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Effect of trifloxystrobin and azoxystrobin on the control of black Sigatoka (Mycosphaerella fijiensis Morelet) on banana and plantain
Crop Protection 21 (2002) 17–23
Effect of trifloxystrobin and azoxystrobin on the control of black Sigatoka (Mycosphaerella fijiensis Morelet) on banana and plantain Luis Pe! rez*, Alexis Herna! ndez, La! zaro Herna! ndez, Michel Pe! rez Instituto de Investigaciones de Sanidad Vegetal (INISAV), Ministerio de Agricultura de Cuba, Gaveta 634, Playa, Havana City 11300, Cuba Received 14 June 2000; received in revised form 31 August 2000; accepted 4 April 2001
Abstract Trials to determine the efficacy of trifloxystrobin (75 and 100 g ai/ha) and azoxystrobin (100 g ai/ha), on the control of black Sigatoka disease caused by Mycosphaerella fijiensis Morelet (M. fijiensis), in small randomized plots of banana and plantain treated from the ground and in commercial banana fields treated from the air, were carried out between 1997 and 1999. Trifloxystrobin at 75 g ai/ha showed a similar efficacy to propiconazole at 100 g ai/ha and benomyl at 150 g ai/ha and was superior to azoxystrobin (100 g ai/ha). Azoxystrobin show a similar efficacy to propiconazole against M. fijiensis populations sensitive to DMIs. The ED50 (ascospore germinative test) of five wild type M. fijiensis populations of different localities to azoxystrobin were between 0.03 and 0.8 mg/ml. Results suggest that monitoring for strobilurin resistance may be conducted using 5 mg/ml as the threshold concentration. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Black Sigatoka; Mycosphaerella fijiensis; Azoxystrobin; Trifloxystrobin; Fungicides
1. Introduction Black Sigatoka disease caused by Mycosphaerella fijiensis Morelet was first reported in Cuba in 1991. To control the disease, triazole (propiconazole, tebuconazole, bitertanol), morpholine (tridemorph) and benzimidazole (benomyl) fungicides aerial treatments were used following a bio-climatic forecasting system of treatment (P!erez et al., 1993; P!erez, 1997). The monitoring of the sensitivity of M. fijiensis populations to DMIs (C14 demethylase inhibitors) and morpholines (D7–D8 isomerase and D14 reductase inhibitors) has been carried out since 1991 (P!erez and Batlle, 1993). In the last two years, an increase in the frequency of populations with a decreased sensitivity (10–100 ) to propiconazole and other DMIs has been observed (P!erez et al., 2000). Due to this, it is necessary to discover new fungicides with a good efficacy against the disease and different biochemical mechanisms of action compared to benzimidazole and DMIs that are currently in use for the control of M. fijiensis. The methoxyacrylates represent a new family of fungicides with a wide spectrum of action against *Corresponding author. E-mail address: [email protected], [email protected] (L. P!erez).
different taxonomic groups of fungi, and different degrees of systemic movement (Godwin et al., 1992). Their biochemical mechanism of action is based on the inhibition of the transport of electrons between cytochrome b and cytochrome C1 in mitochondria, therefore preventing the formation of ATP (Geier et al., 1992; Wiggins and Jager, 1994). Among these compounds are azoxystrobin (Godwin et al., 1992) and trifloxystrobin (Margot et al., 1998). In this paper, the results of the studies to determine the efficacy of trifloxystrobin and azoxystrobin on the control of M. fijiensis populations with different levels of sensitivity to DMIs are reported.
2. Materials and methods 2.1. Field trials 2.1.1. Small plots trials on banana (Musa acuminata AAA) and plantain (Musa spp. AAB) using knapsack application Two trials were carried out in plots conformed by four lines of ten plants each (192 m2), in a randomized block design with four replicates in the locality of Gu. ira
0261-2194/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 5 5 - 2
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
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de Melena. All treatments were carried out as oil in water emulsions (with 7 l of oil and water up to 60 l/ha) with a knapsack mist blower sprayer. The first trial was in 1997 in a plantain field of the cultivar CEMSA 3/4 (AAB) that has never received triazole treatments. The M. fijiensis population sensitivity to propiconazole was determined by the assessment of the ascospore germination tube growth (Smith et al., 1991; P!erez and Batlle, 1993; Chin et al., 1996). The populations of M. fijiensis showed an ED50 o0.001 mg/ ml (sensitive; class 2 of sensitivity according to Chin et al., 1996). Four applications of the following treatments: (1) azoxystrobin 100 g ai/ha, (2) propiconazole 100 g ai/ha and (3) untreated control were made at 12–14 days intervals. The second trial in 1998 was in a field of the cultivar Grand Nain (subgroup Cavendish, AAA); the M. fijiensis population had an ED50 of 0.17 mg/ml (resistant population; class 7 of sensitivity according to Chin et al., 1996). The trial includes the following treatments: (1) propiconazole 100 g ai/ha; (2) azoxystrobin 100 g ai/ha; (3) trifloxystrobin 100 g ai/ ha; (4) trifloxystrobin 75 g ai/ha and (5) untreated control. Four applications were made at intervals of 12–14 days. In both trials the disease severity was evaluated before each treatment according to: (1) The number of the youngest unfurled leaf with ten or more scattered necrotic spots (YLS) (Stover and Dickson, 1970). (2) Severity of the attack on all leaves of ten nonflowered plants/plot according to the international scale of Stover (1971) that was modified by P!erez (1978). In this scale 0=until 10 isolated spots/leaf; 1=until 5% of the leaf area necrotic; 2=from 6% to 15% of leaf area necrotic; 3=from 16% to 33% leaf area necrotic; 4=from 33% to 50% of leaf area necrotic and 5=more than 50% of the leaf area necrotic. An infection index (II) (%) was calculated using the formula of Townsend and Heuberger (Unterstenhoefer, 1963): II% ¼
X
an=6N 100;
ð1Þ
where a is the value of severity of scale, n the number of leaves in each value and N the total number of leaves assessed. The assessments of disease development were carried out for one or two days before each treatment. For ANOVA statistical analysis, data in percentages were transformed to arcsine O%. Media were compared according to the Tukey test.
