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Efficacy of integrating biologicals with fungicides for the suppression of Fusarium wilt of cyclamen
ARTICLE IN PRESS
Crop Protection 23 (2004) 909–914
Efficacy of integrating biologicals with fungicides for the suppression of Fusarium wilt of cyclamen Wade H. Elmera,*, Robert J. McGovernb b
a Connecticut Agricultural Experiment Station, P. O. Box 1106, New Haven, CT 06504, USA Department of Plant Pathology, University of Florida-IFAS, P. O. Box 110680, Gainesville, FL 32611, USA
Received 19 September 2003; received in revised form 27 January 2004; accepted 30 January 2004
Abstract Three series of greenhouse studies were conducted to determine if disease suppression of Fusarium wilt of cyclamen could be enhanced by integrating commercial formulations of beneficial microbes (biologicals) with registered fungicides. When biologicals were used as the first preventative treatment and inoculated a week later, there was no acceptable disease control even when plants were returned to a full conventional chemical fungicide program. However, when biologicals were applied following a fungicide treatment or tank-mixed with a fungicide, there were combinations that yielded significant reductions in the area-under-the-diseaseprogress-curve. These interactions were specific to certain biologicals and certain fungicides, and no pattern could be discerned from the pairings except that fludioxonil combined with a biological generally resulted in the greatest reductions in disease severity when compared to control treatments. The efficacy of integrating biologicals with fungicides has potential, but more studies are required to identify specific combinations between biologicals and fungicides that improve plant health. r 2004 Elsevier Ltd. All rights reserved. Keywords: Fusarium oxysporum f. sp. cyclaminis; Cyclamen persicum; Biological control; Chemical control
1. Introduction Fusarium wilt of cyclamen (Cyclamen persicum) is caused by Fusarium oxysporum f. sp. cyclaminis and is highly destructive and economically limiting to the production of quality cyclamens (Tompkins and Snyder, 1972). The damage caused by this disease has steadily increased in many production facilities (Daughtrey et al., 1995; Matteoni, 1988; Wouldt et al., 1995). Actual losses due to this disease have not been calculated, but its incidence increased during the 1990s to levels that forced several growers in the United States to discontinue production of the crop. In Ontario, losses of 40–50% of the crop have been reported (Gracia-Garza, 2001). Symptoms of the disease include stunting, chlorosis, and unilateral wilt, and eventual death of the plant. Cyclamen infected by F. oxysporum f.sp. cyclaminis develop a characteristic discoloration in the vascular tissue in the corm. The corm remains hard, which *Corresponding author. Tel.: +1-203-974-8503; fax: +1-203-9748502. E-mail address: [email protected] (W.H. Elmer). 0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.01.012
distinguishes the disease from soft rotting bacteria. Fusarium wilt symptoms can appear anytime during the life of the plant. It is not understood if late appearances of symptoms are the result of new inoculum being spread in the greenhouse via infested soil, wind or fungus gnats, or if infections occur early in the seedling stage and remain latent until other factors, such as stress, trigger the onset of symptoms. Isolations from seed and seed debris are rare. Late appearances of Fusarium wilt of cyclamen are particularly problematic for growers in that fungicides applied at this stage often fail to suppress the disease. Thus, fungicides, time, and labor are wasted. Adequate control strategies for Fusarium wilt of cyclamen have not yet been identified. No commercially acceptable resistant germ plasm is currently available (Orlicz-Luthardt, 1998). The registered fungicides azoxystrobin, thiophanate methyl, fludioxonil, and triflumizole are the most effective products available, but they have poor curative properties against this disease. Antagonistic microbes or biologicals have been tested in many laboratories to control Fusarium wilt of cyclamen, but these organisms have not provided the
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same level of suppression as fungicides (Elmer, 2001, 2002; Gracia-Garza, 2001; Minuto et al., 1995; Someya et al., 2000). Most biologicals can delay the onset of Fusarium wilt (Elmer, 2001, 2002), but they ultimately fail to protect the plant so that a marketable crop can be produced. Although most studies that examined biologicals were conducted in systems with artificially elevated, and perhaps unrealistic, inoculum levels, growers have not placed great reliance on biologicals and still use chemical fungicides in their management of Fusarium wilt. Integrating biologicals with chemicals has become an acceptable strategy for many pest systems, but it has not been fully explored for Fusarium wilt of cyclamen. The benefits of this approach include reducing the number of chemical sprays and improving plant growth and quality. There is also motivation on the part of the grower to use biologicals to minimize the cost of fungicide applications, lessen the chance of developing resistance in the pest populations, and avoid potential environmental hazards. Since many fungicides may alter or reduce the efficacy of the beneficial organism, timing of sprays and/or tank mixing become important issues. The objectives of this study were to evaluate different methods for rotating or tank-mixing biologicals with chemical fungicides for the efficacy in suppressing Fusarium wilt of cyclamen.
