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Bacillus subtilis to biological control of postbloom fruit drop caused by Colletotrichum acutatum under field conditions
Scientia Horticulturae 134 (2012) 139–143
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Bacillus subtilis to biological control of postbloom fruit drop caused by Colletotrichum acutatum under field conditions K.C. Kupper a,∗ , F.E. Corrêa b , F.A. de Azevedo a , A.C. da Silva a a b
Centro de Citricultura Citros Sylvio Moreira, Instituto Agronômico, Rod. Anhanguera, Km 158, 13490-970 Cordeirópolis, SP, Brazil Universidade Federal de São Carlos, Rod. Anhanguera, Km 174, Cep 13600-970, Araras, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 May 2011 Received in revised form 9 November 2011 Accepted 17 November 2011 Keywords: Blossom blight Citrus sinensis Flowers
a b s t r a c t The objective of this research was to study the viability of Bacillus subtilis (ACB-69) to control the casual agent in postbloom fruit drop, Colletotrichum acutatum under field conditions. During the 2007/2008 crop season, B. subtilis was tested in 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations on ‘Pera’ sweet orange (Citrus sinensis (L.) Osbeck) plants grafted on Rangpur lime (Citrus limonia Osb.), in Botucatu, São Paulo, Brazil. The same treatments were repeated in the 2008/2009 crop season with and without adding a carbon source (molasses 5%) to ‘Valência’ sweet orange plants grafted on Rangpur lime. Additional experiment was conducted to determine the most appropriate flower growth stage to apply the biocontrol agent. The biological products were applied with an air assisted sprayer in all experiments with the intention of evaluating the practicality of this type of sprayer. A weekly application of B. subtilis (5 × 108 cfu ml−1 ) reduced the quantity of symptomatic flowers by 47% while the chemical treatment only had an 18% reduction. On the other hand, B. subtilis (10% or 1 × 109 cfu ml−1 ) applied weekly or applied one week before to the “green bud bloom” stage (like a preventive control) provided a greater average number of effective fruits (ANEF). The addition of a carbon source at the time of application, did not favor the antagonistic activity of bacteria. In controlling postbloom fruit drop, the best time to apply B. subtilis was observed in the open flower stage when the percentage of symptomatic flowers was evaluated. However, when the bacterium was applied in all flowering stages there was a greater average number of effective fruits. The use of air assisted sprayer helped implement the antagonistic on a commercial scale. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In terms of cultivated area, production and quantity of processed fruits, Brazil is among the largest citrus producers in the world. About 85% of this production goes directly to industrialization in which juice is exported to several countries. These countries mainly include the United States, Japan, Switzerland, China and Belgium (Agricultural, 2009). Though the citrus sector is greatly important, this crop faces serious phytosanitary problems and the postbloom fruit drop (PFD) caused by Colletotrichum acutatum Simmonds is one of the most severe citrus diseases. In Brazil, PFD was first reported in the state of Rio Grande do Sul (Dornelles, 1977) and is now present throughout all cities in São Paulo as well as in other states such as Rio de Janeiro, Paraná, Bahia, Minas Gerais, Goias and Amazonas, causing production losses greater than 80% (de Goes and Kupper, 2002).
∗ Corresponding author. Tel.: +55 19 3546 1399; fax: +55 19 3546 1399. E-mail address: [email protected] (K.C. Kupper). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.11.019
Fungicide applications (benzimidazoles) during the entire bloom period are the predominant control measures used in PFD. However, due to the financial and environmental costs of this type of treatment, along with the increasing restrictions to the presence of residue, additional studies of new alternatives are needed. One important alternative has proven to be through biological control; apart from its ecological coherence, in many cases it possesses an appreciative and sustainable appeal. Bacillus subtilis is among the most studied antagonistic agents used to control phytopathogens which have been predominant in controlling phylloplane and post-harvest diseases (Pusey et al., 1986; Ferreira et al., 1991; Bettiol et al., 1994; Sonoda and Guo, 1996; Kalita et al., 1996; Kupper and Gimenes-Fernandes, 2002; Kupper et al., 2003, 2004, 2005, 2009). Kupper and Gimenes-Fernandes (2002) studied in vitro the antagonistic potential of 64 B. subtilis isolates against C. acutatum and in detached ‘Tahiti’ acid lime flowers in Brazil. According to the authors, all Bacillus isolates produced metabolites capable of inhibiting the mycelial growth of the phytopathogen. This was also true in detached flowers which in some cases exhibited a 100% disease control.