2.1.2. Large scale trials in a commercial banana plantation (Musa acuminata AAA), using aircraft application A trial was carried out in a Grand Nain commercial field of the farm ‘Amistad Cuba–Pa!ıses No! rdicos’, in the locality of Gu. ira de Melena, Havana province, using aerial treatments following biological warnings according to the assessments of the development stage of the disease (Four!e, 1988; P!erez, 1996). The M. fijiensis population had an ED50 to propiconazole of 0.008 mg/ml (very sensitive; class 1 according to the scale of Chin et al., 1996). Three plots of 5.4 ha each were used: plot 1 was treated with trifloxystrobin, while plot 2 was treated with azoxystrobin and plot 3 was the standard (according to the schedule applied in the farm). In two of the fields, four treatments were applied with trifloxystrobin (plot 1) and azoxystrobin (plot 2), at the rates of 75 and 100 g ai/ha respectively, in substitution of the benomyl (150 g ai/ha) and propiconazole (100 g ai/ha) treatments that were carried out in the standard plot (plot 3) on the same day. The rest of the treatments of plots 1 and 2 were applied using the same rotation of the fungicides scheduled by the farm and used in the standard plot. Propiconazole was applied at the rate of 100 g ai/ha, tridemorph at 450 g ai/ha, and benomyl at 150 g ai/ha. All the fungicide treatments were applied as oil in water emulsions, using 7 l of mineral oil, an emulsifier (0.1% of oil volume) and water until 20 l of final spray liquid/ha. The timing of the treatments and the fungicides applied in the three plots are shown in Fig. 2. The treatments were carried out very early in the morning using an AN-2M airplane with a boom fitted with Tee-jet 65–10 nozzles. The stage of evolution of the disease (Four!e, 1988) and the youngest leaf with necrosis (YLS) were assessed weekly in 20 plants in each plot. In week 39, during the most favorable period for the disease to develop, ten leaves in phase A (10 cm long, recent emerged cigar leaf) according to the description of leaf development of Brun (1958), were marked in each treated plot, in ten mature, non-flowered plants that received good spray coverage with the treatments. Ten leaves were marked in the same way in an untreated neighboring plot. In that week, plots 1 and 2 were treated with trifloxystrobin and azoxystrobin respectively, and the standard plot (plot 3), was treated with benomyl, all at the rates already described. The leaves were observed every other day to determine the date of the appearance of the different stages of disease symptoms according to the description of Four!e (1982). The duration in days of the maximal incubation (from leaf emergence at stage A, to the appearance of the first signs of symptoms) and of the transition period (from streaks at stage 1 to necrotic spots at stage 6) was
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
determined by the difference of dates. The obtained data were averaged. 2.2. Evaluation of wild type M. fijiensis isolations for sensitivity to azoxystrobin Samples of leaves from banana and plantain plantations of different localities of Havana and Ciego de Avila provinces were collected. The samples were taken from the 8th to the 10th open leaves with black sigatoka spots at stages 5 and 6 of disease development counting down from the top (youngest unfurled leaf) on ten adult not flowered plants in each place. The fragments of leaves were incubated for 48 h in plastic bags with wetted filter paper inside. After incubation, the fragments were stapled to 8 cm filter paper discs, submerged in distilled water during 5 min and placed on the lids of petri plates. The lids were placed over the petri dishes bases that contained water agar (16 g/l of agar from BIOCEN Laboratories, Bejucal, Cuba), amended with different concentrations (0, 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1 and 5 mg/ml) of azoxystrobin (Amistar 250 G, Zeneca). Ascospores were allowed to discharge for 45 min on the agar surface. The plates were then incubated for 48 h at 25–271C. After incubation, the number of ascospores, which had developed a normal germinative tube was determined by observing 100 ascospores from four replicate plates per concentration. The data from this assessment were used to calculate the percentage of inhibition for each treatment relative to the untreated control. These data were then transformed to probit units (Finney, 1971) and concentrations to log of concentrations. The ED50 for each sampled population was then calculated using the software Probit (developed at the Instituto de Investigaciones de Sanidad Vegetal, INISAV).