2. Material and methods Cyclamen seedlings (Halios series) were supplied by The Hortus Group (Castroville, CA). Seedling plugs were transplanted into 1.0-l plastic pots filled with the potting mix, Promix BX (one plant/pot). In all experiments, plants were inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia/ml) into both sides of corm (10 ml/ plant). Macroconidia were produced on potato–carrot agar (Dhingra and Sinclair, 1985) and washed off with distilled water after 10 days. Plants were treated once/ mo with Hoagland’s fertilizer solution (100 ml/pot). Plants received one application of Orthene Systemic
Concentrate (115 ml/l) and one application of Conserve (Spinosad, 15 ml/l) to control insect pests. Three series of studies were conducted to see if combining biologicals with chemical fungicides would enhance disease suppression. The biologicals and chemicals are currently labeled for Fusarium wilt of cyclamen or are approved as microbial inoculants for greenhouse use. Rates were as suggested by their manufacturers (Table 1). The first study determined whether or not biologicals could serve as the first preventative treatment applied at transplanting. Four biologicals were compared to two fungicides. The experiment had nine treatments (Table 2). One day after planting, treatments were applied as root drenches in 200 ml of distilled water. Plants treated with 200 ml of distilled water served as controls. Seven days later cyclamens were inoculated with the pathogen. Cyclamens in each treatment were then placed on a conventional program where they were treated with thiophanate methyl or fludioxonil for the second treatment 14 days after planting and again 28 days after planting. Two treatments had no fungicides applied; one was inoculated with the pathogen, and one was not. The experimental design was a completely randomized block design. There were five blocks each containing two replicate pots. The experiment was repeated using four blocks (two replicate plants/block). Both experiments were conducted in the spring with day/night temperatures at 18/15 C. The second study determined the usefulness of drenching cyclamen plants with biologicals following a preventative fungicide treatment (Table 2). The biological product Deny was dropped from Study II and MycoStop was included. Plants were treated with thiophanate methyl or fludioxonil and then treated one week later with Actinovate CM/Plus, Companion, PlantShield, MycoStop or distilled water (Control). Treatments were applied as root drenches in 200 ml of distilled water. Plants were inoculated after 14 days with macroconidia of F. oxysporum, f. sp. cyclaminis as described above. Two treatments had no fungicides applied; one was inoculated with the pathogen, and one was not. There were 10 replicate pots/treatment, and the
Table 1 Products and rates used in studies for suppression of Fusarium wilt of cyclamen Active ingredient/microbe (strain) Streptomyces lydicus Thiophanate methyl Bacillus subtilis (GB03) Burkholderia cepacia (type WI) Azoxystrobin Fludioxonil Streptomyces griseoviridis (K61) Trichoderma harzianum (T-22) a
Products sa
Actinovate CM/Plus Cleary’s 3336s 50WP Companions Denys Heritages 50 WP Medallions 50 WP MycoStops PlantShields
Manufacturer/City
Rate of product/l H2O
Natural Industries, Houston, TX Clearys Chemical Corp., Dayton, NJ Growth Products, White Plains, NY Stine Microbial Products, Shawnee, KS Syngenta Prof. Products, Greensboro, NC Syngenta Prof. Products, Greensboro, NC Kemira Agro Helsinki, Finland Bioworks, Inc. Geneva, NY
0.14 g 1.20 g 1.25 ml 1.25 ml 0.90 g 0.24 g 0.01 g 0.90 g
Actinovate Cm/plus is registered as soil inoculant and claims no disease-suppressing activity against Fusarium wilt of cyclamen.
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Table 2 Treatments and times when products were applied for studying combinations of biologicals with chemical fungicides for suppression of Fusarium wilt of cyclamen Study I Day 1
Day 7
1. 2. 3. 4. 5. 6. 7. 8. 9.
Not inoculated Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with
No treatment No treatment No treatment Thiophanate methyl Fludioxonil Actinovate CM/Plus Companion Deny PlantShield
FOCa FOC FOC FOC FOC FOC FOC FOC
Day 14
Day 28
No treatment Thiophanate methyl Fludioxonil Fludioxonil Thiophanate methyl Thiophanate methyl Thiophanate methyl Thiophanate methyl Thiophanate methyl
No treatment Fludioxonil Thiophanate methyl Thiophanate methyl Fludioxonil Fludioxonil Fludioxonil Fludioxonil Fludioxonil
Study II 1. No treatment 2. No treatment 3. Thiophanate methyl 4. Thiophanate methyl 5. Thiophanate methyl 6. Thiophanate methyl 7. Thiophanate methyl 8. Fludioxonil 9. Fludioxonil 10. Fludioxonil 11. Fludioxonil 12. Fludioxonil
No treatment No treatment No treatment Actinovate CM/Plus Companion PlantShield MycoStop No treatment Actinovate CM/Plus Companion PlantShield MycoStop
Not inoculated Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with
Study III 1. Not inoculated 2. Inoculated with FOC 3. Inoculated with FOC 4. Inoculated with FOC 5. Inoculated with FOC 6. Inoculated with FOC 7. Inoculated with FOC 8. Inoculated with FOC 9. Inoculated with FOC 10. Inoculated with FOC
No treatment No treatment Azoxystrobin Azoxystrobin + Actinovate Azoxystrobin + PlantShield Azoxystrobin Fludioxonil Fludioxonil + Actinovate Fludioxonil + PlantShield Fludioxonil
No treatment No treatment No treatment Actinovate CM/Plus PlantShield Azoxystrobin No treatment Actinovate CM/Plus PlantShield Fludioxonil
FOC FOC FOC FOC FOC FOC FOC FOC FOC FOC FOC
a FOC = Fusarium oxysporum f. sp. cyclaminis; plants were inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia/ml) into both sides of corm (10 ml/plant).
experiment was repeated using 12 replicate plants. The experimental designed as a completely randomized block design with two replicates/block. Both experiments were conducted in the summer months with day/ night temperatures at 25/20 C. The third study examined the efficacy of mixing chemical fungicide treatments with one of two biologicals and applying them to plants that had been simultaneously inoculated (Table 2). The chemical fungicide thiophanate methyl was replaced with azoxystrobin. Following these treatments plants were then treated with the same chemical fungicide used initially or with one of biologicals. Plants treated with 200 ml of distilled water served as controls. There were 10 replicate pots/treatment, and the experiment was repeated using 10 replicate plants. Both experiments
were conducted in the fall with day/night temperatures at 18/15 C. Evaluation of disease severity was made every 3 to 5 days over the duration of each experiment by assigning a disease severity rating to each plant. Disease ratings were based on the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/ or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. Area-underthe-disease-progress-curve (AUDPC, days) was computed using the formula AUDPC ¼ SðYi þ Yðiþ1Þ Þ=2ðtðiþ1Þ ti Þ;
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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 were removed, weighed, and then dried in an oven and re-weighed. The percentage of vascular discoloration in the crown was visually estimated by slicing the corm in half with a knife. Symptomatic plants were aseptically cultured for F. oxysporum f. sp. cyclaminis by placing diseased tissue on selective media to re-isolate the pathogen. Data were analyzed using analysis of variance, and means were separated using Bonferroni’s test at P=0.05. Data sets generated from Studies II and III were re-analyzed as factorial experiments after the two control treatments were removed. Single degree of freedom contrasts were conducted when appropriate. All analyses were conducted on SystatV.10 (SPSS Inc., Chicago, IL).