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K.C. Kupper et al. / Scientia Horticulturae 134 (2012) 139–143
According to Kupper et al. (2003), out of the seven B. subtilis isolates tested in controlling PFD under field conditions, the only isolate which differed from the control (non-sprayed plants) and statistically equated to the benomyl fungicide was the ACB-69 isolate, which caused a lower percentage of symptomatic flowers and a greater fruit retention. The objectives of this study were: (i) to test the feasibility of B. subtilis (ACB-69) under field conditions in two different concentrations; (ii) to test the effects of adding a carbon source during the application of the biocontrol agent; (iii) to verify the feasibility of applying the bacteria with an air assisted sprayer on a commercial scale, and finally, (iv) to determine the most appropriate growth stages of citrus flowers to apply the biocontrol agent in order to obtain the best disease control.
2. Materials and methods For this study B. subtilis (ACB-69) was originally isolated from samples of leaves of ‘Valência’ sweet orange (Kupper and GimenesFernandes, 2002).
2.1. Effect of the B. subtilis concentration to control C. acutatum under field conditions (2007/2008 growth season) The experiment was conducted in a 11-year-old grove of ‘Pera’ sweet orange (Citrus sinensis L. Osbeck) grafted on Rangpur lime (C. limonia Osbeck) located in Botucatu, São Paulo, Brazil. To produce antagonist suspensions, B. subtilis (ACB-69) colonies were transferred to glass vessels with a 20 l capacity containing 15 l of medium composing the foliar manure base Ajifol® at 5% (v/v), autoclaved at 120 ◦ C for 30 min, at 1 atm. This medium contains a carbon, nitrogen and mineral source, is low cost and widely used in several citrus orchards, as a spray fertilizer. The medium was inoculated with a bacterial suspension and incubated at room temperature (22 ± 2 ◦ C), for 72 h under constant agitation, in the dark (Kupper and Gimenes-Fernandes, 2002). Afterwards, the inoculum containing 108 cells per ml was sent to the farm’s laboratory where it was distributed in flask with a 200 l capacity containing 50 l of same medium, multiplied under the same conditions of asepsis and incubated for the same amount of time, reaching a final concentration of 1010 cells per ml. The treatments corresponded to the ABC-69 isolate in the 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations which were applied weekly and at 15-day intervals during the blossom period. An additional treatment was conducted by applying of ACB-69 isolate at 10%; another treatment was performed with the ACB-69 isolate, also at 10%, though this time it was applied one week prior to the other treatments totaling five treatments using biological control agents. The chemical control followed the standard strategies already used by the private property along with three applications spaced 15-days apart, from green bud stage. The first application was done using mancozeb + fomaxadone (Midas® ) and the second and third was done using carbendazim (Derosal® ). The plants which correspond to the control only received water applications under the same conditions as cited in the other treatments. Overall, 12 applications of B. subtilis were given to the treatment with ACB-69 applied weekly, 13 applications to the treatment which was applied the bacteria one week before to the other treatments (prior to the “green bud bloom” stage), like a preventive control, and 6 applications to the treatments conducted at 15-day intervals (Table 1). An experimental randomized block design was used with seven treatments and four repetitions. Each experimental plot was composed of 10 plants.
Table 1 Scheme of application of Bacillus subtilis (ACB-69 isolate) at 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations. Treatments
Intervals of applications during the blossom period
Total of applications
1. ACB-69 at 05% 2. ACB-69 at 05% 3. ACB-69 at 10% 4. ACB-69 at 10% 5. ACB-69 at 10%
Weekly Every 15 days Weekly Every 15 days One week before to the other treatments (preventive control) Spaced 15-days apart, from green bud stage Weekly
12 06 12 06 13
6. Chemical 7. Control (water)
03 12
The treatments were applied using an air assisted sprayer, calibrated to provide an application volume of 3800 l/ha, using 8.0 l/plant. The working pressure used was 100 lb/in.2 , with enough motor rotation to provide 540 rpm. The evaluations were based on counting the number of diseased and healthy open flowers in a sample of four branches per plant from the three central plants in the plot. The percentage of diseased flowers followed the methodology adapted from Timmer and Zitko (1996), so that were evaluated up to 100 flowers that had recently opened or were about to open were observed on each branch. One second evaluation was performed 90 days after the first evaluation, accounting for the number of fruit set and calyces retained and/or of the yellowness caused by the disease in order to obtain the average number of effective fruits (ANEF), using the following equation: ANEF = [A/(A + B)] × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease, according to de Goes (1995). 2.2. Effect of carbon additions in the spraying of B. subtilis to control C. acutatum (2008/2009 growth season) The following experiment was conducted on a 15-year-old orchard of ‘Valência’ sweet orange grafted on Rangpur lime (Citrus limonia Osbeck) located in Botucatu, São Paulo, Brazil. The treatments corresponding to the ACB-69 isolate were applied at 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations, with and without the addition of a carbon source applied together the cell suspension; in this case, weekly applications of molasses (a viscous product of the processing of sugar cane) at 5% concentration resulting in a total of four treatments with biological control agents. The plants which correspond to the control only received water applications. Fungicide applications were applied using mancozeb + fomaxadone (Midas® ) and Derosal® , as mentioned in the previous experiment. To produce the antagonist suspensions, the treatment applications and the evaluations followed the same procedures as the previous experiment. An experimental randomized block design was used with eight treatments and four repetitions. Each experimental plot was composed of 10 plants and the evaluations were realized in a sample of four branches per plant from the two central plants in the plot. 2.3. Biological control agent applications and growth stages of flowers (2008/2009 growth season) This experiment was conducted at the Morrinhos Farm in Botucatu, São Paulo, Brazil on a 15-year-old grove of ‘Valência’ sweet orange grafted on Rangpur lime. The trials conducted to test the efficiency of applying the biocontrol agent (ACB-69) (1 × 109 cfu ml−1 ) during different flower growth stages were conducted by spraying the agent at the four
K.C. Kupper et al. / Scientia Horticulturae 134 (2012) 139–143 Table 2 The effect of biological control agents in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Pera’ orange plants under field conditions in Botucatu, São Paulo State, Brazil, during the 2007/2008 season.
