- Email: [email protected]
Effects of acibenzolar-S-methyl on the suppression of Fusarium wilt of cyclamen
ARTICLE IN PRESS
Crop Protection 25 (2006) 671–676 www.elsevier.com/locate/cropro
Effects of acibenzolar-S-methyl on the suppression of Fusarium wilt of cyclamen Wade H. Elmer Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, CT 06504, USA Received 27 April 2005; received in revised form 27 September 2005; accepted 27 September 2005
Abstract Acibenzolar-S-methyl (ASM; Actigard 50 WPs, Syngenta Inc.) was evaluated for its ability to suppress Fusarium wilt of cyclamen in the greenhouse. The effect of applying increasing rates of ASM to cyclamen seedlings grown in potting mix infested with Fusarium oxysporum f. sp. cyclaminis resulted in a negative relationship between estimates of disease progress values (area-under-the-diseaseprogress-curve, AUDPC) and ASM rate. Disease symptoms were still evident in most plants, but some ASM-treated plants remained asymptomatic for the entire period. Dry mass of plants grown in infested potting mix were proportionally increased as ASM concentrations increased. In the absence of the pathogen, however, increasing rates of ASM resulted in linear reductions in dry mass. Flower number and quality were not affected by ASM rate. When seedlings were sprayed with 50 mg a.i. ASM ml1 and then grown in potting mix infested with increasing densities of F. oxysporum f. sp. cyclaminis, disease progress was significantly less than untreated seedlings, but there was variation among repetitions of the experiment. Dry mass was similarly greater than untreated seedlings. The major benefit of ASM was that it delayed the onset of wilt symptoms in most plants for up to 3 weeks and kept a few plants completely asymptomatic. Although applications resulted in no visible phytotoxicity on the leaves, the decline in dry mass may suggest some level of inhibition due to ASM. ASM may still be a useful component of an integrated disease management program for Fusarium wilt of cyclamen. r 2006 Elsevier Ltd. All rights reserved. Keywords: Fusarium oxysporum f. sp. cyclaminis; Cyclamen persicum; SAR
1. Introduction Fusarium wilt of cyclamen (Cyclamen persicum) is caused by Fusarium oxysporum f. sp. cyclaminis and is one of most destructive diseases of cyclamens (Tompkins and Snyder, 1972). Symptoms of the disease include stunting, chlorosis, and unilateral wilt, and eventual death of the plant. The disease has been particularly troublesome over the last 15 years (Daughtrey et al., 1995; Matteoni, 1988; Wouldt et al., 1995). It is not uncommon to have losses that exceed more than 50% of the crop when outbreaks of Fusarium wilt occur (Gracia-Garza, 2001). Currently, host resistance is not available in horticulturally acceptable cultivars (Orlicz-Luthardt, 1998).
Tel.: +1 203 974 8503; fax: +1 203 974 8502.
E-mail address: [email protected]. 0261-2194/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2005.09.010
Management of Fusarium wilt of cyclamen has been difficult because symptoms can appear at any time. The registered fungicides azoxystrobin, thiophanate methyl, fludioxonil, and triflumizole are the most effective products available (Elmer, 2001; Elmer and McGovern, 2004; Gullino et al., 2002), but they have poor curative properties against this disease. Antagonistic microbes or ‘‘biologicals’’ can delay the onset of Fusarium wilt (Elmer and McGovern, 2004; Gracia-Garza, 2001; Minuto et al., 1995; Someya et al., 2000), and improve disease control when they are combined with chemical fungicides (Elmer and McGovern, 2004). The source of inoculum can be from contaminated potting mix, pots, trays, irrigation water, and from fungus gnats (Daughtrey et al., 1995; Matteoni, 1988; Rattink, 1990). It is also possible that cyclamens can be infected as seedlings as a result of contaminated seed and seed debris (Tompkins and Snyder, 1972), but the plant remains
ARTICLE IN PRESS 672
W.H. Elmer / Crop Protection 25 (2006) 671–676
asymptomatic until stressed by heat or drought. Until the major inoculum source has been identified and eradicated, growers must remain under constant alert for disease outbreaks. New strategies for disease management are urgently needed. Acibenzolar-S-methyl [(ASM), Actigard 50 WG, Syngenta, Inc.] is a systemic compound used for the control of downy mildew in leafy vegetables, bacterial leaf spots in tomatoes, and blue mould in tobacco. The compound has minimal fungicidal and bactericidal activity, but induces host plant resistance via a unique mode of action that mimics the natural systemic activated resistance (SAR) response found in most plant species. ASM and its analogues have suppressed Fusarium diseases on some crops (Elmer, 2004, 2005; Benhamou and Be´langer, 1998b, but not others (Ishii et al., 1999). Its efficacy against Fusarium wilt of cyclamen has not been determined. It was reasoned that since ASM can be phytotoxic, applications made early in the production cycle might still suppress disease without causing economic damage. The objectives of this study were to determine the effect of ASM concentrations on cyclamen seedlings and Fusarium wilt and to determine the effect of inoculum rates of F. oxysporum f. sp. cyclaminis on ASM-treated seedlings.