3. Results 3.1. Study I
Table 3 Effect of rotating biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study I) Treatmentsa No treatment (not inoculated) No treatment No treatment–Thiophanate methyl–Fludioxonil No treatment–Fludioxonil– Thiophanate methyl Thiophanate methyl– Fludioxonil–Thiophanate methyl Fludioxonil–Thiophanate methyl–Fludioxonil Actinovate CM/Plus– Thiophanate methyl– Fludioxonil Companion–Thiophanate methyl–Fludioxonil Deny–Thiophanate methyl– Fludioxonil PlantShield–Cleary’s 3336– Fludioxonil
AUDPCb
Dry weight (g)
% VDc
1.53 a
0a
0.19 c 0.16 c
89 bc 92 c
53.8 bc
0.61 bc
76 bc
34.5 b
0.79 b
54 bc
24.0 b
0.72 bc
40 b
75.0 bc
0.16 c
85 bc
74.2 bc
0.24 bc
82 bc
61.5 bc
0.40 bc
76 bc
54.9 bc
0.56 bc
68 bc
7.0 a 105.0 cd 93.9 c
a
Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=18). Symptoms of wilt and chlorosis were noted 3 weeks after inoculation in the no treatment and the no treatment–thiophanate methyl– fludioxonil treatment (data not shown). Although the biologicals delayed the onset and initially retarded the development of disease symptoms, none were effective in suppressing the total AUDPC values when compared to the no treatment control (Table 3). A significant reduction in the AUDPC value was only achieved when the chemical fungicides, thiophanate methyl or fludioxonil, were used preventively and not after inoculation when compared to the no treatment control. Dry weights were significantly increased over the no treatment when plants were treated with thiophanate methyl–fludioxonil–thiophanate methyl. In contrast, vascular discoloration in the corm was only reduced in the fludioxonil–thiophanate methyl–fludioxonil treatment when compared to the no treatment. All other treatments did not differ in their AUDPC values, dry weights, or the percentage of vascular discoloration. 3.2. Study II Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=22). Disease symptoms
Treatments were applied at day 1, 14 and 28 after planting; plants were inoculated on day 7. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c Statistical separations were performed on the arcsine transformation. %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 18 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05.
were first noted 10 days after inoculation in the no treatment control (data not shown). All treatments that included thiophanate methyl and a biological significantly reduced the AUDPC values when compared to controls whereas thiophanate methyl alone failed to reduce the AUDPC value (Table 4). In addition, fludioxonil alone or fludioxonil followed by MycoStop or PlantShield reduce the AUDPC values compared to the control. All other treatments delayed the onset of the disease (data not shown), but did not affect the final AUDPC values. All treatments numerically increased the dry weights relative to the no treatment but only fludioxonil followed by Actinovate CM/Plus, MycoStop or PlantShield produced statistically significant increases. The percentage of vascular discoloration in the corm was significantly reduced by thiophanate methyl followed by Actinovate or Companion, and
ARTICLE IN PRESS W.H. Elmer, R.J. McGovern / Crop Protection 23 (2004) 909–914 Table 4 Effect of rotating biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study II) Treatment (day 1–day 7)a No treatment (Not inoculated) No treatment Thiophanate methyl–No treatment Thiophanate methyl– Actinovate CM/Plus Thiophanate methyl– Companion Thiophanate methyl– MycoStop Thiophanate methyl– PlantShield Fludioxonil–No treatment Fludioxonil–Actinovate CM/Plus Fludioxonil–Companion Fludioxonil–MycoStop Fludioxonil–PlantShield Averaging over fungicides Thiophanate Methyl Fludioxonil
AUDPCb
Dry weight (g)
% VDc,d
1.9 a
0.89 a
0a
81.7 c 32.4 bc
0.03 b 0.32 ab
90 b 53 ab
20.1 ab
0.36 ab
14 a
17.9 ab
0.47 ab
11 a
29.9 ab
0.22 ab
58 ab
22.1 ab
0.34 ab
51 ab
29.3 ab 35.1 bc
0.39 ab 0.51 a
44 ab 23 ab
50.3 bc 8.6 a 15.6 ab
0.19 ab 0.75 a 0.73 a
53 ab 6a 16 a
24.5 a 27.8 a
0.34 a 0.52 b
37 a 28 a
a
Treatments were applied at day 1 and 7; plants were inoculated on day 14. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c Statistical separations were performed on the arcsine transformation. %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 22 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05. Analyses conducted on arcsine transformation; Non-transformed data presented.
Fludioxonil followed by PlantShield or MycoStop. Overall fludioxonil was marginally more effective that thiophanate methyl, but the only significant effect was on increasing dry weight. When the two control treatments (Inoculated and non-inoculated) were removed from the data set, the design could be reanalyzed as a randomized factorial experiment between the two fungicides and the four biologicals. There were no interactions between treatments for the variable AUDPC, but there were significant interactions for dry weight (P = 0.023) and the percent vascular discoloration (P=0.008). Actinovate, MycoStop, and PlantShield increased dry weights when applied following fludioxonil, but had no effect when applied after thiophanate methyl.