3. Results and discussion 3.1. Field trials 3.1.1. Small plot trials on banana (Musa acuminata AAA) and plantain (Musa spp. AAB) using knapsack application 3.1.1.1. Small plot trial on plantain cultivar CEMSA 3/4. The results of the evaluations of the effectiveness of the treatments against black Sigatoka in the trial in small plots of the plantain cultivar CEMSA 3/4 with M. fijiensis populations, are shown in Table 1. Differences in the development of the disease between the treated plots and the untreated control plot, started to be observed after the second treatment. After the third treatment, differences started to be defined among the plots treated with propiconazole and those
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treated with azoxystrobin. The best reduction of the diseased leaf area was obtained with propiconazole. In the plot treated with azoxystrobin, no further increment of the infection index was observed until the end of the trial. No differences were observed in relation to YLS between propiconazole and azoxystrobin treatments. In the last evaluation, a small non-significative difference was observed in favor of propiconazole. 3.1.1.2. Small plot trials on banana cultivar Grand Nain (AAA). The results of the assessments of black Sigatoka development in terms of infection index (%) and YLS at the time of major disease development in the trial in small plots with the cultivar Grand Nain (AAA), are shown in Fig. 1. All treatments significantly reduced disease development. Azoxystrobin at 100 g ai/ha did not show differences with propiconazole on the control of the disease. The treatment with trifloxystrobin at 75 g ai/ha showed two units more of YLS than propiconazole and azoxystrobin which is an important difference in terms of black Sigatoka control. Trifloxystrobin at the rate of 100 g ai/ha allowed the lowest development of the disease without significative differences with the rate of 75 g ai/ha. 3.1.2. Large scale trials in a commercial banana plantation (Musa acuminata AAA), using aircraft application The dates of the applications and the results of the weekly assessments of the stage of disease development (SE4L) are shown in Fig. 2. The stage of evolution or the speed at which the disease develops are primarily used to establish the date of the fungicide applications. Four applications of trifloxystrobin at 75 g ai/ha (plot 1) and azoxystrobin at 100 g ai/ha (plot 2) in comparison with the standard treatments in use in the commercial fields (plot 3) were assessed. The four treatments with trifloxystrobin and azoxystrobin were in weeks 25, 35, 37 and 39. The standard plot was treated at the same dates with propiconazole, tridemorph and benomyl respectively. As shown in Fig. 2, the curves of disease development of the standard plot (plot 3) and of the trifloxystrobin plot (plot 2) are practically identical. Treatments with propiconazol and tridemorph in all the plots in weeks 25 and 30 respectively (Fig. 2) were not very effective and allowed increased development of the disease in the following weeks. These treatments had to be repeated two weeks later, as the disease had not been adequately controlled. During this period intense rains were very favorable to the development of black Sigatoka, and reduced the time for the fungicide to penetrate inside leaves. The YLS values are inversely correlated with the severity of the disease (diseased leaf area; Stover and
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Table 1 Infection index (%) and youngest leaf with necrosis in the plots of the cultivar CEMSA 3/4 (AAB), in 1997, with M. fijiensis populations sensitive to DMIs Assessments Variants
Rate (g ai/ha)
Infection index (%)a b
1 Untreated control Azoxystrobin Propiconazole VC. (%)
F 100 100
c
26.1a 29.6a 33.1a 7.8
YLS
2
3
4
1
2
3
4
16.6a 13.6a 12.3a 7.2
18.2a 12.1b 10.5b 6.8
21.4a 11.4b 5.5c 6.4
6.0a 5.8a 5.6a 5.2
6.1a 6.6a 6.4a 5.2
6.1a 6.6a 6.4a 5.6
5.2a 8.0b 8.4b 5.4
For Anova, the percentages were transformed to arcsine O%. The observations were made every 14 days before the following treatment. c Different letters mean significative differences according to the Tukey test at 5% probability level. a
b
Fig. 1. Infection index and youngest leaf with necrotic spots (YLS) in the different treatments at the moment of major disease development. Grand Nain, Gu. ira de Melena 1998. M. fijiensis population with a reduced sensitivity to DMIs. (Different letters mean significative differences according to the Tukey test at 5% probability level.)
Fig. 2. Efficacy of trifloxystrobin 75 g ai/ha and azoxystrobin 100 g ai/ha on the development of black Sigatoka in banana (Grand Nain, AAA). State of evolution. M. fijiensis populations sensitive to DMIs. Farm Cuba–Pa!ıses No! rdicos, 1999. (Az=azoxystrobin; Tf=trifloxystrobin; Be=benomyl; Tri=tridemorph; P=propiconazole)
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
Dickson, 1971). In the whole course of the trial, there were more than ten functional healthy leaves in the trifloxystrobin and the standard plots (Fig. 3). The trifloxystrobin treated plots and the standard plot showed a similar number of healthy leaves. Starting from week 30, the attack of black Sigatoka began to increase in the plot treated with azoxystrobin. From week 30 to the end of the trial, the plot treated with azoxystrobin had more severe disease (Fig. 2) and affected leaves (Fig. 3), than those of the trifloxystrobin and standard plots. In week 42, this plot showed two to three fewer healthy leaves than the standard and trifloxystrobin plots (YLS, Fig. 3). The average maximal incubation period of the disease (from leaf emergence to the observation of the first stage of the symptoms) in the leaves of marked plants in the
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different plots during treatment in week 39, is shown in Fig. 4. All treatments affected the length of the incubation and transition periods in comparison with the untreated plants. The effect on incubation and transition periods was stronger with benomyl and trifloxystrobin, followed by the treatments with azoxystrobin.
3.1.3. Summary of field trial results Trifloxystrobin and azoxystrobin were effective in the control of black Sigatoka in the trials in small banana and plantain plots treated with mistblower knapsack sprayers and in commercial banana fields aerially treated. Azoxystrobin showed a similar effectiveness to propiconazole against M. fijiensis populations sensitive to this active ingredient. This is in agreement with
Fig. 3. Efficacy of trifloxystrobin 75 g ai/ha and azoxystrobin 100 g ai/ha in the severity of black Sigatoka in banana according to the values of YLS (Grand Nain, AAA). M. fijiensis populations sensitive to DMIs. Farm Cuba–Pa!ıses No! rdicos, 1999.
Fig. 4. Effect of treatments on incubation and transition periods (in days).
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
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Fig. 5. Sensitivity of five wild type M. fijiensis populations to azoxystrobin. Inhibition of ascospore germination at different azoxystrobin concentrations.
Table 2 Sensitivity of five populations of M. fijiensis to azoxystrobin
3.2. Sensitivity of five wild type M. fijiensis populations to azoxystrobin
Populations
Province
ED50 (mg/ml)
ED95 (mg/ml)
R2(5%)
Gu. ira 98.1 B. Esperanza Gu. ines La Cuba 15 La Cuba 17
Havana Havana Havana Ciego de Avila Ciego de Avila
0.12 0.08 0.06 0.2 0.005
1.2 1.2 1.0 1.8 0.12
0.94 0.92 0.96 0.92 0.96
previous results (Guzm!an and Romero, 1997; Figueroa, 1998). There were no differences observed between the treatments with trifloxystrobin at the rates of 75 and 100 g ai/ha. Both trifloxystrobin rates were superior to azoxystrobin at 100 g ai/ha. This was put on evidence not only in the trials in small plots treated from the ground, but also in the trials with aerial treatments as it was reflected on the speed of disease evolution and YLS. Under heavy inoculum pressure of DMI’s sensitive M. fijiensis populations, azoxystrobin showed a lower efficacy than propiconazole on black Sigatoka control. Against populations with a reduced sensitivity to DMIs (ED50=0.17 mg/ml, class 7 according to the scale of Chin et al., 1996) the azoxystrobin treatment showed a better performance than propiconazole. Azoxystrobin and trifloxystrobin fulfill the requirements of the treatments by biological warnings causing immediate regression of the stage of evolution after the applications. Both active ingredients are new alternatives in the strategy to reduce the pressure of selection of M. fijiensis populations resistant to DMIs and benzimidazole fungicides.