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Table 3 Effect of adding a carbon source during pulverization with Bacillus subtilis in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Valência’ orange plants, Botucatu, São Paulo State, Brazil (2008/2009 season).
Treatments
Percentage of flowers with C. acutatum symptoms
ANEFb
Treatments
Percentage of flowers with C. acutatum symptoms
ANEFb
Chemical control ACB-69 at 10% (every 15 days) ACB-69 at 10% (weekly) ACB-69 at 05% (every 15 days) ACB-69 at 10% (preventive control) ACB-69 at 05% (weekly) Control
28.69 ab 27.26 ab 22.75 ab 22.05 b 19.40 b 18.72 b 35.12 aa
51.81 ab 62.69 ab 73.57 a 64.73 ab 76.72 a 57.24 ab 46.50 b
ACB-69 at 10% ACB-69 (10%) + molasses (5%) Chemical control ACB-69 (5%) ACB-69 (5%) + molasses (5%) Control
42.31 ba 35.85 b 56.60 b 61.78 ab 57.75 ab 80.71 a
67.00 a 66.00 a 63.00 a 37.00 b 32.00 bc 26.00 c
a
Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). b ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease.
different flower growth stages previously described by Agustí et al. (2002): (1) closed visible flowers, (green bud), (FGB); (2) elongating flower petals; sepal covering corolla (white bud) (FWB); (3) most flowers with petals forming a hollow ball (HB); and (4) open flowers (FO). These treatments were compared to the (5) standard fungicide treatment (mancozeb + fomaxadone (Midas® ) and Derosal® ) used by the farm and to the (6) control treatment corresponding to the plants which received water applications. A total of nine treatments were tested, considering the applications: (7) in all flowering stages; and in the combinations of flower growth stages: (8) FGB + FWB and (9) FGB + FWB + HB, as described above. The inoculum production, applications and evaluations followed the same procedures as in the previous tests. All the treatments were applied from 20/09/2008 for each development stage, and the next applications were made from 7 after the first application, for each development stage. An experimental randomized block design was used with nine treatments and four repetitions. Each experimental plot was composed of 10 plants and the evaluations were realized in a sample of four branches per plant from the two central plants in the plot. 3. Results and discussion 3.1. Effect of the B. subtilis concentration to control C. acutatum under field conditions (2007/2008 growth season) The data related to how the B. subtilis concentration affects the control of C. acutatum under field conditions was evaluated according to the percentage of PFD symptomatic flowers and average number of effective fruits, as seen in Table 2. The results reveal that the disease occurred with great intensity and that the treatments affected the disease incidence. When the data referring to the percentage of symptomatic flowers was analyzed, it was verified that the only treatments that statistically differed from the control treatment (without disease control) were those which applied the bacteria at 5% concentration with either weekly or every 15 days applications or when the Bacillus (at 10%) was applied one week prior to the other treatments (prior to the “green bud bloom” stage). It was shown that when the plants were treated weekly with ACB-69 (at 5%) the quantity of symptomatic flowers was reduced by 47% while the chemical treatment showed a control efficiency of only 28.7% when compared to the control treatment data. The data presented by the average number of effective fruits showed that the best treatments were obtained when the ACB-69 isolate (at 10%) was applied one week prior to the other treatments and when the same treatment was applied weekly. These treatments did not differ from chemical control. The frequency of
a Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). b ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease.