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3.3. Study III Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=20). Approximately 30 days after inoculation, symptoms appeared in the no treatment control and the azoxystrobin combined with Actinovate treatment (data not shown). All treatments except azoxystrobin + Actinovate followed by Actinovate and azoxystrobin followed by azoxystrobin decreased the AUDPC values when compared to no treatment controls (Table 5). On the contrary, when Actinovate was mixed with fludioxonil and followed by Actinovate, the lowest numerical AUDPC values were observed. None of the treatment combinations were able Table 5 Effect of tank mixing biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study III) Treatment (day 2–day 15)a
AUDPCb
Dry weight (g)
No treatment (not inoculated) No treatment (Inoculated) Azoxystrobin–No treatment (Azoxystrobin + Actinovate CM/Plus)– Actinovate CM/Plus (Azoxystrobin + PlantShield)–PlantShield Azoxystrobin– Azoxystrobin Fludioxonil–No treatment (Fludioxonil + Actinovate CM/Plus)–Actinovate CM/ Plus (Fludioxonil + PlantShield)–PlantShield Fludioxonil–Fludioxonil
12.8 ad
1.30 a
0.0 a
65.2 c 27.8 ab
0.95 a 1.02 a
20.8 a 17.5 a
58.8 bc
1.11 a
22.5 a
36.5 ab
0.94 a
17.0 a
42.7 bc
0.88 a
32.5 a
21.2 ab 4.0 a
1.02 a 1.04 a
11.5 a 0.5 a
25.5 ab
0.93 a
15.8 a
38.3 ab
0.90 a
25.0 a
22.2 a 41.4 a
0.97 a 0.99 a
13.2 a 22.4 a
Averaging over fungicides Fludioxonil Azoxystrobin
% VDc
a Treatments were applied at day 2, 15 and 29; plants were inoculated on day 1. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 20 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05.
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to affect the dry weights or the percentage of vascular discoloration in the corms when compared to controls. When the two control treatments (inoculated and noninoculated) were removed from the data set, the design could again be re-analyzed as a factorial between the two fungicides and the four biologicals. There were no interactions between treatments for the variable AUDPC, dry weight, or vascular discoloration. Azoxystrobin and fludioxonil were comparable in reducing the AUDPC and the percentage of vascular discoloration and increasing dry weight.
biologicals have potential as part of strategy that includes sanitation and fungicides, but more research is needed to determine how to best use them most efficaciously in cyclamen management (Gracia-Garza, 2001; Minuto et al., 2002; Someya et al., 2000). Fludioxonil provided the best protection of Fusarium wilt of cyclamen of the three fungicides used in this study, but statistical differences with the other chemicals were marginal. Although we did not directly compare azoxystrobin with thiophanate methyl, azoxystrobin is reported to have more efficacy than the benzimidiazoles groups that contain Thiophanate Methyl (Gullino et al., 2002).
4. Discussion Since the advent of commercial preparations of biological control, researchers have experimented with many of the products to suppress Fusarium wilt of cyclamen. These researchers concluded that most biologicals are effective in delaying the onset of disease and can provide some suppression, but rarely provide the level of protection that is equal to that obtained with chemical fungicides (Elmer, 2001, 2002; Gracia-Garza, 2001; Minuto et al., 1995). When biologicals were used in the current study as the preventative treatment prior to inoculation, they provided no acceptable control even though the plants were then placed on a regime of chemical fungicides. This study also demonstrates the ineffectiveness of applying any treatment to cyclamens once disease symptoms have appeared. Vascular infections by F. oxysporum f. sp. cyclaminis are very difficult to cure, and thus, reinforce the important strategy of preventing infection. We recognized that our inoculation pressure may have been much higher than found in typical greenhouse operations and that this may not reflect the efficacy of these products under lower inoculum densities. Nevertheless, applying biologicals after a preventative chemical fungicide was applied showed promise for suppression of the disease, but unexplained combinations between biologicals and fungicides were noted. Differences in the combination treatments might be expected since each biological control agent might respond differently to different chemicals. The active organisms in Actinovate and MycoStop are actinomycetes, whereas in Companion and Deny, they are bacteria. The active microbe in PlantShield is a fungus. Although tank-mixing these organisms is allowed for a wide variety of chemistries, it appears that different combination of organisms and chemicals do vary in their effectiveness against Fusarium wilt of cyclamen. Our research also demonstrated that, in general, when biologicals were rotated following chemical fungicides, the level of disease suppression was better than that achieved by the chemical alone. These conclusions agree with others who reported that
Acknowledgements This study was supported in part by funds from the American Floral Endowment Fund. The authors thank Elizabeth O’Dowd for technical assistance and The Hortus Group (Castorville, CA) for cyclamen seedlings.