Evaluation of disease severity was made every 3–5 d over the duration of each experiment by assigning a disease severity rating to each plant. Disease ratings were based on the scale: 0 ¼ no disease observed, 1 ¼ slight stunting, 2 ¼ slight stunting and chlorosis of leaves, 3 ¼ o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 ¼ o11–25% of the plant with wilt symptoms, 5 ¼ 26–50% of the plant with wilt symptoms, and 6 ¼ 51–100% of the plant with wilt symptoms or plant death. Area-under-the-disease-progress-curve (AUDPC, days) was calculated using the formula X AUDPC ¼ ðY i þ Y ðiþ1Þ Þ=2ðtðiþ1Þ ti Þ,
2. Materials and methods
Based on the aforementioned studies, 50 mg a.i. of ASM ml1 was chosen for all future experiments. Forty seedlings (15–17-weeks old) with 7–10 leaves were sprayed to run-off with ASM in plug trays. Another 40 seedlings were sprayed with distilled H2O to serve as controls. One week later, seedlings were treated again and then transplanted into 10-cm plastic pots filled with ProMix BX as described before only that the potting mix was infested with increasing amounts of millet colonized by F. oxysporum f. sp. cyclaminis. Millet inoculum was prepared by adding agar plugs colonized by the pathogen to flasks filled with twice-autoclaved millet seeds (100 g 200 ml1 H2O). Flasks were incubated at room temperature and shaken daily for 14 d to promote even growth. The contents were first dried in autoclaved paper bags, then ground in a Wiley mill. The milled product was passed through a 40-mesh sieve and stored in glass jars at 4 1C. Millet inoculum was rotary incorporated into potting mix for 20 min using a cement mixer at the rate of 0, 0.25, 0.50, and 1.0 g of millet inoculum l1 of potting mix. This rate resulted in approximately 0, 0.44, 0.62, or 1.00 colonyforming units (CFU) of F. oxysporum f. sp. cyclaminis ( 105 ml1 potting mix), respectively, as determined by dilution plating onto Komada’s selective media (Komada, 1975). Plants were arranged on a greenhouse bench in a randomized block design. There were 10 replicate plants per treatment. Disease severity data were recorded as described above and leaves plus petioles were weighed and recorded. The number of flowers/plant was recorded. The experiment was repeated 4 months later. Data from repeated experiments were combined when experiment treatment effects were not significant, but
2.1. Effect of ASM concentration on Fusarium wilt Cyclamen seedlings (‘Scarlet Red’ Halios series) (15–17 weeks old) with approximately 7–10 leaves per seedling were supplied by the Hortus Group (Castroville, CA) and were transplanted into 0.5-l plastic pots filled with the potting mix ProMix BX (one plant/pot) and set on benches in a research greenhouse. One day after transplanting, seedlings were sprayed to run-off with ASM at the rate of 0, 10, 25, or 50 mg ml1. The applications were re-applied 1 week later. Plants were then inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia ml1) into the potting mix on both sides of the corm (10 ml/plant). Since macroconidia have greater disease-causing ability than microconidia (Elmer, 1985), only macroconidia were enumerated. Microconidia, however, were present at an approximate 1:10 macroconidia/microconidia ratio. Conidia were produced on potato–carrot agar (Dhingra and Sinclair, 1985) and washed off with distilled water after 10 d. Plants were treated once a month with Hoagland’s fertilizer solution (100 ml/pot). Plants received one application of Orthene Systemic Concentrate (115 ml l1) and one application of Conserve (Spinosad, 15 ml l1) to control insect pests. Plants were placed on greenhouse benches with temperatures adjusted to 25 1C day and 20 1C night under sodium vapour lamps set for 12-h d photoperiod. The experimental design was a completely randomized block design with ten replicates pot treatments. The experiment was repeated 14 months later.
where Yi is the disease rating at time ti. Plants were grown to flowering. At final harvest when plants were commercially marketable, they were destructively harvested. Leaves plus petioles were removed, weighed, and then dried in an oven and re-weighed. Flower number also was recorded. 2.2. Effect of ASM on cyclamen grown in potting mix with increasing inoculum densities of F. oxysporum f. sp. cyclaminis
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
presented individually when there was interaction. Linear regression analysis was used to fit, test, and compare equations. All analyses were conducted on SystatV.10 (SPSS Inc., Chicago, IL).
Disease severity rating
6
3. Results 3.1. Effect of ASM concentration on Fusarium wilt of cyclamen
160
5 4
Nontreated control ..................
Acibenzolar-s-methyl (50 µg ml-1)
3 2 1 0 0
20
40
60
80
100
Days after transplanting Fig. 2. Effect of acibenzolar-S-methyl (50 mg ml1) on the development of disease severity of cyclamens grown in potting mix infested with Fusarium oxysporum f. sp. cyclaminis.
Noninfested (control) soil Soil infested with FOC
1.4 1.2 Foliar dry mass (g)
Data for the two trials were not homogenous for the AUDPC values, so the experiments are presented separately (Figs. 1a and b). The variance was due, in part, to the slower rate of disease progress in plants in the second experiment. Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants whereas symptoms of wilt and chlorosis were noted 3 weeks after inoculation in the No ASM treatment. For the two experiments, ASM concentrations were inversely correlated with AUDPC values (Figs. 1a and b), and best fitted by linear regression. Although the highest applied rate of ASM did not completely eliminate disease development, the time of symptom onset was dramatically affected. ASM
673
r2 = 0.35, P = 0.004 1.0
r2 = 0.15, P = 0.05
0.8 0.6 0.4
140
0.2
120
0.0
100
0
80
50
60
Fig. 3. Effect of acibenzolar-S-methyl concentrations on the dry mass of cyclamen grown in non-infested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis.
60 40 AUDPC (days)
10 20 30 40 Acibenzolar -s-methyl (µg ml-1)
20 0 Noninfested (control) soil Soil infested with FOC
140 120
r2= 0.15, P = 0.017
100 80 60 40 20
applied at 50 mg a.i. ml1 delayed symptom development by approximately 20 d (Fig. 2). Since the dry mass interaction term between the experiments and treatments was not significant, data sets were combined (n ¼ 20). The dry mass of the aboveground tissue of plants inoculated with F. oxysporum f. sp. cyclaminis increased linearly as ASM concentrations increased (Fig. 3). However, ASM decreased the dry mass of the non-inoculated control plants by 36%, but did not affect flower number or quality (data not shown). There was no visible phytotoxic damage on the leaves.