application seems to cause more effects in the disease control and a single application before at the initial flowering stage provided good results. As for the applied methodologies evaluated, the percentage of symptomatic flowers used by Timmer and Zitko (1996), directly evaluates the effects of the disease, while the ANEF method proposed by de Goes (1995), apart from the direct effects of the disease also includes the number of fruit set. Considering the above, the results presented in this study reported that the treatment composted of an additional application favored the plant giving it protection to the pathogen attacks and consequently providing a greater number of fruit set. Kupper et al. (2003) reported that while developing the experiment using seven B. subtilis isolates and tree isolates from different Trichoderma species with cell concentrations of 1 × 107 cells per ml during the blossom period to control C. acutatum, the ACB-69 isolate differed from the control, statistically equating to the benomyl, causing a lower percentage of symptomatic flowers and a greater number of effective fruits. When this study, which was performed using a manual sprayer, is compared to the work of these authors, a greater audacity in terms of test installation is revealed, in this study was emphasized the possibility of the biocontrol agent to be applied to citrus plants on a commercial scale and with an air assisted spray. Several studies are found in literature proving the production of toxic metabolites with B. subtilis (Huang and Chang, 1975; Baker et al., 1983; Mckeen et al., 1986; Arras and Arru, 1997), which can inhibit the germination of spores or the growth of many fungi (Cubeta et al., 1985; Bettiol and Kimati, 1989, 1990; Kupper et al., 2003) which could affect the Bacillus’s own cells when in greater concentrations on the action site of the bio-fungicide broth. In this context, the higher concentration of B. subtilis in the broth did not reduce the biologic product efficiency. This was proven in the results obtained by the average number of effective fruits (Table 2), where the weekly application or the application that took place one week prior to the indicated period for the control of the disease, proved itself efficient in controlling C. acutatum. 3.2. Effect of carbon additions in the spraying of B. subtilis to control C. acutatum (2008/2009 growth season) The data presented in Table 3 shows that treatments where a sugar source was added during the application of a biological control agent did not statistically differ from the treatments where only the bacteria at a 10% concentration was applied. This was seen when evaluating the percentage of diseased flowers showing an efficiency of 48–56% in relation to the control. When the average number of effective fruits was evaluated, the addition of molasses to the Bacillus (at 10%) application also
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Table 4 Effect of timing of application of Bacillus subtilis on the development flowers, in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Valência’ orange plants, Botucatu, São Paulo State, Brazil, during 2008/2009 season. Treatments
Percentage of flowers with C. acutatum symptoms
ANEFc
Chemical control All flowering stagesd Hollow ball (HB) Green bud (FGB) FGB + FWB Open flower (FO) FGB + FWB + HB White bud (FWB) Control
10.54 ca , b 17.10 abc 20.43 ab 15.84 abc 15.06 abc 14.45 bc 15.94 abc 15.85 abc 21.73 a
86.00 ab 75.00 ab 66.75 bc 59.00 bcd 57.75 cd 51.25 cd 49.25 d 42.00 d 48.00 d
a
Data transformed to: ar sen sqrt (x + 0.5). Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). c ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease. d Flowering stages: visible flower still closed and green bud (FGB); flower petals elongating, sepal involving corolla and white bud (FWB); most flowers with petals forming a hollow bud (FHB) and Flowers open (FO). b
showed to be indifferent. With the evaluation methodology used, the best treatments were: ACB-69 applications at a 10% concentration, ACB-69 (10%) + molasses (5%) and the chemical control, with percentages of the average number of effective fruits varying from 63 to 67%, not recording statistical differences between the cited treatments. The positive results of B. subtilis reducing the percentage of symptomatic flowers and enabling a greater number of effective fruits, suggest that ACB-69 is a potential agent in the biological control of postbloom fruit drop and that the addition of a carbon source during application was completely innocuous to the antagonism of the bacteria (Table 3). 