References 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, W.H., 2001. The influence of nonpathogenic Fusarium species and Pseudomonas fluorescens on Fusarium wilt of cyclamen. 2000: Biol. Cult. Tests Control Plant Dis. 16, O40. Elmer, W.H., 2002. The influence of microbial inoculants and chemical fungicides of Fusarium wilt of cyclamen. 2001: Biol. Cult. Tests Control Plant Dis. 17, O06. 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. Matteoni, J.A., 1988. Diseases of cyclamen in Ontario from 1983 to 1987. Can. Plant Dis. Survey 68, 84. Minuto, A., Migheli, Q., Garibaldi, A., 1995. Evaluation of antagonistic strains of Fuarium spp. in the biological and integrated control of Fusarium wilt of cyclamen. Crop Prot. 14, 221–226. Minuto, A., Bruzzone, C., Tinivella, F., Gullino, L., Garibaldi, A., 2002. Biological control in cyclamen. Colture Protette 31 (12), 85–95 (as cited in the Rev. Plant Pathol.2003, 82, 736). Orlicz-Luthardt, A., 1998. Studies on the resistance of cyclamen to Fusarium wilt. Beit. zur Zuchtungsforsch. . 4, 48–49. 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 23 (2004) 909–914
Efficacy of integrating biologicals with fungicides for the suppression of Fusarium wilt of cyclamen Wade H. Elmera,*, Robert J. McGovernb b
a Connecticut Agricultural Experiment Station, P. O. Box 1106, New Haven, CT 06504, USA Department of Plant Pathology, University of Florida-IFAS, P. O. Box 110680, Gainesville, FL 32611, USA
Received 19 September 2003; received in revised form 27 January 2004; accepted 30 January 2004
Abstract Three series of greenhouse studies were conducted to determine if disease suppression of Fusarium wilt of cyclamen could be enhanced by integrating commercial formulations of beneficial microbes (biologicals) with registered fungicides. When biologicals were used as the first preventative treatment and inoculated a week later, there was no acceptable disease control even when plants were returned to a full conventional chemical fungicide program. However, when biologicals were applied following a fungicide treatment or tank-mixed with a fungicide, there were combinations that yielded significant reductions in the area-under-the-diseaseprogress-curve. These interactions were specific to certain biologicals and certain fungicides, and no pattern could be discerned from the pairings except that fludioxonil combined with a biological generally resulted in the greatest reductions in disease severity when compared to control treatments. The efficacy of integrating biologicals with fungicides has potential, but more studies are required to identify specific combinations between biologicals and fungicides that improve plant health. r 2004 Elsevier Ltd. All rights reserved. Keywords: Fusarium oxysporum f. sp. cyclaminis; Cyclamen persicum; Biological control; Chemical control
1. Introduction Fusarium wilt of cyclamen (Cyclamen persicum) is caused by Fusarium oxysporum f. sp. cyclaminis and is highly destructive and economically limiting to the production of quality cyclamens (Tompkins and Snyder, 1972). The damage caused by this disease has steadily increased in many production facilities (Daughtrey et al., 1995; Matteoni, 1988; Wouldt et al., 1995). Actual losses due to this disease have not been calculated, but its incidence increased during the 1990s to levels that forced several growers in the United States to discontinue production of the crop. In Ontario, losses of 40–50% of the crop have been reported (Gracia-Garza, 2001). Symptoms of the disease include stunting, chlorosis, and unilateral wilt, and eventual death of the plant. Cyclamen infected by F. oxysporum f.sp. cyclaminis develop a characteristic discoloration in the vascular tissue in the corm. The corm remains hard, which *Corresponding author. Tel.: +1-203-974-8503; fax: +1-203-9748502. E-mail address: [email protected] (W.H. Elmer). 0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.01.012
distinguishes the disease from soft rotting bacteria. Fusarium wilt symptoms can appear anytime during the life of the plant. It is not understood if late appearances of symptoms are the result of new inoculum being spread in the greenhouse via infested soil, wind or fungus gnats, or if infections occur early in the seedling stage and remain latent until other factors, such as stress, trigger the onset of symptoms. Isolations from seed and seed debris are rare. Late appearances of Fusarium wilt of cyclamen are particularly problematic for growers in that fungicides applied at this stage often fail to suppress the disease. Thus, fungicides, time, and labor are wasted. Adequate control strategies for Fusarium wilt of cyclamen have not yet been identified. No commercially acceptable resistant germ plasm is currently available (Orlicz-Luthardt, 1998). The registered fungicides azoxystrobin, thiophanate methyl, fludioxonil, and triflumizole are the most effective products available, but they have poor curative properties against this disease. Antagonistic microbes or biologicals have been tested in many laboratories to control Fusarium wilt of cyclamen, but these organisms have not provided the
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same level of suppression as fungicides (Elmer, 2001, 2002; Gracia-Garza, 2001; Minuto et al., 1995; Someya et al., 2000). Most biologicals can delay the onset of Fusarium wilt (Elmer, 2001, 2002), but they ultimately fail to protect the plant so that a marketable crop can be produced. Although most studies that examined biologicals were conducted in systems with artificially elevated, and perhaps unrealistic, inoculum levels, growers have not placed great reliance on biologicals and still use chemical fungicides in their management of Fusarium wilt. Integrating biologicals with chemicals has become an acceptable strategy for many pest systems, but it has not been fully explored for Fusarium wilt of cyclamen. The benefits of this approach include reducing the number of chemical sprays and improving plant growth and quality. There is also motivation on the part of the grower to use biologicals to minimize the cost of fungicide applications, lessen the chance of developing resistance in the pest populations, and avoid potential environmental hazards. Since many fungicides may alter or reduce the efficacy of the beneficial organism, timing of sprays and/or tank mixing become important issues. The objectives of this study were to evaluate different methods for rotating or tank-mixing biologicals with chemical fungicides for the efficacy in suppressing Fusarium wilt of cyclamen.
