Ralph BakerColorado State University, Fort Collins

Measures to ControlFusarium and Phialophora WiltPathogens of Carnations

Carnation plant losses due to thevascular wilt pathogens Fusariumoxysporum f. sp. dianthi and Phialophoracinerescens (Fig. 1) were reduced ingreenhouses in the United States manyyears ago with the advent of pathogen-free operations producing massivequantities of vegetatively propagated

0191-2917/80/08074307/$03.00/0©1980 American Phytopathological Society

material. Because wilt pathogens weredisseminated within the vascular system,the "cultured cutting" technique was usedto obtain pathogen-free propagativematerial. In the 1960s, however,procedures for growing gnotobiotic oreven germfree plants from shoot tips wereadopted in the industry (1). For the firsttime, rooted cuttings free of all knownpathogens of carnation could be suppliedto commercial growers. Eventually,

advanced methods of tissue cultureprovided even more efficiency in rapidmultiplication of "clean" propagativematerial (5).

In the past, growers transplanted thesepathogen-free cuttings into raisedbenches. Between crops, the benches andsoil were steamed according to rigorousprotocols, and any inoculum remainingfrom the previous crop was eradicated.Integrated control measures involving

Fig. 1. Symptoms of the vascular wilt diseases of carnation: (A) Fusarlum wilt. External symptoms often develop on one side. Stems andleaves on the afflicted side are yellow, with a light brown vascular discoloration at the base of the stem. The vascular system on the upperpart of the stem Is chalky white. (B) Phialophora wilt. Plants gradually wilt, and leaves fade and become straw color. The vascular system Ischocolate brown, much darker than with Fusarlum wilt. (From original paintings by Eleanor J. Baker highlighting the differences Insymptom expression between the two diseases.)

Plant Disease/August 1980 743

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Fig. 2. Disease Incidence among carnation cultivars planted In soil Infested with Fusarlumoxysporum f. sp. dianthi after 1 year; 90 plants of each cultivar were divided Into threereplicates.

beds with carnations in the United States,in the recently developing industry inColombia, and in Great Britain. AtColorado State University over the lastdecade, innovative control measures andthose reported in and out of the literaturehave been tested for their potential inrectifying this situation. This articlereviews the progress of those attempts.

Resistance as a StrategyDevelopment of carnation cultivars

resistant to the wilt pathogens providesan opportunity for disease control. Thecommercial cultivars appear to havevarying degrees of resistance, andattempts to breed for resistance have metwith some success (3). A fortunatesituation, which may be more than acoincidence, is that resistance in abreeding selection operates for bothPhialophora and Fusarium wilt (19).

Fig. 2 gives an example of the range ofhost response to infection by F.oxysporum f. sp. dianthi encountered incultivars grown in commercial green-houses. These ratings confirm growerobservations that among the mostcommonly grown flower colors, pinkcultivars are most susceptible, red mostresistant, and white intermediate orvariable. Even within selections of thesame color, however, evidence ofvariability suggests that resistance differsslightly among clones of the same cultivar(Table 1).

This situation is interesting from thestandpoint of the genetic mechanismscontributing to resistance or suscepti-bility. Most standard carnation cultivarscurrently in commercial production hereand elsewhere were selected directly orindirectly from a single parent, thecultivar William Sim. (The lineage ofthese cultivars was contributed by Stacket al [20].) All the cultivars listed in Fig. 2and Table 1 are Sim cultivars. Thus,mutations not only alter growth habitand flower color but also, in more subtleways, influence resistance. This situationbecomes even more intriguing becausecultivars derived from William Sim arepericlinal chimeras (11). Presumably, allSim cultivars have similar geneticcomposition in interior tissues, includingthe vascular system, and differ in externaltissues responsible for flower color.

In spite of evidence that disease controlthrough resistance is of potential benefit,this strategy has not been adopted bycommercial carnation growers. The first

qu'un pas" (Napoleon).Conventional raised greenhouse

benches made from redwood inevitablydeteriorated, and replacement materialswere either no longer available orexpensive. The same problems arose innew construction, but with properfloricultural practices, plants could begrown in the soil on the greenhouse"floor"-in the so-called ground beds.This procedure was much less expensive,and a better quality crop could beproduced. In many cases, however,inoculum from the era before adequatecontrol measures survived in the soil onthe greenhouse floor. Surface steaming ofthe ground beds with the conventionallyused Thomas method (11) did noteradicate inoculum at depths below heatpenetration or in the compacted aislesbetween beds. Therefore, transplantingpathogen-free propagative material tosuch beds no longer insured a continuinghealthy crop. The same difficulty wasexperienced with other greenhouse crops.