0 0
10
20
30
Acibenzolar -s-methyl (µg
40
50
ml-1)
Fig. 1. Effect of acibenzolar-S-methyl concentrations on the area-underthe-disease-progress-curve (AUDPC, days) of cyclamens grown in noninfested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
3.2. Effect of ASM on cyclamen grown in potting mix with increasing inoculum densities of F. oxysporum f. sp. cyclaminis Data for the two trials were not homogenous for the AUDPC values, so the experiments are presented separately (Figs. 4a and b). In the first trial, the AUDPC values
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
674
80
2.0 No acibenzolar-s-methyl .................... r2 = 0.38, P < 0.001 Acibenzolar-s-methyl (50 µg ml-1) _________ r2 = 0.57, P < 0.001
40
1.0
20
0.5
0
60
No acibenzolar-s-methyl .................. Acibenzolar-s-methyl (50 µg ml-1) ________
1.5
Dry foliar mass (g)
AUDPC (days)
60
No acibenzolar-s-methyl --------- r2 = 0.30, P = 0.025
0.0 No acibenzolar-s-methyl --------- r2 = 0.65, P < 0.001 Acibenzolar-s-methyl (50 µg ml-1) ______ r2 = 0.48, P < 0.001
1.5
ml-1)
Acibenzolar-s-methyl (50 µg ______ r2 = 0.57, P < 0.001
1.0
40 0.5 20 0.0 0
0.0 0.0
0.2
0.4
0.6
0.8
1.0
Millet Inoculum ofF. oxysporum f. sp. cyclaminis (g l-1 potting mix) Fig. 4. Effect of acibenzolar-S-methyl concentrations on the area-underthe-disease-progress-curve (AUDPC, days) of cyclamens grown in noninfested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
from ASM-treated plants were best fitted by a curvilinear equation as inoculum increased whereas the AUDPC values of non-treated plants were fitted best by a linear relationship (Fig. 4a). The greatest difference between ASM-treated and untreated controls was observed at the higher inoculum densities. At the highest level, ASMtreated plants had lower AUDPC values. In the second experiment, both treated and non-treated plants produced AUDPC values that increased curvilinearly as inoculum increased (Fig. 4b). Plants treated with ASM did not develop symptoms until inoculum levels reached 0.5 g l1 potting mix whereas non-treated plants developed severe disease symptoms at lower inoculum densities. As before, the disease-suppressing effect of ASM under higher inoculum densities was due to the delay in disease onset (data not shown). The dry mass data from the two experiments were not homogenous, so each experiment is presented separately (Figs. 5a and b). In the first experiment, the dry mass of ASM-treated and untreated plants declined curvilinearly, but the dry mass of treated plants were greater at higher inoculum densities than untreated plants (Fig. 5a). However, ASM-treated plants grown in non-infested potting mix were 65% less in dry mass than untreated plants.
0.2
0.4
0.6
0.8
1.0
1.2
Millet inoculum of F. oxysporum f. sp. cyclaminis (g l-1potting mix) Fig. 5. Effect of acibenzolar-S-methyl concentrations on the dry mass of cyclamens grown in non-infested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
Trends were similar in the second experiment except that ASM-treated plants had significantly more dry mass when grown in potting mix with millet inoculum levels of 0.25 and 5.0 g l1 potting mix as opposed to higher levels in the first experiment (Fig. 5b). 4. Discussion Fusarium wilt of cyclamen can be a devastating disease and can result in over 50% loss (Gracia-Garza, 2001; Matteoni, 1988). Given the lack of cultivar resistance (Orlicz-Luthardt, 1998), ASM treatments were evaluated as a preventative management strategy. ASM has no effect on the pathogen, but activates SAR mechanisms. When increasing rates of ASM were applied, disease severity declined proportionately, but symptoms in all plants were never completely eliminated. The major benefit of ASM was the delay in the mean onset of symptoms up to 3 weeks when compared to controls, but the number of asymptomatic plants at the end of the second study was 20% greater in plants grown in soil at the highest infestation and sprayed with 50 a.i. mg ml1 than unsprayed plants (data not shown). It may be reasonable to assume that ASM was successful in preventing infection in these plants, thus
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
giving additional time for growers to apply preventative fungicides. Future studies need to examine the application of preventative fungicides following ASM treatment to determine the combinations provide superior control that either products alone. Combinations between ASM and chemical fungicides were effective in suppressing Fusarium corm rot of gladiolus (Elmer, 2005). Matheron and Porchas (2002) also found that foliar applications of ASM suppressed Phytophthora blight on bell pepper, but this treatment alone could not provide complete disease suppression. Although no foliar damage was observed increasing rates of ASM grown in the absence of F. oxysporum f. sp. cyclaminis were associated with reduced dry weights, and this effect could be indicative of phytotoxicity. Reports of phytotoxicity have been made for pineapple (Chinnasri and Sipes, 2005) and tobacco (Cole, 1999). However, no phytotoxicity was reported on apple (Brisset et al., 2000), asparagus, (W.H. Elmer, unpublished data), bell pepper (Matheron and Porchas, 2002), and several woody ornamentals (Ali et al., 2000). In the current study, the deleterious effects were only observed as reduced growth and not foliar damage, therefore it might be possible to overcome the reductions in dry mass via fertilization. In addition, there was no reduction in flower number or quality. Cost of ASM application is fairly expensive (roughly $2.00 USD g1 Actigard 50 WP), but when applied to low acreage seedling plugs, the cost may be practical. ASM is currently registered under the commercial label Actigards (Syngenta Crop Production) for use on tobacco, tomato, spinach, and brassica for the control of fungal and bacterial leaf diseases. The product has not been approved for suppression of any soilborne diseases on any crops. ASM has suppressed Fusarium diseases on some crops (Elmer, 2004); Benhamou and Be´langer, 1998b), but not others (Ishii et al., 1999). In addition, this product has shown some efficacy in suppressing root disease on woody ornamentals (Ali et al., 2000), nematodes on pineapple (Chinnasri and Sipes, 2005), Phytophthora blight on bell pepper (Matheron and Porchas, 2002), and Pythium root rot in cucumber (Benhamou and Be´langer, 1998a). Although ASM rarely provided complete suppression, studies on its use and the utility of SAR to increase root health should be pursued further as another disease management strategy in crop production.