The ED50 and ED95 to azoxystrobin of M. fijiensis populations never previously treated with strobilurin fungicides are shown in Table 2. The values of ED50 were in the range from 0.12 to 0.2 mg/ml and the ED95 values are very close to 1 mg/ml. Differences were observed in sensitivity response between populations that were most marked at low concentrations of azoxystrobin (Fig. 5). When the responses of ascospore populations collected were compared at 1 and 5 mg/ml, no differences in sensitivity were apparent. The results suggest that all M. fijiensis isolates were sensitive to azoxystrobin. If we consider the ED95 values of the isolates and the curves of responses to the different concentrations of azoxystrobin it could be recommended that monitoring should be carried out using 1 and 5 mg/ml of azoxystrobin as baseline for sensitivity. Resistance to strobilurin fungicides has been detected in a number of different fungal pathogens (K.M. Chin, personal communication) and lately in M. fijiensis populations from banana plantations in Costa Rica (Wirz and Chin, 2000). The resistance is characterized by high resistance factors and competitive resistant strains that persist in the population in the absence of selection pressure. Resistance in strobilurin and related (fomadoxone) fungicides is qualitative, controlled by a single gene (Geier et al., 1992; Wiggins and Jager, 1994). Due to this, it is mandatory to keep a constant surveillance of the changes in sensitivity in M. fijiensis populations. In the future, concentrations of 1 and 5 mg/ ml of azoxystrobin can be used for monitoring the shift in the sensitivity of M. fijiensis populations. However,
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this recommendation should be used in an advisory sense and a higher number of isolates should be tested to determine a baseline for sensitivity.
4. Conclusions 1. Trifloxystrobin at 75 g ai/ha and azoxystrobin at 100 g ai/ha are new alternatives for the management of DMI’s sensitive and resistant M. fijiensis populations in banana and plantain. They fulfill the requirements of the treatments following biological warnings. 2. Trifloxystrobin at the used rates in all trials carried out showed activity against M. fijiensis superior to azoxystrobin. It showed a similar efficacy to propiconazole in the presence of a population sensitive to DMIs. The rate of 75 g ai/ha did not show a significant difference from the rate of 100 g ai/ha. 3. The treatment with trifloxystrobin at 75 g ai/ha increased the incubation and transition periods when compared with untreated plants. 4. There was a variation of responses at low azoxystrobin concentrations among different M. fijiensis populations but they were stable at 1 and 5 mg/ml. These results suggest that all M. fijiensis isolates tested were sensitive to the azoxystrobin. 5. Results suggest that rates of 1 and 5 mg/ml can be used to monitor changes of sensitivity in populations being treated with azoxystrobin. It is recommended to test a larger sample of populations to provide an accurate baseline of M. fijiensis sensitivity to azoxystrobin.
References Brun, J., 1958. Etude sur laction des fongicides huileux dans la lutte contre la Cercosporiose. Fruits 13, 13–17. Chin, K., Arroyo, T., Forster, B., Steden, C., 1996. Sensitivity of Mycosphaerella fijiensis to demethylation-inhibitors in Central America: testing methodology and cross resistance. Proceedings of the XII ACORBAT Meeting, October 28–November 1. Dominican Republic, pp. 1–5. Figueroa, L., 1998. Bankit 25 SC (azoxystrobin) and Anvil 25 SC (hexaconazole)+Turbocharger as penetrant in the control of black Sigatoka (Mycosphaerella fijiensis) on bananas. Proceedings of the XIII ACORBAT Meeting, November 23–29. Ecuador, pp. 400–401. Finney, D.J., 1971. Probit Analysis, 3rd Edition. Cambridge University Press, Cambridge, 333 pp. Four!e, E., 1982. Les Cercosporiose du bananier et leur traitement. Comportement des vari!et!es. Etude de la sensibilit!e vari!etale des bananiers et plantains a Mycosphaerella fijiensis Morelet au Gabon (maladie de raies noires). I. Incubation et e! volution de la maladie. II. Etude de quelques param"etres. Fruits 37, 749–754. Four!e, E., 1988. Strat!egies de lutte contre la Cercospora noire des bananiers et des plantains provoqu!es par Mycosphaerella fijiensis
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Morelet. L’avertissement biologique au Cameroun. Evaluation des possibilit!es dam!elioration. Fruits 43, 269–274. Geier, B.M., Sch.agger, H., Brandt, U., Colson, A.M., Von Jagow, G., 1992. Point mutation in cytochrome b of yeast ubihydroquinone: cytochrome c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit. Eur J Biochem 208, 375–380. Godwin, J.R., Anthony, J.M., Clough, J.M., Godfrey, C.R.A., 1992. ICI 5504: a novel, broad spectrum systemic (beta-methoxyacrilate fungicide). Proceedings of the Brighton Crop Protection Conference on Pest and Diseases, Vol. 1, pp. 435–442. Guzm!an, M.Y, Romero, R., 1997. Comparacio! n de los fungicidas azoxystrobin, propiconazole y difenoconazole en el control de la Sigatoka negra (Mycosphaerella fijiensis Morelet) en banano(Musa AAA). Corbana 22, 49–59. P!erez, L., 1978. Control de Mycosphaerella musicola Leach (Sigatoka) con aceite y mezclas de fungicidas en aceite. Agrotecnia de Cuba 10, 83–94. P!erez, L., 1996. Manual para el manejo integrado de Sigatoka negra (Mycosphaerella fijiensis Morelet) y Sigatoka amarilla (Mycosphaerella musicola Leach ex Mulder) en banano y pl!atano. Proyecto FAOFMinisterio de Agricultura. TCP/CUB/4454. La Habana, p. 54. P!erez, L., 1997. Black Sigatoka disease control in banana and plantains plantations in Cuba. Management of the disease based on an integrated approach. INFOMUSA 7, 27–30. P!erez, L., Batlle, A., 1993. Monitoring procedure to determine propiconazole resistance in Mycosphaerella fijiensis Morelet. Proceedings of the 6th International Congress of Plant Pathology, Montreal, p. 90. P!erez, L., Batlle, A., Hern!andez, A., Trujillo, R., Alvarez, C., M!endez, A., 2000. Evolution of Mycosphaerella fijiensis Morelet populations sensitivity to fungicides in banana in Cuba. Abstracts of Papers presented at Acorbat 2000, San Juan, Puerto Rico, July 31– August 4. P!erez, L., Mauri, F., Barranco, B., Garc!