The vascular wilts have thus becomethe most important diseases in ground

clean stock, soil treatment, and sanitationvirtually eliminated the diseases, and theybecame oddities. Recently, however, thissublime situation has deteriorated.Indeed, "Du sublime au ridicule il n'y a

744 Plant DiseaseNol. 64 No. 8

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reason advanced by a plant pathologistmight be the evidence for the existence ofvarious races of the pathogens (10) thatcould overcome resistance. In a practicalsense, however, the reasons are related tofloricultural rather than pathologicalconsiderations. Management of commer-cial pathogen-free floricultural programsconsiders the most important operationalfactor to be maintenance of qualityselections of cultivars (11). Selectionsthat do not adhere to the rigorousstandards of plant and flower quality areconstantly being eliminated; routinely,50% or more are eliminated each year. Bythe time resistant selections are detected,they may no longer exist in the trade.Thus, development and detection ofresistant cultivars and clones, a time-consuming and expensive operation, inmost cases is wasted effort. Even ifbreeding succeeds in developing resistantlines, floricultural characteristics mustmeet or exceed the quality of existingcultivars, an extremely difficult objectiveeven for breeders not selecting forresistance.

At present, the use of resistance as acontrol strategy for extensive applicationin the industry is not practical.Fortunately, the vascular wilt diseases areusually distributed in a relatively smallarea within a greenhouse range. Wheredisease is a problem, the grower can beadvised to plant red-flowered cultivars,because the incubation period for diseasedevelopment is longer for red than forpink or white cultivars.

Eradicating Inoculum in SoilEradication of inoculum from ground

beds is no small problem. In situationssimilar to those encountered in thecarnation industry, F. oxysporum f. sp.lycopersici was recovered from soil atdepths to 90 cm (8).

Soil fumigation with methyl bromidecontrolled vascular wilt disease ofcarnation (2), but bromide residues in soilare toxic to carnations and must beleached out with copious amounts ofwater. Although the research was notformal, English growers reportedfavorable results from treating "wiltpatches" with metham sodium followedby steaming (7). These possibilities forcontrolling vascular wilts have beenevaluated during the past 6 years incommercial greenhouses in Colorado.Using the method of the English growers,metham sodium was applied as a soil

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Fig. 3. (A) Losses In carnations due to Fusarium wilt in the second year after transplantingwhen beds were treated with fumigants. All plots were steamed after fumigation. Each plotcontained 210 plants, with three replicates. o---* steam only, *...o methyl bromide (1.36kg/6.51 m2) and steam, *-e metham sodium (946 ml/9.3 m2) and steam. (B) Diagrammaticrepresentation to scale of data obtained in plots in A. Loss over time is presented above orbelow each plot. Figures in parentheses are total losses in the methyl bromide-treated plotin replicate I and in the control (steamed only) plot in replicate II by June. In each case,dark areas on the bench diagram to the left represent dead plants in the preceding cropand those to the right, dead plants after 2 years, at the end of the experiment. Checkeredareas indicate that disease was scattered through that section of the plot. CK = control, V =

metham sodium, MB = methyl bromide.

Plant Disease/August 1980 745

drench (946 ml/9.3 m) to plots (6.1 mXl.06 m) at the ends of ground beds inwhich losses from Fusarium wilt hadbeen extensive. Carnations from thepreceding planting, transplanted 2 yearsbefore, were still in the beds. Treatedareas were watered thoroughly to aidpenetration of the fumigant and provide aseal for trapping evolved volatiles. Then,5 days later, the plants were uprooted andremoved, the beds were cultivated, andpreparations were made for steaming.

In other plots, 1 day before steaming,methyl bromide was applied at a rate of1.36 kg/6.51 m2 of soil surface andconfined with a plastic cover. The coverwas left in place for 24 hours.

All plots, including nontreated controls,were steamed with the Thomas method.Disease losses were observed over a 2-year period (Fig. 3A). Steaming may havereduced bromide residue to below toxiclevels, since no injury was observed inplots treated with methyl bromide.Losses from Fusarium wilt were not asgreat initially in plots with this treatment;at the end of 2 years, however, littledifference was found in comparison withcontrols given only conventional steamtreatment. Disease was not seen in plotstreated with metham sodium and steamuntil the end of the experiment.

Experiments such as this one have theadvantage of being performed undercommercial conditions, and results maybe readily applicable to the industry as awhole. Some parameters cannot beadjusted to uniformity, however. Forinstance, even distribution of inoculum isdifficult to insure. Obviously, growerswill not allow the inclusion of nontreatedinoculated controls. Such disadvantagescan be at least partially overcome byplotting, in diagrammatic form, lossdistribution before treatments wereapplied and disease incidence at the endof the experiment (Fig. 3B). Study of thediagram suggests that 1) inoculum was

eradicated consistently when methamsodium and steam were applied and 2) alarge proportion of the loss with thistreatment (Fig. 3A) originated fromdisease in bed 7 (third relicate) from anadjacent nontreated area. In some plots,

steam alone or combined with methylbromide apparently eradicated inoculum.