2. Material and methods Cyclamen seedlings (Halios series) were supplied by The Hortus Group (Castroville, CA). Seedling plugs were transplanted into 1.0-l plastic pots filled with the potting mix, Promix BX (one plant/pot). In all experiments, plants were inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia/ml) into both sides of corm (10 ml/ plant). Macroconidia were produced on potato–carrot agar (Dhingra and Sinclair, 1985) and washed off with distilled water after 10 days. Plants were treated once/ mo with Hoagland’s fertilizer solution (100 ml/pot). Plants received one application of Orthene Systemic
Concentrate (115 ml/l) and one application of Conserve (Spinosad, 15 ml/l) to control insect pests. Three series of studies were conducted to see if combining biologicals with chemical fungicides would enhance disease suppression. The biologicals and chemicals are currently labeled for Fusarium wilt of cyclamen or are approved as microbial inoculants for greenhouse use. Rates were as suggested by their manufacturers (Table 1). The first study determined whether or not biologicals could serve as the first preventative treatment applied at transplanting. Four biologicals were compared to two fungicides. The experiment had nine treatments (Table 2). One day after planting, treatments were applied as root drenches in 200 ml of distilled water. Plants treated with 200 ml of distilled water served as controls. Seven days later cyclamens were inoculated with the pathogen. Cyclamens in each treatment were then placed on a conventional program where they were treated with thiophanate methyl or fludioxonil for the second treatment 14 days after planting and again 28 days after planting. Two treatments had no fungicides applied; one was inoculated with the pathogen, and one was not. The experimental design was a completely randomized block design. There were five blocks each containing two replicate pots. The experiment was repeated using four blocks (two replicate plants/block). Both experiments were conducted in the spring with day/night temperatures at 18/15 C. The second study determined the usefulness of drenching cyclamen plants with biologicals following a preventative fungicide treatment (Table 2). The biological product Deny was dropped from Study II and MycoStop was included. Plants were treated with thiophanate methyl or fludioxonil and then treated one week later with Actinovate CM/Plus, Companion, PlantShield, MycoStop or distilled water (Control). Treatments were applied as root drenches in 200 ml of distilled water. Plants were inoculated after 14 days with macroconidia of F. oxysporum, f. sp. cyclaminis as described above. Two treatments had no fungicides applied; one was inoculated with the pathogen, and one was not. There were 10 replicate pots/treatment, and the
Table 1 Products and rates used in studies for suppression of Fusarium wilt of cyclamen Active ingredient/microbe (strain) Streptomyces lydicus Thiophanate methyl Bacillus subtilis (GB03) Burkholderia cepacia (type WI) Azoxystrobin Fludioxonil Streptomyces griseoviridis (K61) Trichoderma harzianum (T-22) a
Products sa
Actinovate CM/Plus Cleary’s 3336s 50WP Companions Denys Heritages 50 WP Medallions 50 WP MycoStops PlantShields
Manufacturer/City
Rate of product/l H2O
Natural Industries, Houston, TX Clearys Chemical Corp., Dayton, NJ Growth Products, White Plains, NY Stine Microbial Products, Shawnee, KS Syngenta Prof. Products, Greensboro, NC Syngenta Prof. Products, Greensboro, NC Kemira Agro Helsinki, Finland Bioworks, Inc. Geneva, NY
0.14 g 1.20 g 1.25 ml 1.25 ml 0.90 g 0.24 g 0.01 g 0.90 g
Actinovate Cm/plus is registered as soil inoculant and claims no disease-suppressing activity against Fusarium wilt of cyclamen.
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Table 2 Treatments and times when products were applied for studying combinations of biologicals with chemical fungicides for suppression of Fusarium wilt of cyclamen Study I Day 1
Day 7
1. 2. 3. 4. 5. 6. 7. 8. 9.
Not inoculated Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with
No treatment No treatment No treatment Thiophanate methyl Fludioxonil Actinovate CM/Plus Companion Deny PlantShield
FOCa FOC FOC FOC FOC FOC FOC FOC
Day 14
Day 28
No treatment Thiophanate methyl Fludioxonil Fludioxonil Thiophanate methyl Thiophanate methyl Thiophanate methyl Thiophanate methyl Thiophanate methyl
No treatment Fludioxonil Thiophanate methyl Thiophanate methyl Fludioxonil Fludioxonil Fludioxonil Fludioxonil Fludioxonil
Study II 1. No treatment 2. No treatment 3. Thiophanate methyl 4. Thiophanate methyl 5. Thiophanate methyl 6. Thiophanate methyl 7. Thiophanate methyl 8. Fludioxonil 9. Fludioxonil 10. Fludioxonil 11. Fludioxonil 12. Fludioxonil
No treatment No treatment No treatment Actinovate CM/Plus Companion PlantShield MycoStop No treatment Actinovate CM/Plus Companion PlantShield MycoStop
Not inoculated Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with Inoculated with
Study III 1. Not inoculated 2. Inoculated with FOC 3. Inoculated with FOC 4. Inoculated with FOC 5. Inoculated with FOC 6. Inoculated with FOC 7. Inoculated with FOC 8. Inoculated with FOC 9. Inoculated with FOC 10. Inoculated with FOC
No treatment No treatment Azoxystrobin Azoxystrobin + Actinovate Azoxystrobin + PlantShield Azoxystrobin Fludioxonil Fludioxonil + Actinovate Fludioxonil + PlantShield Fludioxonil
No treatment No treatment No treatment Actinovate CM/Plus PlantShield Azoxystrobin No treatment Actinovate CM/Plus PlantShield Fludioxonil
FOC FOC FOC FOC FOC FOC FOC FOC FOC FOC FOC
a FOC = Fusarium oxysporum f. sp. cyclaminis; plants were inoculated by pipetting 5 ml of a conidial suspension of F. oxysporum f. sp. cyclaminis (4000 macroconidia/ml) into both sides of corm (10 ml/plant).
experiment was repeated using 12 replicate plants. The experimental designed as a completely randomized block design with two replicates/block. Both experiments were conducted in the summer months with day/ night temperatures at 25/20 C. The third study examined the efficacy of mixing chemical fungicide treatments with one of two biologicals and applying them to plants that had been simultaneously inoculated (Table 2). The chemical fungicide thiophanate methyl was replaced with azoxystrobin. Following these treatments plants were then treated with the same chemical fungicide used initially or with one of biologicals. Plants treated with 200 ml of distilled water served as controls. There were 10 replicate pots/treatment, and the experiment was repeated using 10 replicate plants. Both experiments
were conducted in the fall with day/night temperatures at 18/15 C. Evaluation of disease severity was made every 3 to 5 days over the duration of each experiment by assigning a disease severity rating to each plant. Disease ratings were based on the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/ or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. Area-underthe-disease-progress-curve (AUDPC, days) was computed using the formula AUDPC ¼ SðYi þ Yðiþ1Þ Þ=2ðtðiþ1Þ ti Þ;
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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 were removed, weighed, and then dried in an oven and re-weighed. The percentage of vascular discoloration in the crown was visually estimated by slicing the corm in half with a knife. Symptomatic plants were aseptically cultured for F. oxysporum f. sp. cyclaminis by placing diseased tissue on selective media to re-isolate the pathogen. Data were analyzed using analysis of variance, and means were separated using Bonferroni’s test at P=0.05. Data sets generated from Studies II and III were re-analyzed as factorial experiments after the two control treatments were removed. Single degree of freedom contrasts were conducted when appropriate. All analyses were conducted on SystatV.10 (SPSS Inc., Chicago, IL).