ıa G., 1993. Efficacy of EBI’s fungicides in the control of Mycosphaerella fijiensis Morelet on banana and plantains with treatments based on stage of evolution of the disease (biological warnings) in Cuba. Proceedings of the International Congress of Plant Pathology, Montreal, p. 55. Margot, P., Huggenberger, F., Amrein, J., Weiss, B., 1998. CGA 279202, a new broad-spectrum strobilurin fungicide. Proceedings of Brighton Crop Protection Conference on Pests a Diseases, UK, November 16–19, Vol. 2, pp. 375–382. Smith, C.M., Johnson, L.E.B., Shillingford, C.A., 1991. In vitro methods for monitoring flusilazole sensitivity of Mycosphaerella fijiensis (Black Sigatoka of bananas). In: FRAC methods for monitoring fungicide resistance. Fungicide Resistance Action Committe of GIFAP. EPPO Bulletin 21, 291–354. Stover, R.H., 1971. A proposed international scale for evaluating intensity of banana leaf spot. Trop Agric Trin 48, 185–196. Stover, R.H., Dickson, J.D., 1970. Leaf spot of banana caused by Mycosphaerella musicola Leach. Methods of measuring spotting prevalence and severity. Trop Agric Trin 47, 289–302. Unterstenhoefer, G., 1963. Las bases para los ensayos fitosanitarios de campo. Pflanzentchuz - Nachrichten Bayer 16, 89–176. Wiggins, T.E., Jager, B.J., 1994. Mode of action of the new methoxyacrilate antifungal agent ICI 5504. Biochem. Soc. Transac. 22, 685. Wirz, M., Chin, K.M., 2000. Detection and characterization of strobilurin resistant individuals of Black Sigatoka (Mycosphaerella fijiensis Morelet) in Costa Rica. Abstracts of Papers of XIV ACORBAT Meeting, San Juan, Puerto Rico, July 31–August 4, p. 65.
Effect of trifloxystrobin and azoxystrobin on the control of black Sigatoka (Mycosphaerella fijiensis Morelet) on banana and plantain Luis Pe! rez*, Alexis Herna! ndez, La! zaro Herna! ndez, Michel Pe! rez Instituto de Investigaciones de Sanidad Vegetal (INISAV), Ministerio de Agricultura de Cuba, Gaveta 634, Playa, Havana City 11300, Cuba Received 14 June 2000; received in revised form 31 August 2000; accepted 4 April 2001
Abstract Trials to determine the efficacy of trifloxystrobin (75 and 100 g ai/ha) and azoxystrobin (100 g ai/ha), on the control of black Sigatoka disease caused by Mycosphaerella fijiensis Morelet (M. fijiensis), in small randomized plots of banana and plantain treated from the ground and in commercial banana fields treated from the air, were carried out between 1997 and 1999. Trifloxystrobin at 75 g ai/ha showed a similar efficacy to propiconazole at 100 g ai/ha and benomyl at 150 g ai/ha and was superior to azoxystrobin (100 g ai/ha). Azoxystrobin show a similar efficacy to propiconazole against M. fijiensis populations sensitive to DMIs. The ED50 (ascospore germinative test) of five wild type M. fijiensis populations of different localities to azoxystrobin were between 0.03 and 0.8 mg/ml. Results suggest that monitoring for strobilurin resistance may be conducted using 5 mg/ml as the threshold concentration. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Black Sigatoka; Mycosphaerella fijiensis; Azoxystrobin; Trifloxystrobin; Fungicides
1. Introduction Black Sigatoka disease caused by Mycosphaerella fijiensis Morelet was first reported in Cuba in 1991. To control the disease, triazole (propiconazole, tebuconazole, bitertanol), morpholine (tridemorph) and benzimidazole (benomyl) fungicides aerial treatments were used following a bio-climatic forecasting system of treatment (P!erez et al., 1993; P!erez, 1997). The monitoring of the sensitivity of M. fijiensis populations to DMIs (C14 demethylase inhibitors) and morpholines (D7–D8 isomerase and D14 reductase inhibitors) has been carried out since 1991 (P!erez and Batlle, 1993). In the last two years, an increase in the frequency of populations with a decreased sensitivity (10–100 ) to propiconazole and other DMIs has been observed (P!erez et al., 2000). Due to this, it is necessary to discover new fungicides with a good efficacy against the disease and different biochemical mechanisms of action compared to benzimidazole and DMIs that are currently in use for the control of M. fijiensis. The methoxyacrylates represent a new family of fungicides with a wide spectrum of action against *Corresponding author. E-mail address: [email protected], [email protected] (L. P!erez).
different taxonomic groups of fungi, and different degrees of systemic movement (Godwin et al., 1992). Their biochemical mechanism of action is based on the inhibition of the transport of electrons between cytochrome b and cytochrome C1 in mitochondria, therefore preventing the formation of ATP (Geier et al., 1992; Wiggins and Jager, 1994). Among these compounds are azoxystrobin (Godwin et al., 1992) and trifloxystrobin (Margot et al., 1998). In this paper, the results of the studies to determine the efficacy of trifloxystrobin and azoxystrobin on the control of M. fijiensis populations with different levels of sensitivity to DMIs are reported.