Acknowledgements This study was supported in part by funds from the American Floral Endowment Fund. The author thanks Elizabeth O’Dowd and Joan Bravo for technical assistance, the Hortus Group (Castroville, CA) for cyclamen seedlings and Syngenta Crop Protection Inc. for samples of Actigards 50 WP.
675
References Ali, Z., Smith, I., Guest, D.I., 2000. Combinations of potassium phosphonate and Bion (acibenzolar-S-methyl) reduce root infection and dieback of Pinus radiata, Banksia integrifolia and Isopogon cuneatus caused by Phytophthora cinnamomi. Australas. Plant Pathol. 29, 59–63. Benhamou, N., Be´langer, R.R., 1998a. Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response. Plant J. 14, 13–21. Benhamou, N., Be´langer, R.R., 1998b. Benzothiadiazole-mediated induced resistance to Fusarium oxysporum f. sp. radicis-lycopersici in tomato. Plant Physiol. 118, 1203–1212. Brisset, M.-B., Cesbron, S.S., Thomson, S.V., Paulin, J.P., 2000. Acibenzolar-S-methyl induces the accumulation of defense-related enzymes in apple and protects from fire blight. Eur. J. Plant Pathol. 106, 529–536. Chinnasri, B., Sipes, K., 2005. Effect of a systemic acquired resistance inducer on nematodes infecting pineapple. Acta Hortic. 666, 213–222. Cole, D.L., 1999. The efficacy of acibenzolar-S-methyl, an inducer of systemic acquired resistance, against bacterial and fungal diseases of tobacco. Crop Prot. 18, 267–273. Daughtrey, M.L., Wick, R.L., Peterson, J.L., (Eds.), 1995. Compendium of Flowering Potted Plant Diseases. APS Press, St. Paul, MN, 90pp. Dhingra, O.D., Sinclair, J.B., 1985. Basic Plant Pathology Methods. CRC Press, Inc., Boca Raton, FL, 355pp. Elmer, 1985. The ecology and control of Fusarium yellows of celery. Ph.D. Dissertation, Michigan State University, East Lansing, MI. Elmer, W.H., 2001. The influence of nonpathogenic Fusarium species and Pseudomonas fluorescens on Fusarium wilt of cyclamen. Biol. Cult. Tests Control Plant Dis. 16, O40. Elmer, W.H., 2004. Effect of fungicides and combinations of biologicals and acibenzolar-S-methyl on Fusarium rot of gladiolus. Biol. Cult. Tests Control Plant Dis. 19, O007. Elmer, W.H., 2005. Effect of fungicide dips with and without Actigard 50WP on Fusarium corm rot of gladiolus. Fungicide and Nematicide Tests 60, OT, 015. Elmer, W.H., McGovern, R.J., 2004. Influence of combining biological products with chemical fungicides for control of Fusarium wilt of cyclamen. Crop Prot. 23, 909–914. Gracia-Garza, J.A., 2001. Fusarium oxysporum Schlechtendahl f. sp. cyclaminis Gerlach, Fusarium wilt of cyclamen (Hypomycetes). In: Mason, P., Huber, J. (Eds.), Biological Control Programmes in Canada 1981–2000. CABI Press, Surrey UK, pp. 452–456. Gullino, L., Minuto, A., Gilardi, G., Garibaldi, A., 2002. Efficacy of azoxystrobin and other strobilurins against Fusarium wilts of carnation, cyclamen and Paris daisy. Crop Prot. 21, 57–61. Ishii, H., Tomita, Y., Horio, T., Narusaka, Y., Nakazawa, Y., Nishimura, K., Iwamoto, S., 1999. Induced resistance of acibenzolar-S-methyl (CGA 245704) to cucumber and Japanese pear diseases. Eur. J. Plant Pathol. 105, 77–85. Komada, H., 1975. Development of selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Prot. Res. 8, 114–124. Matteoni, J.A., 1988. Diseases of cyclamen in Ontario from 1983 to 1987. Can. Plant Dis. Surv. 68, 84. Matheron, M.E., Porchas, M., 2002. Suppression of Phytophthora root and crown rot on pepper plants treated with acibenzolar-S-methyl. Plant Dis. 86, 292–297. Minuto, A., Migheli, Q., Garibaldi, A., 1995. Evaluation of antagonistic strains of Fusarium spp. in the biological and integrated control of Fusarium wilt of cyclamen. Crop Prot. 14, 221–226. Orlicz-Luthardt, A., 1998. Studies on the resistance of cyclamen to Fusarium wilt. Beit. Zu¨chtungsforsch. 4, 48–49.
ARTICLE IN PRESS 676
W.H. Elmer / Crop Protection 25 (2006) 671–676
Rattink, H., 1990. Epidemiology of Fusarium wilt of cyclamen in ebb and flow. Neth. J. Plant Pathol. 96, 171–177. Someya, N., Kataoka, N., Komagata, T., Hirayae, K., Hibi, T., Akutsu, K., 2000. Biological control of cyclamen soil diseases by Serratia marcescens strain B2. Plant Dis. 84, 334–340.
Tompkins, C.M., Snyder, W.C., 1972. Cyclamen wilt in California and its control. Plant Dis. Rep. 56, 493–497. Wouldt, L.P., Neuvel, A., Sikkema, A., van Grinsven, M.Q.J.M., de Milliano, W.A.J., 1995. Genetic variation in Fusarium oxysporum from cyclamen. Phytopathology 85, 1348–1355.