3.3. Biological control agent applications and growth stages of flowers (2008/2009 growth season) Experiments have proven that even under controlled conditions during pulverization, the desired control level is not always obtained. Among some of the possible factors that could influence the effectiveness of treatments are the applications of biological products, the time of pulverization and the respective flowering stage. In relation to the data in Table 4, it was shown that the best time to apply the B. subtilis ACB-69 isolate to control the disease was during the open flower stage, presenting approximately a 34% reduction of symptomatic flowers in relation to the without disease control treatment. This treatment did not differ from the chemical control which presented a control efficiency of approximately 52% in relation to the without disease control treatment. According to Kupper et al. (2003) antibiosis seems to be a major mechanism involved in the interaction between B. subtilis, especially the ACB-69 isolate and C. acutatum. Previous work related that ACB-69 isolate was one of the most effective in producing anti-fungal substances and in quantities sufficient to inhibit the germination of C. acutatum (Kupper et al., 2009). Consequently, the high activity of this isolate seems related principally to the preventive action of infection by C. acutatum considering to be pathogen spore germination an important stage of the disease cycle. In this context, when the disease control is evaluated using the method of diseased flower percentages (Timmer and Zitko, 1996), it is assumed that the best time to apply a bacterial application is during the open flower stage as shown in this research. Considering that the open flower stage is the stage in which most of the pathogen sporulation occurs, and since the environmental
conditions are favorable, this is also the stage where the flower is most susceptible to fungus attacks. According to Zulfiqar et al. (1996), after the initial infection of C. actutatum in citrus flowers, the petals are rapidly colonized and the first symptoms appear in less than 48 h, therefore the antagonist action of B. subtilis by antibiosis is essential in this phase in order to suppress the disease under natural conditions. The efficiency of the ACB-69 isolate in comparison to the standard fungicide treatment, once again confirmed the potential that was previously verified in vitro, in detached flowers (Kupper and Gimenes-Fernandes, 2002; Kupper et al., 2003) and under field conditions (Kupper et al., 2003). When the average number of effective fruits was evaluated, it was verified that the best results were obtained with the chemical product application and with the bacterial application during all flowering stages, having an average fruit set quantity of 86% and 75%, respectively. Several factors should be taken into consideration in an attempt to explain the necessity of applying B. subtilis during all flowering stages. First, in respect to the etiologic behavior of the pathogen, one of the survival forms of C. acutatum is by appresorias (Agostini et al., 1992; Zulfiqar et al., 1996), which germinate and produce hyphae and conidia on the surface of the leaves when in the presence of humidity and petal extracts (Timmer et al., 1994), such conditions were present during all trials; second, a long period of citrus plants blooming in the Botucatu area, exposing them to a longer period of susceptibility when compared with other regions and, third, the action mechanism used by the bacteria, the antibiosis, as previously mentioned which requires an immediate action in the presence of the phytopathogen. The long period of exposure of the flowers associated with a relatively short infection period and long periods of rain during the flowering stage, make it more difficult to plan and execute pulverizations with chemical products causing great difficulties to control the pathogen, which under favorable environmental conditions reproduce themselves intensely with consequent symptom expressions. Under these circumstances the disease manifests itself exponentially which does not permit delays in control programs even when adequate fungicides are used (de Goes et al., 2008). Considering that the bacteria was obtained in the same habitat and therefore adapted to the same conditions of the phytopathogen, the chances of establishing and exercising its antagonist activities are greater due to the above circumstances cited. On the other hand, the results obtained from this experiment diverge from the work with chemical products which are cited in literature. According to de Goes et al. (2008), the fungicides carbendazim and folpet were efficient in controlling postbloom fruit drop when they were applied in ‘green bud bloom’ and ‘hollow ball’ stages under field conditions; as for Roberto and Borges (2001), the best control was obtained with benomyl, when sweet orange plants were pulverized in the predominant ‘white round’ blossom stage until the ‘hollow ball’ stage in precocious flowering. The results obtained from this experiment also differ from others applications with B. subtilis (ACB-69) performed in previous seasons (Kupper et al., 2003, 2009). According to these authors, the best control was obtained with ACB-69, when sweet orange plants were pulverized in green bloom and hollow ball stages under field conditions. The extended bloom period in the Botucatu area was favorable for pathogen development as open flowers are more susceptible to the pathogens. Under these circumstances, and especially under elevated inoculums pressure and favorable environmental conditions, the application of biological product for flower protection during all bloom period was essential. Perhaps these reasons could explain the divergences found in this work, when compared with results from other authors.