2. Materials and methods 2.1. Field trials 2.1.1. Small plots trials on banana (Musa acuminata AAA) and plantain (Musa spp. AAB) using knapsack application Two trials were carried out in plots conformed by four lines of ten plants each (192 m2), in a randomized block design with four replicates in the locality of Gu. ira
0261-2194/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 5 5 - 2
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
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de Melena. All treatments were carried out as oil in water emulsions (with 7 l of oil and water up to 60 l/ha) with a knapsack mist blower sprayer. The first trial was in 1997 in a plantain field of the cultivar CEMSA 3/4 (AAB) that has never received triazole treatments. The M. fijiensis population sensitivity to propiconazole was determined by the assessment of the ascospore germination tube growth (Smith et al., 1991; P!erez and Batlle, 1993; Chin et al., 1996). The populations of M. fijiensis showed an ED50 o0.001 mg/ ml (sensitive; class 2 of sensitivity according to Chin et al., 1996). Four applications of the following treatments: (1) azoxystrobin 100 g ai/ha, (2) propiconazole 100 g ai/ha and (3) untreated control were made at 12–14 days intervals. The second trial in 1998 was in a field of the cultivar Grand Nain (subgroup Cavendish, AAA); the M. fijiensis population had an ED50 of 0.17 mg/ml (resistant population; class 7 of sensitivity according to Chin et al., 1996). The trial includes the following treatments: (1) propiconazole 100 g ai/ha; (2) azoxystrobin 100 g ai/ha; (3) trifloxystrobin 100 g ai/ ha; (4) trifloxystrobin 75 g ai/ha and (5) untreated control. Four applications were made at intervals of 12–14 days. In both trials the disease severity was evaluated before each treatment according to: (1) The number of the youngest unfurled leaf with ten or more scattered necrotic spots (YLS) (Stover and Dickson, 1970). (2) Severity of the attack on all leaves of ten nonflowered plants/plot according to the international scale of Stover (1971) that was modified by P!erez (1978). In this scale 0=until 10 isolated spots/leaf; 1=until 5% of the leaf area necrotic; 2=from 6% to 15% of leaf area necrotic; 3=from 16% to 33% leaf area necrotic; 4=from 33% to 50% of leaf area necrotic and 5=more than 50% of the leaf area necrotic. An infection index (II) (%) was calculated using the formula of Townsend and Heuberger (Unterstenhoefer, 1963): II% ¼
X
an=6N 100;
ð1Þ
where a is the value of severity of scale, n the number of leaves in each value and N the total number of leaves assessed. The assessments of disease development were carried out for one or two days before each treatment. For ANOVA statistical analysis, data in percentages were transformed to arcsine O%. Media were compared according to the Tukey test.
2.1.2. Large scale trials in a commercial banana plantation (Musa acuminata AAA), using aircraft application A trial was carried out in a Grand Nain commercial field of the farm ‘Amistad Cuba–Pa!ıses No! rdicos’, in the locality of Gu. ira de Melena, Havana province, using aerial treatments following biological warnings according to the assessments of the development stage of the disease (Four!e, 1988; P!erez, 1996). The M. fijiensis population had an ED50 to propiconazole of 0.008 mg/ml (very sensitive; class 1 according to the scale of Chin et al., 1996). Three plots of 5.4 ha each were used: plot 1 was treated with trifloxystrobin, while plot 2 was treated with azoxystrobin and plot 3 was the standard (according to the schedule applied in the farm). In two of the fields, four treatments were applied with trifloxystrobin (plot 1) and azoxystrobin (plot 2), at the rates of 75 and 100 g ai/ha respectively, in substitution of the benomyl (150 g ai/ha) and propiconazole (100 g ai/ha) treatments that were carried out in the standard plot (plot 3) on the same day. The rest of the treatments of plots 1 and 2 were applied using the same rotation of the fungicides scheduled by the farm and used in the standard plot. Propiconazole was applied at the rate of 100 g ai/ha, tridemorph at 450 g ai/ha, and benomyl at 150 g ai/ha. All the fungicide treatments were applied as oil in water emulsions, using 7 l of mineral oil, an emulsifier (0.1% of oil volume) and water until 20 l of final spray liquid/ha. The timing of the treatments and the fungicides applied in the three plots are shown in Fig. 2. The treatments were carried out very early in the morning using an AN-2M airplane with a boom fitted with Tee-jet 65–10 nozzles. The stage of evolution of the disease (Four!e, 1988) and the youngest leaf with necrosis (YLS) were assessed weekly in 20 plants in each plot. In week 39, during the most favorable period for the disease to develop, ten leaves in phase A (10 cm long, recent emerged cigar leaf) according to the description of leaf development of Brun (1958), were marked in each treated plot, in ten mature, non-flowered plants that received good spray coverage with the treatments. Ten leaves were marked in the same way in an untreated neighboring plot. In that week, plots 1 and 2 were treated with trifloxystrobin and azoxystrobin respectively, and the standard plot (plot 3), was treated with benomyl, all at the rates already described. The leaves were observed every other day to determine the date of the appearance of the different stages of disease symptoms according to the description of Four!e (1982). The duration in days of the maximal incubation (from leaf emergence at stage A, to the appearance of the first signs of symptoms) and of the transition period (from streaks at stage 1 to necrotic spots at stage 6) was
L. Pe!rez et al. / Crop Protection 21 (2002) 17–23
determined by the difference of dates. The obtained data were averaged. 2.2. Evaluation of wild type M. fijiensis isolations for sensitivity to azoxystrobin Samples of leaves from banana and plantain plantations of different localities of Havana and Ciego de Avila provinces were collected. The samples were taken from the 8th to the 10th open leaves with black sigatoka spots at stages 5 and 6 of disease development counting down from the top (youngest unfurled leaf) on ten adult not flowered plants in each place. The fragments of leaves were incubated for 48 h in plastic bags with wetted filter paper inside. After incubation, the fragments were stapled to 8 cm filter paper discs, submerged in distilled water during 5 min and placed on the lids of petri plates. The lids were placed over the petri dishes bases that contained water agar (16 g/l of agar from BIOCEN Laboratories, Bejucal, Cuba), amended with different concentrations (0, 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1 and 5 mg/ml) of azoxystrobin (Amistar 250 G, Zeneca). Ascospores were allowed to discharge for 45 min on the agar surface. The plates were then incubated for 48 h at 25–271C. After incubation, the number of ascospores, which had developed a normal germinative tube was determined by observing 100 ascospores from four replicate plates per concentration. The data from this assessment were used to calculate the percentage of inhibition for each treatment relative to the untreated control. These data were then transformed to probit units (Finney, 1971) and concentrations to log of concentrations. The ED50 for each sampled population was then calculated using the software Probit (developed at the Instituto de Investigaciones de Sanidad Vegetal, INISAV).