Crop Protection 25 (2006) 671–676 www.elsevier.com/locate/cropro
Effects of acibenzolar-S-methyl on the suppression of Fusarium wilt of cyclamen Wade H. Elmer Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, CT 06504, USA Received 27 April 2005; received in revised form 27 September 2005; accepted 27 September 2005
Abstract Acibenzolar-S-methyl (ASM; Actigard 50 WPs, Syngenta Inc.) was evaluated for its ability to suppress Fusarium wilt of cyclamen in the greenhouse. The effect of applying increasing rates of ASM to cyclamen seedlings grown in potting mix infested with Fusarium oxysporum f. sp. cyclaminis resulted in a negative relationship between estimates of disease progress values (area-under-the-diseaseprogress-curve, AUDPC) and ASM rate. Disease symptoms were still evident in most plants, but some ASM-treated plants remained asymptomatic for the entire period. Dry mass of plants grown in infested potting mix were proportionally increased as ASM concentrations increased. In the absence of the pathogen, however, increasing rates of ASM resulted in linear reductions in dry mass. Flower number and quality were not affected by ASM rate. When seedlings were sprayed with 50 mg a.i. ASM ml1 and then grown in potting mix infested with increasing densities of F. oxysporum f. sp. cyclaminis, disease progress was significantly less than untreated seedlings, but there was variation among repetitions of the experiment. Dry mass was similarly greater than untreated seedlings. The major benefit of ASM was that it delayed the onset of wilt symptoms in most plants for up to 3 weeks and kept a few plants completely asymptomatic. Although applications resulted in no visible phytotoxicity on the leaves, the decline in dry mass may suggest some level of inhibition due to ASM. ASM may still be a useful component of an integrated disease management program for Fusarium wilt of cyclamen. r 2006 Elsevier Ltd. All rights reserved. Keywords: Fusarium oxysporum f. sp. cyclaminis; Cyclamen persicum; SAR
1. Introduction Fusarium wilt of cyclamen (Cyclamen persicum) is caused by Fusarium oxysporum f. sp. cyclaminis and is one of most destructive diseases of cyclamens (Tompkins and Snyder, 1972). Symptoms of the disease include stunting, chlorosis, and unilateral wilt, and eventual death of the plant. The disease has been particularly troublesome over the last 15 years (Daughtrey et al., 1995; Matteoni, 1988; Wouldt et al., 1995). It is not uncommon to have losses that exceed more than 50% of the crop when outbreaks of Fusarium wilt occur (Gracia-Garza, 2001). Currently, host resistance is not available in horticulturally acceptable cultivars (Orlicz-Luthardt, 1998).
Tel.: +1 203 974 8503; fax: +1 203 974 8502.
E-mail address: [email protected]. 0261-2194/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2005.09.010
Management of Fusarium wilt of cyclamen has been difficult because symptoms can appear at any time. The registered fungicides azoxystrobin, thiophanate methyl, fludioxonil, and triflumizole are the most effective products available (Elmer, 2001; Elmer and McGovern, 2004; Gullino et al., 2002), but they have poor curative properties against this disease. Antagonistic microbes or ‘‘biologicals’’ can delay the onset of Fusarium wilt (Elmer and McGovern, 2004; Gracia-Garza, 2001; Minuto et al., 1995; Someya et al., 2000), and improve disease control when they are combined with chemical fungicides (Elmer and McGovern, 2004). The source of inoculum can be from contaminated potting mix, pots, trays, irrigation water, and from fungus gnats (Daughtrey et al., 1995; Matteoni, 1988; Rattink, 1990). It is also possible that cyclamens can be infected as seedlings as a result of contaminated seed and seed debris (Tompkins and Snyder, 1972), but the plant remains
ARTICLE IN PRESS 672
W.H. Elmer / Crop Protection 25 (2006) 671–676
asymptomatic until stressed by heat or drought. Until the major inoculum source has been identified and eradicated, growers must remain under constant alert for disease outbreaks. New strategies for disease management are urgently needed. Acibenzolar-S-methyl [(ASM), Actigard 50 WG, Syngenta, Inc.] is a systemic compound used for the control of downy mildew in leafy vegetables, bacterial leaf spots in tomatoes, and blue mould in tobacco. The compound has minimal fungicidal and bactericidal activity, but induces host plant resistance via a unique mode of action that mimics the natural systemic activated resistance (SAR) response found in most plant species. ASM and its analogues have suppressed Fusarium diseases on some crops (Elmer, 2004, 2005; Benhamou and Be´langer, 1998b, but not others (Ishii et al., 1999). Its efficacy against Fusarium wilt of cyclamen has not been determined. It was reasoned that since ASM can be phytotoxic, applications made early in the production cycle might still suppress disease without causing economic damage. The objectives of this study were to determine the effect of ASM concentrations on cyclamen seedlings and Fusarium wilt and to determine the effect of inoculum rates of F. oxysporum f. sp. cyclaminis on ASM-treated seedlings.