3. Results 3.1. Study I
Table 3 Effect of rotating biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study I) Treatmentsa No treatment (not inoculated) No treatment No treatment–Thiophanate methyl–Fludioxonil No treatment–Fludioxonil– Thiophanate methyl Thiophanate methyl– Fludioxonil–Thiophanate methyl Fludioxonil–Thiophanate methyl–Fludioxonil Actinovate CM/Plus– Thiophanate methyl– Fludioxonil Companion–Thiophanate methyl–Fludioxonil Deny–Thiophanate methyl– Fludioxonil PlantShield–Cleary’s 3336– Fludioxonil
AUDPCb
Dry weight (g)
% VDc
1.53 a
0a
0.19 c 0.16 c
89 bc 92 c
53.8 bc
0.61 bc
76 bc
34.5 b
0.79 b
54 bc
24.0 b
0.72 bc
40 b
75.0 bc
0.16 c
85 bc
74.2 bc
0.24 bc
82 bc
61.5 bc
0.40 bc
76 bc
54.9 bc
0.56 bc
68 bc
7.0 a 105.0 cd 93.9 c
a
Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=18). Symptoms of wilt and chlorosis were noted 3 weeks after inoculation in the no treatment and the no treatment–thiophanate methyl– fludioxonil treatment (data not shown). Although the biologicals delayed the onset and initially retarded the development of disease symptoms, none were effective in suppressing the total AUDPC values when compared to the no treatment control (Table 3). A significant reduction in the AUDPC value was only achieved when the chemical fungicides, thiophanate methyl or fludioxonil, were used preventively and not after inoculation when compared to the no treatment control. Dry weights were significantly increased over the no treatment when plants were treated with thiophanate methyl–fludioxonil–thiophanate methyl. In contrast, vascular discoloration in the corm was only reduced in the fludioxonil–thiophanate methyl–fludioxonil treatment when compared to the no treatment. All other treatments did not differ in their AUDPC values, dry weights, or the percentage of vascular discoloration. 3.2. Study II Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=22). Disease symptoms
Treatments were applied at day 1, 14 and 28 after planting; plants were inoculated on day 7. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c Statistical separations were performed on the arcsine transformation. %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 18 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05.
were first noted 10 days after inoculation in the no treatment control (data not shown). All treatments that included thiophanate methyl and a biological significantly reduced the AUDPC values when compared to controls whereas thiophanate methyl alone failed to reduce the AUDPC value (Table 4). In addition, fludioxonil alone or fludioxonil followed by MycoStop or PlantShield reduce the AUDPC values compared to the control. All other treatments delayed the onset of the disease (data not shown), but did not affect the final AUDPC values. All treatments numerically increased the dry weights relative to the no treatment but only fludioxonil followed by Actinovate CM/Plus, MycoStop or PlantShield produced statistically significant increases. The percentage of vascular discoloration in the corm was significantly reduced by thiophanate methyl followed by Actinovate or Companion, and
ARTICLE IN PRESS W.H. Elmer, R.J. McGovern / Crop Protection 23 (2004) 909–914 Table 4 Effect of rotating biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study II) Treatment (day 1–day 7)a No treatment (Not inoculated) No treatment Thiophanate methyl–No treatment Thiophanate methyl– Actinovate CM/Plus Thiophanate methyl– Companion Thiophanate methyl– MycoStop Thiophanate methyl– PlantShield Fludioxonil–No treatment Fludioxonil–Actinovate CM/Plus Fludioxonil–Companion Fludioxonil–MycoStop Fludioxonil–PlantShield Averaging over fungicides Thiophanate Methyl Fludioxonil
AUDPCb
Dry weight (g)
% VDc,d
1.9 a
0.89 a
0a
81.7 c 32.4 bc
0.03 b 0.32 ab
90 b 53 ab
20.1 ab
0.36 ab
14 a
17.9 ab
0.47 ab
11 a
29.9 ab
0.22 ab
58 ab
22.1 ab
0.34 ab
51 ab
29.3 ab 35.1 bc
0.39 ab 0.51 a
44 ab 23 ab
50.3 bc 8.6 a 15.6 ab
0.19 ab 0.75 a 0.73 a
53 ab 6a 16 a
24.5 a 27.8 a
0.34 a 0.52 b
37 a 28 a
a
Treatments were applied at day 1 and 7; plants were inoculated on day 14. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c Statistical separations were performed on the arcsine transformation. %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 22 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05. Analyses conducted on arcsine transformation; Non-transformed data presented.
Fludioxonil followed by PlantShield or MycoStop. Overall fludioxonil was marginally more effective that thiophanate methyl, but the only significant effect was on increasing dry weight. When the two control treatments (Inoculated and non-inoculated) were removed from the data set, the design could be reanalyzed as a randomized factorial experiment between the two fungicides and the four biologicals. There were no interactions between treatments for the variable AUDPC, but there were significant interactions for dry weight (P = 0.023) and the percent vascular discoloration (P=0.008). Actinovate, MycoStop, and PlantShield increased dry weights when applied following fludioxonil, but had no effect when applied after thiophanate methyl.