3. Results and discussion 3.1. Field trials 3.1.1. Small plot trials on banana (Musa acuminata AAA) and plantain (Musa spp. AAB) using knapsack application 3.1.1.1. Small plot trial on plantain cultivar CEMSA 3/4. The results of the evaluations of the effectiveness of the treatments against black Sigatoka in the trial in small plots of the plantain cultivar CEMSA 3/4 with M. fijiensis populations, are shown in Table 1. Differences in the development of the disease between the treated plots and the untreated control plot, started to be observed after the second treatment. After the third treatment, differences started to be defined among the plots treated with propiconazole and those
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treated with azoxystrobin. The best reduction of the diseased leaf area was obtained with propiconazole. In the plot treated with azoxystrobin, no further increment of the infection index was observed until the end of the trial. No differences were observed in relation to YLS between propiconazole and azoxystrobin treatments. In the last evaluation, a small non-significative difference was observed in favor of propiconazole. 3.1.1.2. Small plot trials on banana cultivar Grand Nain (AAA). The results of the assessments of black Sigatoka development in terms of infection index (%) and YLS at the time of major disease development in the trial in small plots with the cultivar Grand Nain (AAA), are shown in Fig. 1. All treatments significantly reduced disease development. Azoxystrobin at 100 g ai/ha did not show differences with propiconazole on the control of the disease. The treatment with trifloxystrobin at 75 g ai/ha showed two units more of YLS than propiconazole and azoxystrobin which is an important difference in terms of black Sigatoka control. Trifloxystrobin at the rate of 100 g ai/ha allowed the lowest development of the disease without significative differences with the rate of 75 g ai/ha. 3.1.2. Large scale trials in a commercial banana plantation (Musa acuminata AAA), using aircraft application The dates of the applications and the results of the weekly assessments of the stage of disease development (SE4L) are shown in Fig. 2. The stage of evolution or the speed at which the disease develops are primarily used to establish the date of the fungicide applications. Four applications of trifloxystrobin at 75 g ai/ha (plot 1) and azoxystrobin at 100 g ai/ha (plot 2) in comparison with the standard treatments in use in the commercial fields (plot 3) were assessed. The four treatments with trifloxystrobin and azoxystrobin were in weeks 25, 35, 37 and 39. The standard plot was treated at the same dates with propiconazole, tridemorph and benomyl respectively. As shown in Fig. 2, the curves of disease development of the standard plot (plot 3) and of the trifloxystrobin plot (plot 2) are practically identical. Treatments with propiconazol and tridemorph in all the plots in weeks 25 and 30 respectively (Fig. 2) were not very effective and allowed increased development of the disease in the following weeks. These treatments had to be repeated two weeks later, as the disease had not been adequately controlled. During this period intense rains were very favorable to the development of black Sigatoka, and reduced the time for the fungicide to penetrate inside leaves. The YLS values are inversely correlated with the severity of the disease (diseased leaf area; Stover and
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Table 1 Infection index (%) and youngest leaf with necrosis in the plots of the cultivar CEMSA 3/4 (AAB), in 1997, with M. fijiensis populations sensitive to DMIs Assessments Variants
Rate (g ai/ha)
Infection index (%)a b
1 Untreated control Azoxystrobin Propiconazole VC. (%)
F 100 100
c
26.1a 29.6a 33.1a 7.8
YLS
2
3
4
1
2
3
4
16.6a 13.6a 12.3a 7.2
18.2a 12.1b 10.5b 6.8
21.4a 11.4b 5.5c 6.4
6.0a 5.8a 5.6a 5.2
6.1a 6.6a 6.4a 5.2
6.1a 6.6a 6.4a 5.6
5.2a 8.0b 8.4b 5.4
For Anova, the percentages were transformed to arcsine O%. The observations were made every 14 days before the following treatment. c Different letters mean significative differences according to the Tukey test at 5% probability level. a
b
Fig. 1. Infection index and youngest leaf with necrotic spots (YLS) in the different treatments at the moment of major disease development. Grand Nain, Gu. ira de Melena 1998. M. fijiensis population with a reduced sensitivity to DMIs. (Different letters mean significative differences according to the Tukey test at 5% probability level.)
Fig. 2. Efficacy of trifloxystrobin 75 g ai/ha and azoxystrobin 100 g ai/ha on the development of black Sigatoka in banana (Grand Nain, AAA). State of evolution. M. fijiensis populations sensitive to DMIs. Farm Cuba–Pa!ıses No! rdicos, 1999. (Az=azoxystrobin; Tf=trifloxystrobin; Be=benomyl; Tri=tridemorph; P=propiconazole)
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Dickson, 1971). In the whole course of the trial, there were more than ten functional healthy leaves in the trifloxystrobin and the standard plots (Fig. 3). The trifloxystrobin treated plots and the standard plot showed a similar number of healthy leaves. Starting from week 30, the attack of black Sigatoka began to increase in the plot treated with azoxystrobin. From week 30 to the end of the trial, the plot treated with azoxystrobin had more severe disease (Fig. 2) and affected leaves (Fig. 3), than those of the trifloxystrobin and standard plots. In week 42, this plot showed two to three fewer healthy leaves than the standard and trifloxystrobin plots (YLS, Fig. 3). The average maximal incubation period of the disease (from leaf emergence to the observation of the first stage of the symptoms) in the leaves of marked plants in the
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different plots during treatment in week 39, is shown in Fig. 4. All treatments affected the length of the incubation and transition periods in comparison with the untreated plants. The effect on incubation and transition periods was stronger with benomyl and trifloxystrobin, followed by the treatments with azoxystrobin.