Evaluation of disease severity was made every 3–5 d over the duration of each experiment by assigning a disease severity rating to each plant. Disease ratings were based on the scale: 0 ¼ no disease observed, 1 ¼ slight stunting, 2 ¼ slight stunting and chlorosis of leaves, 3 ¼ o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 ¼ o11–25% of the plant with wilt symptoms, 5 ¼ 26–50% of the plant with wilt symptoms, and 6 ¼ 51–100% of the plant with wilt symptoms or plant death. Area-under-the-disease-progress-curve (AUDPC, days) was calculated using the formula X AUDPC ¼ ðY i þ Y ðiþ1Þ Þ=2ðtðiþ1Þ ti Þ,
2. Materials and methods
Based on the aforementioned studies, 50 mg a.i. of ASM ml1 was chosen for all future experiments. Forty seedlings (15–17-weeks old) with 7–10 leaves were sprayed to run-off with ASM in plug trays. Another 40 seedlings were sprayed with distilled H2O to serve as controls. One week later, seedlings were treated again and then transplanted into 10-cm plastic pots filled with ProMix BX as described before only that the potting mix was infested with increasing amounts of millet colonized by F. oxysporum f. sp. cyclaminis. Millet inoculum was prepared by adding agar plugs colonized by the pathogen to flasks filled with twice-autoclaved millet seeds (100 g 200 ml1 H2O). Flasks were incubated at room temperature and shaken daily for 14 d to promote even growth. The contents were first dried in autoclaved paper bags, then ground in a Wiley mill. The milled product was passed through a 40-mesh sieve and stored in glass jars at 4 1C. Millet inoculum was rotary incorporated into potting mix for 20 min using a cement mixer at the rate of 0, 0.25, 0.50, and 1.0 g of millet inoculum l1 of potting mix. This rate resulted in approximately 0, 0.44, 0.62, or 1.00 colonyforming units (CFU) of F. oxysporum f. sp. cyclaminis ( 105 ml1 potting mix), respectively, as determined by dilution plating onto Komada’s selective media (Komada, 1975). Plants were arranged on a greenhouse bench in a randomized block design. There were 10 replicate plants per treatment. Disease severity data were recorded as described above and leaves plus petioles were weighed and recorded. The number of flowers/plant was recorded. The experiment was repeated 4 months later. Data from repeated experiments were combined when experiment treatment effects were not significant, but
2.1. Effect of ASM concentration on Fusarium wilt Cyclamen seedlings (‘Scarlet Red’ Halios series) (15–17 weeks old) with approximately 7–10 leaves per seedling were supplied by the Hortus Group (Castroville, CA) and were transplanted into 0.5-l plastic pots filled with the potting mix ProMix BX (one plant/pot) and set on benches in a research greenhouse. One day after transplanting, seedlings were sprayed to run-off with ASM at the rate of 0, 10, 25, or 50 mg ml1. The applications were re-applied 1 week later. Plants were then inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia ml1) into the potting mix on both sides of the corm (10 ml/plant). Since macroconidia have greater disease-causing ability than microconidia (Elmer, 1985), only macroconidia were enumerated. Microconidia, however, were present at an approximate 1:10 macroconidia/microconidia ratio. Conidia were produced on potato–carrot agar (Dhingra and Sinclair, 1985) and washed off with distilled water after 10 d. Plants were treated once a month with Hoagland’s fertilizer solution (100 ml/pot). Plants received one application of Orthene Systemic Concentrate (115 ml l1) and one application of Conserve (Spinosad, 15 ml l1) to control insect pests. Plants were placed on greenhouse benches with temperatures adjusted to 25 1C day and 20 1C night under sodium vapour lamps set for 12-h d photoperiod. The experimental design was a completely randomized block design with ten replicates pot treatments. The experiment was repeated 14 months later.
where Yi is the disease rating at time ti. Plants were grown to flowering. At final harvest when plants were commercially marketable, they were destructively harvested. Leaves plus petioles were removed, weighed, and then dried in an oven and re-weighed. Flower number also was recorded. 2.2. Effect of ASM on cyclamen grown in potting mix with increasing inoculum densities of F. oxysporum f. sp. cyclaminis
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
presented individually when there was interaction. Linear regression analysis was used to fit, test, and compare equations. All analyses were conducted on SystatV.10 (SPSS Inc., Chicago, IL).
Disease severity rating
6
3. Results 3.1. Effect of ASM concentration on Fusarium wilt of cyclamen
160
5 4
Nontreated control ..................
Acibenzolar-s-methyl (50 µg ml-1)
3 2 1 0 0
20
40
60
80
100
Days after transplanting Fig. 2. Effect of acibenzolar-S-methyl (50 mg ml1) on the development of disease severity of cyclamens grown in potting mix infested with Fusarium oxysporum f. sp. cyclaminis.
Noninfested (control) soil Soil infested with FOC
1.4 1.2 Foliar dry mass (g)
Data for the two trials were not homogenous for the AUDPC values, so the experiments are presented separately (Figs. 1a and b). The variance was due, in part, to the slower rate of disease progress in plants in the second experiment. Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants whereas symptoms of wilt and chlorosis were noted 3 weeks after inoculation in the No ASM treatment. For the two experiments, ASM concentrations were inversely correlated with AUDPC values (Figs. 1a and b), and best fitted by linear regression. Although the highest applied rate of ASM did not completely eliminate disease development, the time of symptom onset was dramatically affected. ASM
673
r2 = 0.35, P = 0.004 1.0
r2 = 0.15, P = 0.05
0.8 0.6 0.4
140
0.2
120
0.0
100
0
80
50
60
Fig. 3. Effect of acibenzolar-S-methyl concentrations on the dry mass of cyclamen grown in non-infested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis.
60 40 AUDPC (days)
10 20 30 40 Acibenzolar -s-methyl (µg ml-1)
20 0 Noninfested (control) soil Soil infested with FOC
140 120
r2= 0.15, P = 0.017
100 80 60 40 20
applied at 50 mg a.i. ml1 delayed symptom development by approximately 20 d (Fig. 2). Since the dry mass interaction term between the experiments and treatments was not significant, data sets were combined (n ¼ 20). The dry mass of the aboveground tissue of plants inoculated with F. oxysporum f. sp. cyclaminis increased linearly as ASM concentrations increased (Fig. 3). However, ASM decreased the dry mass of the non-inoculated control plants by 36%, but did not affect flower number or quality (data not shown). There was no visible phytotoxic damage on the leaves.