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de Goes, A., 1995. Queda prematura de frutos cítricos: Caracterizac¸ão do agente causal, Colletotricum gloeosporioides Penz. (SENSU ARX, 1957), e controle da doenc¸a. 143f. Ph.D. Thesis. Universidade de São Paulo, Piracicaba/SP. de Goes, A., Garrido, R.B.O., Reis, R.F., Baldassari, R.B., Soares, M.A., 2008. Evaluation of fungicide applications to sweet orange at different flowering stages for control of postbloom fruit drop caused by Colletotrichum acutatum. Crop Prot. 27, 71–76. de Goes, A., Kupper, K.C., 2002. Controle das doenc¸as causadas por fungos e bactérias na cultura dos citros. In: Zambolim, L. (Ed.), Manejo Integrado: Fruteiras Tropicais-Doenc¸as e Pragas. Vic¸osa, Brazil, pp. 353–412. Huang, T., Chang, M., 1975. Studies on xanthobacidin, a new antibiotic from Bacillus subtilis active against Xanthomonas. Bot. Bull. Acad. Sin. 16, 137–148. Kalita, P., Bora, L.C., Bhagabati, K.N., 1996. Phylloplane microflora of citrus and their role in management of citrus canker. Indian Phytopathol. 49, 234–237. Kupper, K.C., Bellotte, J.A., de Goes, A., 2009. Controle alternativo de Colletotrichum acutatum, agente causal da queda prematura dos frutos cítricos. Rev. Bras. Frut. 31, 1004–1015. Kupper, K.C., Bettiol, W., Corrêa, E.B., Moretto, C., 2004. Potencialidade de Bacillus subtilis e Trichoderma spp. como agentes de biocontrole de Guignardia citricarpa. Fitopatol. Bras. 29, 129. Kupper, K.C., Bettiol, W., Corrêa, E.B., Moretto, C., de Goes, A., 2005. Controle de Guignardia citricarpa, agente causal da mancha preta dos frutos cítricos, através de Bacillus subtilis e Trichoderma spp. In: Libro de Resúmenes: XII Congreso Latinoamericano de Fitopatologia – III Taller de la Asociación Argentina de Fitopatólogos, (Abstract). Kupper, K.C., Gimenes-Fernandes, N., 2002. Isolamento e selec¸ão de Bacillus spp. para o controle de Colletotrichum acutatum em flores destacadas de lima ácida ‘Tahiti’. Summa Phytopathol. 28, 292–295. Kupper, K.C., Gimenes-Fernandes, N., de Goes, A., 2003. Controle biológico de Colletotrichum acutatum, agente causal da queda prematura dos frutos cítricos. Fitopatol. Bras. 28, 251–257. Mckeen, C.D., Reilly, C.C., Pusey, P.I., 1986. Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology 76, 136–139. Pusey, P.L., Wilson, C.L., Hotchkiss, M.W., Franklin, J.D., 1986. Compatibility of Bacillus subtilis for postharvest control of peach brown rot with comercial fruit waxes, dicloran, and cold-storage conditions. Plant Dis. 70, 587–590. Roberto, S.R., Borges, A.V., 2001. Efeito do estágio de desenvolvimento das flores e da aplicac¸ão de fungicidas no controle da podridão floral dos citros. Rev. Bras. Frut. 23, 306–309. Sonoda, R.M., Guo, Z., 1996. Effect of spray applications of Bacillus subtilis on postbloom drop of citrus. Phytopathology 86, 52. Timmer, L.W., Agostini, J.P., Zitko, S.E., Zulfiqar, M., 1994. Postbloom fruit drop, an increasingly prevalent disease of citrus in the Americas. Plant Dis. 78, 329–334. Timmer, L.W., Zitko, S.E., 1996. Evaluation of a model for prediction of postbloom fruit drop of citrus. Plant Dis. 80, 380–383. Zulfiqar, M., Brlansky, R.H., Timmer, L.W., 1996. Infection of flower and vegetative tissues of citrus by Colletotrichum acutatum and C. gloeosporioides. Mycologia 88, 121–128.
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Bacillus subtilis to biological control of postbloom fruit drop caused by Colletotrichum acutatum under field conditions K.C. Kupper a,∗ , F.E. Corrêa b , F.A. de Azevedo a , A.C. da Silva a a b
Centro de Citricultura Citros Sylvio Moreira, Instituto Agronômico, Rod. Anhanguera, Km 158, 13490-970 Cordeirópolis, SP, Brazil Universidade Federal de São Carlos, Rod. Anhanguera, Km 174, Cep 13600-970, Araras, SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 May 2011 Received in revised form 9 November 2011 Accepted 17 November 2011 Keywords: Blossom blight Citrus sinensis Flowers
a b s t r a c t The objective of this research was to study the viability of Bacillus subtilis (ACB-69) to control the casual agent in postbloom fruit drop, Colletotrichum acutatum under field conditions. During the 2007/2008 crop season, B. subtilis was tested in 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations on ‘Pera’ sweet orange (Citrus sinensis (L.) Osbeck) plants grafted on Rangpur lime (Citrus limonia Osb.), in Botucatu, São Paulo, Brazil. The same treatments were repeated in the 2008/2009 crop season with and without adding a carbon source (molasses 5%) to ‘Valência’ sweet orange plants grafted on Rangpur lime. Additional experiment was conducted to determine the most appropriate flower growth stage to apply the biocontrol agent. The biological products were applied with an air assisted sprayer in all experiments with the intention of evaluating the practicality of this type of sprayer. A weekly application of B. subtilis (5 × 108 cfu ml−1 ) reduced the quantity of symptomatic flowers by 47% while the chemical treatment only had an 18% reduction. On the other hand, B. subtilis (10% or 1 × 109 cfu ml−1 ) applied weekly or applied one week before to the “green bud bloom” stage (like a preventive control) provided a greater average number of effective fruits (ANEF). The addition of a carbon source at the time of application, did not favor the antagonistic activity of bacteria. In controlling postbloom fruit drop, the best time to apply B. subtilis was observed in the open flower stage when the percentage of symptomatic flowers was evaluated. However, when the bacterium was applied in all flowering stages there was a greater average number of effective fruits. The use of air assisted sprayer helped implement the antagonistic on a commercial scale. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In terms of cultivated area, production and quantity of processed fruits, Brazil is among the largest citrus producers in the world. About 85% of this production goes directly to industrialization in which juice is exported to several countries. These countries mainly include the United States, Japan, Switzerland, China and Belgium (Agricultural, 2009). Though the citrus sector is greatly important, this crop faces serious phytosanitary problems and the postbloom fruit drop (PFD) caused by Colletotrichum acutatum Simmonds is one of the most severe citrus diseases. In Brazil, PFD was first reported in the state of Rio Grande do Sul (Dornelles, 1977) and is now present throughout all cities in São Paulo as well as in other states such as Rio de Janeiro, Paraná, Bahia, Minas Gerais, Goias and Amazonas, causing production losses greater than 80% (de Goes and Kupper, 2002).