3.1.3. Summary of field trial results Trifloxystrobin and azoxystrobin were effective in the control of black Sigatoka in the trials in small banana and plantain plots treated with mistblower knapsack sprayers and in commercial banana fields aerially treated. Azoxystrobin showed a similar effectiveness to propiconazole against M. fijiensis populations sensitive to this active ingredient. This is in agreement with
Fig. 3. Efficacy of trifloxystrobin 75 g ai/ha and azoxystrobin 100 g ai/ha in the severity of black Sigatoka in banana according to the values of YLS (Grand Nain, AAA). M. fijiensis populations sensitive to DMIs. Farm Cuba–Pa!ıses No! rdicos, 1999.
Fig. 4. Effect of treatments on incubation and transition periods (in days).
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Fig. 5. Sensitivity of five wild type M. fijiensis populations to azoxystrobin. Inhibition of ascospore germination at different azoxystrobin concentrations.
Table 2 Sensitivity of five populations of M. fijiensis to azoxystrobin
3.2. Sensitivity of five wild type M. fijiensis populations to azoxystrobin
Populations
Province
ED50 (mg/ml)
ED95 (mg/ml)
R2(5%)
Gu. ira 98.1 B. Esperanza Gu. ines La Cuba 15 La Cuba 17
Havana Havana Havana Ciego de Avila Ciego de Avila
0.12 0.08 0.06 0.2 0.005
1.2 1.2 1.0 1.8 0.12
0.94 0.92 0.96 0.92 0.96
previous results (Guzm!an and Romero, 1997; Figueroa, 1998). There were no differences observed between the treatments with trifloxystrobin at the rates of 75 and 100 g ai/ha. Both trifloxystrobin rates were superior to azoxystrobin at 100 g ai/ha. This was put on evidence not only in the trials in small plots treated from the ground, but also in the trials with aerial treatments as it was reflected on the speed of disease evolution and YLS. Under heavy inoculum pressure of DMI’s sensitive M. fijiensis populations, azoxystrobin showed a lower efficacy than propiconazole on black Sigatoka control. Against populations with a reduced sensitivity to DMIs (ED50=0.17 mg/ml, class 7 according to the scale of Chin et al., 1996) the azoxystrobin treatment showed a better performance than propiconazole. Azoxystrobin and trifloxystrobin fulfill the requirements of the treatments by biological warnings causing immediate regression of the stage of evolution after the applications. Both active ingredients are new alternatives in the strategy to reduce the pressure of selection of M. fijiensis populations resistant to DMIs and benzimidazole fungicides.
The ED50 and ED95 to azoxystrobin of M. fijiensis populations never previously treated with strobilurin fungicides are shown in Table 2. The values of ED50 were in the range from 0.12 to 0.2 mg/ml and the ED95 values are very close to 1 mg/ml. Differences were observed in sensitivity response between populations that were most marked at low concentrations of azoxystrobin (Fig. 5). When the responses of ascospore populations collected were compared at 1 and 5 mg/ml, no differences in sensitivity were apparent. The results suggest that all M. fijiensis isolates were sensitive to azoxystrobin. If we consider the ED95 values of the isolates and the curves of responses to the different concentrations of azoxystrobin it could be recommended that monitoring should be carried out using 1 and 5 mg/ml of azoxystrobin as baseline for sensitivity. Resistance to strobilurin fungicides has been detected in a number of different fungal pathogens (K.M. Chin, personal communication) and lately in M. fijiensis populations from banana plantations in Costa Rica (Wirz and Chin, 2000). The resistance is characterized by high resistance factors and competitive resistant strains that persist in the population in the absence of selection pressure. Resistance in strobilurin and related (fomadoxone) fungicides is qualitative, controlled by a single gene (Geier et al., 1992; Wiggins and Jager, 1994). Due to this, it is mandatory to keep a constant surveillance of the changes in sensitivity in M. fijiensis populations. In the future, concentrations of 1 and 5 mg/ ml of azoxystrobin can be used for monitoring the shift in the sensitivity of M. fijiensis populations. However,
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this recommendation should be used in an advisory sense and a higher number of isolates should be tested to determine a baseline for sensitivity.
4. Conclusions 1. Trifloxystrobin at 75 g ai/ha and azoxystrobin at 100 g ai/ha are new alternatives for the management of DMI’s sensitive and resistant M. fijiensis populations in banana and plantain. They fulfill the requirements of the treatments following biological warnings. 2. Trifloxystrobin at the used rates in all trials carried out showed activity against M. fijiensis superior to azoxystrobin. It showed a similar efficacy to propiconazole in the presence of a population sensitive to DMIs. The rate of 75 g ai/ha did not show a significant difference from the rate of 100 g ai/ha. 3. The treatment with trifloxystrobin at 75 g ai/ha increased the incubation and transition periods when compared with untreated plants. 4. There was a variation of responses at low azoxystrobin concentrations among different M. fijiensis populations but they were stable at 1 and 5 mg/ml. These results suggest that all M. fijiensis isolates tested were sensitive to the azoxystrobin. 5. Results suggest that rates of 1 and 5 mg/ml can be used to monitor changes of sensitivity in populations being treated with azoxystrobin. It is recommended to test a larger sample of populations to provide an accurate baseline of M. fijiensis sensitivity to azoxystrobin.
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