0 0
10
20
30
Acibenzolar -s-methyl (µg
40
50
ml-1)
Fig. 1. Effect of acibenzolar-S-methyl concentrations on the area-underthe-disease-progress-curve (AUDPC, days) of cyclamens grown in noninfested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
3.2. Effect of ASM on cyclamen grown in potting mix with increasing inoculum densities of F. oxysporum f. sp. cyclaminis Data for the two trials were not homogenous for the AUDPC values, so the experiments are presented separately (Figs. 4a and b). In the first trial, the AUDPC values
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
674
80
2.0 No acibenzolar-s-methyl .................... r2 = 0.38, P < 0.001 Acibenzolar-s-methyl (50 µg ml-1) _________ r2 = 0.57, P < 0.001
40
1.0
20
0.5
0
60
No acibenzolar-s-methyl .................. Acibenzolar-s-methyl (50 µg ml-1) ________
1.5
Dry foliar mass (g)
AUDPC (days)
60
No acibenzolar-s-methyl --------- r2 = 0.30, P = 0.025
0.0 No acibenzolar-s-methyl --------- r2 = 0.65, P < 0.001 Acibenzolar-s-methyl (50 µg ml-1) ______ r2 = 0.48, P < 0.001
1.5
ml-1)
Acibenzolar-s-methyl (50 µg ______ r2 = 0.57, P < 0.001
1.0
40 0.5 20 0.0 0
0.0 0.0
0.2
0.4
0.6
0.8
1.0
Millet Inoculum ofF. oxysporum f. sp. cyclaminis (g l-1 potting mix) Fig. 4. Effect of acibenzolar-S-methyl concentrations on the area-underthe-disease-progress-curve (AUDPC, days) of cyclamens grown in noninfested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
from ASM-treated plants were best fitted by a curvilinear equation as inoculum increased whereas the AUDPC values of non-treated plants were fitted best by a linear relationship (Fig. 4a). The greatest difference between ASM-treated and untreated controls was observed at the higher inoculum densities. At the highest level, ASMtreated plants had lower AUDPC values. In the second experiment, both treated and non-treated plants produced AUDPC values that increased curvilinearly as inoculum increased (Fig. 4b). Plants treated with ASM did not develop symptoms until inoculum levels reached 0.5 g l1 potting mix whereas non-treated plants developed severe disease symptoms at lower inoculum densities. As before, the disease-suppressing effect of ASM under higher inoculum densities was due to the delay in disease onset (data not shown). The dry mass data from the two experiments were not homogenous, so each experiment is presented separately (Figs. 5a and b). In the first experiment, the dry mass of ASM-treated and untreated plants declined curvilinearly, but the dry mass of treated plants were greater at higher inoculum densities than untreated plants (Fig. 5a). However, ASM-treated plants grown in non-infested potting mix were 65% less in dry mass than untreated plants.
0.2
0.4
0.6
0.8
1.0
1.2
Millet inoculum of F. oxysporum f. sp. cyclaminis (g l-1potting mix) Fig. 5. Effect of acibenzolar-S-methyl concentrations on the dry mass of cyclamens grown in non-infested potting mix or potting mix infested with Fusarium oxysporum f. sp. cyclaminis. Fig. 1a (upper panel) ¼ Experiment 1, Fig. 1b (lower panel) ¼ Experiment 2.
Trends were similar in the second experiment except that ASM-treated plants had significantly more dry mass when grown in potting mix with millet inoculum levels of 0.25 and 5.0 g l1 potting mix as opposed to higher levels in the first experiment (Fig. 5b). 4. Discussion Fusarium wilt of cyclamen can be a devastating disease and can result in over 50% loss (Gracia-Garza, 2001; Matteoni, 1988). Given the lack of cultivar resistance (Orlicz-Luthardt, 1998), ASM treatments were evaluated as a preventative management strategy. ASM has no effect on the pathogen, but activates SAR mechanisms. When increasing rates of ASM were applied, disease severity declined proportionately, but symptoms in all plants were never completely eliminated. The major benefit of ASM was the delay in the mean onset of symptoms up to 3 weeks when compared to controls, but the number of asymptomatic plants at the end of the second study was 20% greater in plants grown in soil at the highest infestation and sprayed with 50 a.i. mg ml1 than unsprayed plants (data not shown). It may be reasonable to assume that ASM was successful in preventing infection in these plants, thus
ARTICLE IN PRESS W.H. Elmer / Crop Protection 25 (2006) 671–676
giving additional time for growers to apply preventative fungicides. Future studies need to examine the application of preventative fungicides following ASM treatment to determine the combinations provide superior control that either products alone. Combinations between ASM and chemical fungicides were effective in suppressing Fusarium corm rot of gladiolus (Elmer, 2005). Matheron and Porchas (2002) also found that foliar applications of ASM suppressed Phytophthora blight on bell pepper, but this treatment alone could not provide complete disease suppression. Although no foliar damage was observed increasing rates of ASM grown in the absence of F. oxysporum f. sp. cyclaminis were associated with reduced dry weights, and this effect could be indicative of phytotoxicity. Reports of phytotoxicity have been made for pineapple (Chinnasri and Sipes, 2005) and tobacco (Cole, 1999). However, no phytotoxicity was reported on apple (Brisset et al., 2000), asparagus, (W.H. Elmer, unpublished data), bell pepper (Matheron and Porchas, 2002), and several woody ornamentals (Ali et al., 2000). In the current study, the deleterious effects were only observed as reduced growth and not foliar damage, therefore it might be possible to overcome the reductions in dry mass via fertilization. In addition, there was no reduction in flower number or quality. Cost of ASM application is fairly expensive (roughly $2.00 USD g1 Actigard 50 WP), but when applied to low acreage seedling plugs, the cost may be practical. ASM is currently registered under the commercial label Actigards (Syngenta Crop Production) for use on tobacco, tomato, spinach, and brassica for the control of fungal and bacterial leaf diseases. The product has not been approved for suppression of any soilborne diseases on any crops. ASM has suppressed Fusarium diseases on some crops (Elmer, 2004); Benhamou and Be´langer, 1998b), but not others (Ishii et al., 1999). In addition, this product has shown some efficacy in suppressing root disease on woody ornamentals (Ali et al., 2000), nematodes on pineapple (Chinnasri and Sipes, 2005), Phytophthora blight on bell pepper (Matheron and Porchas, 2002), and Pythium root rot in cucumber (Benhamou and Be´langer, 1998a). Although ASM rarely provided complete suppression, studies on its use and the utility of SAR to increase root health should be pursued further as another disease management strategy in crop production.
