tap water agar (2 1).Parent sclerotia on agar produced a massof mycelium that developed into asecondary sclerotium. There was littledoubt that the secondary sclerotiaproduced in soil and on agar haddeveloped by the same process (21). In

Table 2. Effect of vertical distance betweenseed and sclerotia of Sclerotinia sclerotiorumon incidence of wilt in sunflowerx

Distance of sclerotiaabove or below seedy

(cm)

4 (above)0 (seed level)5 (below)

15 (below)25 (below)

Wilted plantsz(%)

18 bc52 a16 bc21 b

8c

Table 1. Effect of plant spacing on time and efficiency of Scierotinia sclerotiorum to spread fromprimary infection locus' (PIL) and cause wilt in sunflower

Within-row plant spacing (cm)

Variable 10 20 30 40

Efficiency of spreadb 9/12 3/7 5/9 1/9

Time (wk) for plant-to-plant spread of pathogenRange 1-5 1-5 3-7Mean 1.5 2.6 4.3 4.0

No. wilted plants neighboring PILsRangec 0-8 0-5 0-2 0-1Meand 3.3 1.7 0.7 0.1

Primary infection locus = first of usually a series of plants to develop wilt as a result of infectionfrom sclerotia.

bNo. PIL/total, from which pathogen spread to adjacent plants.Based on all PIL of each plant spacing.

dBased on totals of 40, 12, 6, and 1 plant and on all PIL of respective spacings.

xSeed, 5 cm deep; rows, 90 cm apart; within-

row spacing, 30 cm.'Ten sclerotia per site of infestation.'Data based on 126 plants per treatment; six

replicates. Figures followed by same letter donot differ significantly (Duncan's multiplerange test, P = 0.05).

Table 3. Effect of horizontal distance betweenseed and sclerotia of Sclerotinia sclerotiorumon incidence of wilt in sunflowerx

Distance betweenseed and sclerotia y Wilted plantsz

(cm) (%)0 57 a

10 18 b20 0 c30 3 bc

'Rows, 90 cm apart; within-row spacing,

60 cm.'Five sclerotia per site, deposited at seed level(5 cm). At 0 cm, one site; at 10, 20, and 30cm,four sites in crosswise pattern.

'Data based on 56-60 plants per treatment; sixreplicates. Figures followed by same letter donot differ significantly (Duncan's multiplerange test, P = 0.05).

Plant Disease/January 1980 83