Another experiment that essentiallyrepeated the one described but omittedmethyl bromide treatment also indicatedgood control of Fusarium wilt with the

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Fig. 4. Losses in carnations due toFusarium wilt when beds were treated withmetham sodium (946 ml/9.3 m2) ordrenched with ethephon (10 mg/plant atmonthly Intervals). Each plot contained525 plants and was steamed before bedswere planted. Ethephon treatments werediscontinued after 350 days because ofphytotoxicity. o-o steam only, 9-emetham sodium and steam, 0-0 steamfollowed by drenches of ethephon.

746 Plant Disease/Vol. 64 No. 8

Fig. 5. (A) Losses in carnations due to Phialophora wilt when beds were treated withmetham sodium (946 ml/9.3 m2) and/or steam. The break in the curve between 500 and 550days for the metham sodium treatment indicates elimination of one replicate. Data for thattreatment after 550 days Include only two replicates. o-o steam only, -e metham sodiumand steam, 0-0 metham sodium only. (B) Diagrammatic representation to scale of dataIllustrated in A for those plots where metham sodium plus steam and steam alone wereapplied. Roman numerals indicate replicate numbers. Dark areas are locations where allplants died. Letters at the base of each plot: A, loss in preceding crop; B, lossapproximately 1 year after benches were planted; C, loss at completion of experimentapproximately 2 years after transplanting. Figures on plots refer to actual numbers ofplants with symptoms of Fusarium wilt at the end of the experiment.

metham sodium treatment combinedwith steaming (Fig. 4).

Another large experiment involvingover 14,000 plants was established to testthe efficacy of the metham sodiumtreatment. Losses in the preceding cropwere due primarily to P. cinerescens. Animportant element in this experiment, notused in the preceding test, was theintroduction of steam through two tiles,each 10 cm in diameter, running thelength of all beds and buried approxi-mately 60 cm below the soil surface. Theresults (Fig. 5A) indicate that treatmentwith metham sodium alone is noteffective. They also suggest that steamapplied at a 60-cm depth effectivelyeradicates a large proportion of theinoculum and that treatment withmetham sodium has no additional value.

The graphic presentation (Fig. 5B)suggests that inoculum was apparentlyeradicated in some areas treated with

metham sodium and steam or steamalone but not in others. New centers ofinfection were apparent when eithertreatment was used. In areas in whicheradication was not complete, subsequentdisease proportions were usually as greatas or even greater than those in thepreceding crop.

These results confirm the experience ofBritish growers (7): combined methamsodium and steam treatment is moreeffective than steam alone when theThomas steaming method is used. Steamalone applied at depths sufficient toeradicate inoculum also provides control.

Using Systemic Fungicidesand Growth Regulators

In his elegant review of the status ofresearch on control of the wilt pathogens(6), Erwin defines general principlesrelated to the properties and character-istics of systemic fungicides with specificapplication to ornamentals. The 2-substituted benzimidazoles are effectivein controlling the vascular wilt pathogens.They are insoluble but hydrolyze tomethyl-l1-benzimidazole carbamate inplant tissue. The fungitoxicant must be incontinuous supply in the xylem fluid tokeep the parasite suppressed; this is bestaccomplished by allowing root uptake ofthe chemical. Thus, soil application ofsystemic fungicides should be preferredfor controlling the vascular wilt pathogensof carnation. Numerous reports fromHolland (17), France (15), Germany (4),and Great Britain (7,9) suggest that soilapplications of appropriate systemicfungicides control Fusarium andPhialophora wilt diseases of carnation. Inthe intensive type of agriculture associatedwith carnations, this control, althoughexpensive, is usually cost-effective.

Typical results of controllingPhialophora wilt with benomyl and

thiophanate-methyl are shown in Fig. 6.After 364 days, loss was approximately40% in control plots or in those treatedwith ethazole but less than 10% in thosetreated with benomyl or thiophanate-methyl. Systemic fungicides may also beused for Fusarium wilt; typical resultsof treatment with benomyl are shown inFig. 7.

The amount of control achieved inthese examples is impressive, especiallysince the fungicide was incorporated intothe soil only once, at the time oftransplanting. In practice, drenchesshould be repeated to maintain aneffective concentration of the fungicide inthe soil while beds are producing flowers.With carnations, this period is usually 2years and the total amount of, forexample, benomyl applied may be from4.5 to 6 kg a.i./90 m per year.

Growth regulators have been suggestedas chemotherapeutic agents for vascularwilt (6). The recommendations of Orionand Hoestra (14) were followed whenapplying ethephon to the soil as a part ofthe test illustrated in Fig. 6. In anothertreatment, plants were sprayed withnaphthaleneacetic acid (13). Applicationof ethephon (see also Fig. 4) ornaphthaleneacetic acid had no effect ondevelopment of Fusarium wilt.