913
3.3. Study III Symptoms typical of Fusarium wilt were not observed in non-inoculated control plants. An insignificant F test for the interaction term between the treatments and experimental repetition allowed the data from both experiments to be combined (n=20). Approximately 30 days after inoculation, symptoms appeared in the no treatment control and the azoxystrobin combined with Actinovate treatment (data not shown). All treatments except azoxystrobin + Actinovate followed by Actinovate and azoxystrobin followed by azoxystrobin decreased the AUDPC values when compared to no treatment controls (Table 5). On the contrary, when Actinovate was mixed with fludioxonil and followed by Actinovate, the lowest numerical AUDPC values were observed. None of the treatment combinations were able Table 5 Effect of tank mixing biologicals with chemical fungicides to suppress Fusarium wilt of cyclamen (Study III) Treatment (day 2–day 15)a
AUDPCb
Dry weight (g)
No treatment (not inoculated) No treatment (Inoculated) Azoxystrobin–No treatment (Azoxystrobin + Actinovate CM/Plus)– Actinovate CM/Plus (Azoxystrobin + PlantShield)–PlantShield Azoxystrobin– Azoxystrobin Fludioxonil–No treatment (Fludioxonil + Actinovate CM/Plus)–Actinovate CM/ Plus (Fludioxonil + PlantShield)–PlantShield Fludioxonil–Fludioxonil
12.8 ad
1.30 a
0.0 a
65.2 c 27.8 ab
0.95 a 1.02 a
20.8 a 17.5 a
58.8 bc
1.11 a
22.5 a
36.5 ab
0.94 a
17.0 a
42.7 bc
0.88 a
32.5 a
21.2 ab 4.0 a
1.02 a 1.04 a
11.5 a 0.5 a
25.5 ab
0.93 a
15.8 a
38.3 ab
0.90 a
25.0 a
22.2 a 41.4 a
0.97 a 0.99 a
13.2 a 22.4 a
Averaging over fungicides Fludioxonil Azoxystrobin
% VDc
a Treatments were applied at day 2, 15 and 29; plants were inoculated on day 1. b Area-under-the-disease-progress-curve (AUDPC, days); disease ratings recorded weekly using the scale: 0 = no disease observed, 1 = slight stunting, 2 = slight stunting and chlorosis of leaves, 3 = o10% of the leaves showing chlorosis and/or 10% of the plant with wilt symptoms, 4 = o11–25% of the plant with wilt symptoms, 5 = 26–50% of the plant with wilt symptoms, and 6 = 51–100% of the plant with wilt symptoms or plant death. c %VD is the percent vascular discoloration in the corm recorded at the end of the experiment; statistical separations were performed on the arcsine transformation. d Values represent the means of 20 plants; values in columns followed by differing letters are significantly different according to Bonferroni’s Test at P=0.05.
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to affect the dry weights or the percentage of vascular discoloration in the corms when compared to controls. When the two control treatments (inoculated and noninoculated) were removed from the data set, the design could again be re-analyzed as a factorial between the two fungicides and the four biologicals. There were no interactions between treatments for the variable AUDPC, dry weight, or vascular discoloration. Azoxystrobin and fludioxonil were comparable in reducing the AUDPC and the percentage of vascular discoloration and increasing dry weight.
biologicals have potential as part of strategy that includes sanitation and fungicides, but more research is needed to determine how to best use them most efficaciously in cyclamen management (Gracia-Garza, 2001; Minuto et al., 2002; Someya et al., 2000). Fludioxonil provided the best protection of Fusarium wilt of cyclamen of the three fungicides used in this study, but statistical differences with the other chemicals were marginal. Although we did not directly compare azoxystrobin with thiophanate methyl, azoxystrobin is reported to have more efficacy than the benzimidiazoles groups that contain Thiophanate Methyl (Gullino et al., 2002).
4. Discussion Since the advent of commercial preparations of biological control, researchers have experimented with many of the products to suppress Fusarium wilt of cyclamen. These researchers concluded that most biologicals are effective in delaying the onset of disease and can provide some suppression, but rarely provide the level of protection that is equal to that obtained with chemical fungicides (Elmer, 2001, 2002; Gracia-Garza, 2001; Minuto et al., 1995). When biologicals were used in the current study as the preventative treatment prior to inoculation, they provided no acceptable control even though the plants were then placed on a regime of chemical fungicides. This study also demonstrates the ineffectiveness of applying any treatment to cyclamens once disease symptoms have appeared. Vascular infections by F. oxysporum f. sp. cyclaminis are very difficult to cure, and thus, reinforce the important strategy of preventing infection. We recognized that our inoculation pressure may have been much higher than found in typical greenhouse operations and that this may not reflect the efficacy of these products under lower inoculum densities. Nevertheless, applying biologicals after a preventative chemical fungicide was applied showed promise for suppression of the disease, but unexplained combinations between biologicals and fungicides were noted. Differences in the combination treatments might be expected since each biological control agent might respond differently to different chemicals. The active organisms in Actinovate and MycoStop are actinomycetes, whereas in Companion and Deny, they are bacteria. The active microbe in PlantShield is a fungus. Although tank-mixing these organisms is allowed for a wide variety of chemistries, it appears that different combination of organisms and chemicals do vary in their effectiveness against Fusarium wilt of cyclamen. Our research also demonstrated that, in general, when biologicals were rotated following chemical fungicides, the level of disease suppression was better than that achieved by the chemical alone. These conclusions agree with others who reported that
Acknowledgements This study was supported in part by funds from the American Floral Endowment Fund. The authors thank Elizabeth O’Dowd for technical assistance and The Hortus Group (Castorville, CA) for cyclamen seedlings.
References 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, W.H., 2001. The influence of nonpathogenic Fusarium species and Pseudomonas fluorescens on Fusarium wilt of cyclamen. 2000: Biol. Cult. Tests Control Plant Dis. 16, O40. Elmer, W.H., 2002. The influence of microbial inoculants and chemical fungicides of Fusarium wilt of cyclamen. 2001: Biol. Cult. Tests Control Plant Dis. 17, O06. 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. Matteoni, J.A., 1988. Diseases of cyclamen in Ontario from 1983 to 1987. Can. Plant Dis. Survey 68, 84. Minuto, A., Migheli, Q., Garibaldi, A., 1995. Evaluation of antagonistic strains of Fuarium spp. in the biological and integrated control of Fusarium wilt of cyclamen. Crop Prot. 14, 221–226. Minuto, A., Bruzzone, C., Tinivella, F., Gullino, L., Garibaldi, A., 2002. Biological control in cyclamen. Colture Protette 31 (12), 85–95 (as cited in the Rev. Plant Pathol.2003, 82, 736). Orlicz-Luthardt, A., 1998. Studies on the resistance of cyclamen to Fusarium wilt. Beit. zur Zuchtungsforsch. . 4, 48–49. 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.