∗ Corresponding author. Tel.: +55 19 3546 1399; fax: +55 19 3546 1399. E-mail address: [email protected] (K.C. Kupper). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.11.019
Fungicide applications (benzimidazoles) during the entire bloom period are the predominant control measures used in PFD. However, due to the financial and environmental costs of this type of treatment, along with the increasing restrictions to the presence of residue, additional studies of new alternatives are needed. One important alternative has proven to be through biological control; apart from its ecological coherence, in many cases it possesses an appreciative and sustainable appeal. Bacillus subtilis is among the most studied antagonistic agents used to control phytopathogens which have been predominant in controlling phylloplane and post-harvest diseases (Pusey et al., 1986; Ferreira et al., 1991; Bettiol et al., 1994; Sonoda and Guo, 1996; Kalita et al., 1996; Kupper and Gimenes-Fernandes, 2002; Kupper et al., 2003, 2004, 2005, 2009). Kupper and Gimenes-Fernandes (2002) studied in vitro the antagonistic potential of 64 B. subtilis isolates against C. acutatum and in detached ‘Tahiti’ acid lime flowers in Brazil. According to the authors, all Bacillus isolates produced metabolites capable of inhibiting the mycelial growth of the phytopathogen. This was also true in detached flowers which in some cases exhibited a 100% disease control.
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According to Kupper et al. (2003), out of the seven B. subtilis isolates tested in controlling PFD under field conditions, the only isolate which differed from the control (non-sprayed plants) and statistically equated to the benomyl fungicide was the ACB-69 isolate, which caused a lower percentage of symptomatic flowers and a greater fruit retention. The objectives of this study were: (i) to test the feasibility of B. subtilis (ACB-69) under field conditions in two different concentrations; (ii) to test the effects of adding a carbon source during the application of the biocontrol agent; (iii) to verify the feasibility of applying the bacteria with an air assisted sprayer on a commercial scale, and finally, (iv) to determine the most appropriate growth stages of citrus flowers to apply the biocontrol agent in order to obtain the best disease control.
2. Materials and methods For this study B. subtilis (ACB-69) was originally isolated from samples of leaves of ‘Valência’ sweet orange (Kupper and GimenesFernandes, 2002).
2.1. Effect of the B. subtilis concentration to control C. acutatum under field conditions (2007/2008 growth season) The experiment was conducted in a 11-year-old grove of ‘Pera’ sweet orange (Citrus sinensis L. Osbeck) grafted on Rangpur lime (C. limonia Osbeck) located in Botucatu, São Paulo, Brazil. To produce antagonist suspensions, B. subtilis (ACB-69) colonies were transferred to glass vessels with a 20 l capacity containing 15 l of medium composing the foliar manure base Ajifol® at 5% (v/v), autoclaved at 120 ◦ C for 30 min, at 1 atm. This medium contains a carbon, nitrogen and mineral source, is low cost and widely used in several citrus orchards, as a spray fertilizer. The medium was inoculated with a bacterial suspension and incubated at room temperature (22 ± 2 ◦ C), for 72 h under constant agitation, in the dark (Kupper and Gimenes-Fernandes, 2002). Afterwards, the inoculum containing 108 cells per ml was sent to the farm’s laboratory where it was distributed in flask with a 200 l capacity containing 50 l of same medium, multiplied under the same conditions of asepsis and incubated for the same amount of time, reaching a final concentration of 1010 cells per ml. The treatments corresponded to the ABC-69 isolate in the 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations which were applied weekly and at 15-day intervals during the blossom period. An additional treatment was conducted by applying of ACB-69 isolate at 10%; another treatment was performed with the ACB-69 isolate, also at 10%, though this time it was applied one week prior to the other treatments totaling five treatments using biological control agents. The chemical control followed the standard strategies already used by the private property along with three applications spaced 15-days apart, from green bud stage. The first application was done using mancozeb + fomaxadone (Midas® ) and the second and third was done using carbendazim (Derosal® ). The plants which correspond to the control only received water applications under the same conditions as cited in the other treatments. Overall, 12 applications of B. subtilis were given to the treatment with ACB-69 applied weekly, 13 applications to the treatment which was applied the bacteria one week before to the other treatments (prior to the “green bud bloom” stage), like a preventive control, and 6 applications to the treatments conducted at 15-day intervals (Table 1). An experimental randomized block design was used with seven treatments and four repetitions. Each experimental plot was composed of 10 plants.