Apparently, chemical control (otherthan fumigants) of the vascular wilts ofcarnation has been feasible only withsystemic fungicides applied to soil.Unfortunately, strains of the pathogensare resistant to these agents (12,21).

Antitranspirants-Hypotheticalbut Not Practical Measure

A unique method of controllingFusarium wilt of carnations is presentedhere not because of its application tocommercial situations but because itseems to confirm certain features of the

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Fig. 7. Control of Fusarlum wilt by mixingbenomyl In soil before transplantingrooted carnation cuttings Into soil Infestedwith Fusarlum oxysporum f. sp. dianthi.Other plots were either treated with sprays(every 10 days) of naphthaleneacetic acid(10 g g/mI of water) or drenched withethephon solutions (10 mg/plant eachmonth). o-o Inoculated control, 9-naphthaleneacetic acid, X-X ethephon,0-0 benomyl.

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Fig. 8. Incidence of Fusarium wilt the second year after transplanting In two carnationcultivars sprayed to runoff at weekly Intervals with phenylmercuric acetate (PMA) at twoconcentrations. The standard errors shown as vertical brackets were computed fromoriginal data. 0-0 nontreated control, 9-9 1 1ig/ml PMA, X-X 5 pg/ml PMA.

Plant Disease/August 1980 747

Ralph Baker

Dr. Baker received his Ph.D. at theUniversity of California (Berkeley) in1954. Since then, he has taught anddone research at Colorado StateUniversity, Fort Collins, except forappointments as visiting professor atthe University of California (Berkeley)during 1963-1964 and as a NationalScience Foundation Senior Post Doc-toral Fellow at Cambridge University,England, during 1968-1969. His originalresearch responsibility in Coloradowas to develop control measures fordiseases of ornamentals; this resultedin establishment of large pathogen-free operations for propagatingcarnations in the state. His otherresearch interests involve the modelingof phenomena associated with theecology of plant pathogens in soil,especially those connected withbiological control. He also dabbles inspace biology.

mechanisms involved in diseaseexpression. Wilting is thought to beinduced by impediment of watertranspiration in the host's vascularsystem. Therefore, chemicals that reducetranspiration (18) and, hypothetically,could reduce stress on hosts infected withthe vascular wilt pathogens were testedfor their ability to extend the incubationperiod required for symptom expression.

Four antitranspirants were screened inpreliminary tests. One, phenylmercuricacetate (PMA), exerted some degree ofcontrol and was selected for further tests.CSU Pink Sim was lightly sprayedbiweekly at 0, 20, and 40 jug a.i. PMA/ mlof water. After 1 year, wilt symptomswere seen in 60, 42, and 28% of the plants,respectively. The experiment wasrepeated, this time using the relativelyresistant CSU Red Sim as well as thesusceptible CSU Pink Sim. Significantcontrol was obtained with PMA for CSUPink but not for Red Sim (Fig. 8).

Attempts to detect fungitoxic entitiesin plants treated with PMA were notsuccessful. Thus, the hypothesis that themechanism of control was associatedwith the ability of the antitranspirant toreduce stress on the host was notdisproved. The inability of PMA toinfluence the incubation period ofFusarium wilt on the resistant cultivarsuggests another hypothesis. CSU RedSim may be resistant because the tissue inthe periclinal chimera regulating stomatalaperture reduces transpiration-and,correspondingly, stress-of the host.Applications of an antitranspirant, whichcloses stomata, to such a cultivar provideno additional relief from transpirationalstress.

Control with PMA is not spectacular.Also, PMA contains mercury andtherefore is not available to agri-culturalists. These factors preclude its useas a practical control measure.

Biological Control, a PossibilityThe Salinas Valley in California

contains a Metz fine sandy loam soil thatis suppressive to Futzarium wilt diseases.This soil (600 gm/m 2 ) was added toground beds in a commercial greenhousewith a history of loss from Fusarium wilt.Carnations were planted, and lossesinduced by F. oxysporum f. sp. dianthiwere observed over a 2-year period. Atthat time, losses were reduced 60%(Fig. 9).

The mechanism apparently is associat-ed with a biocontrol agent in thesuppressive soil. Experimental evidencesuggests that the factor may be in thebacterial community, and, indeed,certain component Pseudomonas spp.,added at l0' cells/gm, confer suppres-siveness to soils that were conducivebefore treatment (16). The possibility ofusing this type of biological control incommercial applications is beinginvestigated.

Fig. 9. All the carnations In the plot on the right, planted In a ground bed steamed 18months previously, have died and shown symptoms of Fusarlum wilt. Plants In the plot onthe left are without symptoms to the line where 600 gm/m' of Metz fine sandy loam, aFusarlum-suppressive soil, had been added at the time of transplanting.

748 Plant DiseaseNol. 64 No. 8