Acknowledgements This study was supported in part by funds from the American Floral Endowment Fund. The author thanks Elizabeth O’Dowd and Joan Bravo for technical assistance, the Hortus Group (Castroville, CA) for cyclamen seedlings and Syngenta Crop Protection Inc. for samples of Actigards 50 WP.
675
References Ali, Z., Smith, I., Guest, D.I., 2000. Combinations of potassium phosphonate and Bion (acibenzolar-S-methyl) reduce root infection and dieback of Pinus radiata, Banksia integrifolia and Isopogon cuneatus caused by Phytophthora cinnamomi. Australas. Plant Pathol. 29, 59–63. Benhamou, N., Be´langer, R.R., 1998a. Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response. Plant J. 14, 13–21. Benhamou, N., Be´langer, R.R., 1998b. Benzothiadiazole-mediated induced resistance to Fusarium oxysporum f. sp. radicis-lycopersici in tomato. Plant Physiol. 118, 1203–1212. Brisset, M.-B., Cesbron, S.S., Thomson, S.V., Paulin, J.P., 2000. Acibenzolar-S-methyl induces the accumulation of defense-related enzymes in apple and protects from fire blight. Eur. J. Plant Pathol. 106, 529–536. Chinnasri, B., Sipes, K., 2005. Effect of a systemic acquired resistance inducer on nematodes infecting pineapple. Acta Hortic. 666, 213–222. Cole, D.L., 1999. The efficacy of acibenzolar-S-methyl, an inducer of systemic acquired resistance, against bacterial and fungal diseases of tobacco. Crop Prot. 18, 267–273. Daughtrey, M.L., Wick, R.L., Peterson, J.L., (Eds.), 1995. Compendium of Flowering Potted Plant Diseases. APS Press, St. Paul, MN, 90pp. Dhingra, O.D., Sinclair, J.B., 1985. Basic Plant Pathology Methods. CRC Press, Inc., Boca Raton, FL, 355pp. Elmer, 1985. The ecology and control of Fusarium yellows of celery. Ph.D. Dissertation, Michigan State University, East Lansing, MI. Elmer, W.H., 2001. The influence of nonpathogenic Fusarium species and Pseudomonas fluorescens on Fusarium wilt of cyclamen. Biol. Cult. Tests Control Plant Dis. 16, O40. Elmer, W.H., 2004. Effect of fungicides and combinations of biologicals and acibenzolar-S-methyl on Fusarium rot of gladiolus. Biol. Cult. Tests Control Plant Dis. 19, O007. Elmer, W.H., 2005. Effect of fungicide dips with and without Actigard 50WP on Fusarium corm rot of gladiolus. Fungicide and Nematicide Tests 60, OT, 015. Elmer, W.H., McGovern, R.J., 2004. Influence of combining biological products with chemical fungicides for control of Fusarium wilt of cyclamen. Crop Prot. 23, 909–914. Gracia-Garza, J.A., 2001. Fusarium oxysporum Schlechtendahl f. sp. cyclaminis Gerlach, Fusarium wilt of cyclamen (Hypomycetes). In: Mason, P., Huber, J. (Eds.), Biological Control Programmes in Canada 1981–2000. CABI Press, Surrey UK, pp. 452–456. Gullino, L., Minuto, A., Gilardi, G., Garibaldi, A., 2002. Efficacy of azoxystrobin and other strobilurins against Fusarium wilts of carnation, cyclamen and Paris daisy. Crop Prot. 21, 57–61. Ishii, H., Tomita, Y., Horio, T., Narusaka, Y., Nakazawa, Y., Nishimura, K., Iwamoto, S., 1999. Induced resistance of acibenzolar-S-methyl (CGA 245704) to cucumber and Japanese pear diseases. Eur. J. Plant Pathol. 105, 77–85. Komada, H., 1975. Development of selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Prot. Res. 8, 114–124. Matteoni, J.A., 1988. Diseases of cyclamen in Ontario from 1983 to 1987. Can. Plant Dis. Surv. 68, 84. Matheron, M.E., Porchas, M., 2002. Suppression of Phytophthora root and crown rot on pepper plants treated with acibenzolar-S-methyl. Plant Dis. 86, 292–297. Minuto, A., Migheli, Q., Garibaldi, A., 1995. Evaluation of antagonistic strains of Fusarium spp. in the biological and integrated control of Fusarium wilt of cyclamen. Crop Prot. 14, 221–226. Orlicz-Luthardt, A., 1998. Studies on the resistance of cyclamen to Fusarium wilt. Beit. Zu¨chtungsforsch. 4, 48–49.
ARTICLE IN PRESS 676
W.H. Elmer / Crop Protection 25 (2006) 671–676
Rattink, H., 1990. Epidemiology of Fusarium wilt of cyclamen in ebb and flow. Neth. J. Plant Pathol. 96, 171–177. Someya, N., Kataoka, N., Komagata, T., Hirayae, K., Hibi, T., Akutsu, K., 2000. Biological control of cyclamen soil diseases by Serratia marcescens strain B2. Plant Dis. 84, 334–340.
Tompkins, C.M., Snyder, W.C., 1972. Cyclamen wilt in California and its control. Plant Dis. Rep. 56, 493–497. Wouldt, L.P., Neuvel, A., Sikkema, A., van Grinsven, M.Q.J.M., de Milliano, W.A.J., 1995. Genetic variation in Fusarium oxysporum from cyclamen. Phytopathology 85, 1348–1355.