Table 1 Scheme of application of Bacillus subtilis (ACB-69 isolate) at 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations. Treatments
Intervals of applications during the blossom period
Total of applications
1. ACB-69 at 05% 2. ACB-69 at 05% 3. ACB-69 at 10% 4. ACB-69 at 10% 5. ACB-69 at 10%
Weekly Every 15 days Weekly Every 15 days One week before to the other treatments (preventive control) Spaced 15-days apart, from green bud stage Weekly
12 06 12 06 13
6. Chemical 7. Control (water)
03 12
The treatments were applied using an air assisted sprayer, calibrated to provide an application volume of 3800 l/ha, using 8.0 l/plant. The working pressure used was 100 lb/in.2 , with enough motor rotation to provide 540 rpm. The evaluations were based on counting the number of diseased and healthy open flowers in a sample of four branches per plant from the three central plants in the plot. The percentage of diseased flowers followed the methodology adapted from Timmer and Zitko (1996), so that were evaluated up to 100 flowers that had recently opened or were about to open were observed on each branch. One second evaluation was performed 90 days after the first evaluation, accounting for the number of fruit set and calyces retained and/or of the yellowness caused by the disease in order to obtain the average number of effective fruits (ANEF), using the following equation: ANEF = [A/(A + B)] × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease, according to de Goes (1995). 2.2. Effect of carbon additions in the spraying of B. subtilis to control C. acutatum (2008/2009 growth season) The following experiment was conducted on a 15-year-old orchard of ‘Valência’ sweet orange grafted on Rangpur lime (Citrus limonia Osbeck) located in Botucatu, São Paulo, Brazil. The treatments corresponding to the ACB-69 isolate were applied at 5% (5 × 108 cfu ml−1 ) and 10% (1 × 109 cfu ml−1 ) concentrations, with and without the addition of a carbon source applied together the cell suspension; in this case, weekly applications of molasses (a viscous product of the processing of sugar cane) at 5% concentration resulting in a total of four treatments with biological control agents. The plants which correspond to the control only received water applications. Fungicide applications were applied using mancozeb + fomaxadone (Midas® ) and Derosal® , as mentioned in the previous experiment. To produce the antagonist suspensions, the treatment applications and the evaluations followed the same procedures as the previous experiment. An experimental randomized block design was used with eight treatments and four repetitions. Each experimental plot was composed of 10 plants and the evaluations were realized in a sample of four branches per plant from the two central plants in the plot. 2.3. Biological control agent applications and growth stages of flowers (2008/2009 growth season) This experiment was conducted at the Morrinhos Farm in Botucatu, São Paulo, Brazil on a 15-year-old grove of ‘Valência’ sweet orange grafted on Rangpur lime. The trials conducted to test the efficiency of applying the biocontrol agent (ACB-69) (1 × 109 cfu ml−1 ) during different flower growth stages were conducted by spraying the agent at the four
K.C. Kupper et al. / Scientia Horticulturae 134 (2012) 139–143 Table 2 The effect of biological control agents in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Pera’ orange plants under field conditions in Botucatu, São Paulo State, Brazil, during the 2007/2008 season.
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Table 3 Effect of adding a carbon source during pulverization with Bacillus subtilis in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Valência’ orange plants, Botucatu, São Paulo State, Brazil (2008/2009 season).
Treatments
Percentage of flowers with C. acutatum symptoms
ANEFb
Treatments
Percentage of flowers with C. acutatum symptoms
ANEFb
Chemical control ACB-69 at 10% (every 15 days) ACB-69 at 10% (weekly) ACB-69 at 05% (every 15 days) ACB-69 at 10% (preventive control) ACB-69 at 05% (weekly) Control
28.69 ab 27.26 ab 22.75 ab 22.05 b 19.40 b 18.72 b 35.12 aa
51.81 ab 62.69 ab 73.57 a 64.73 ab 76.72 a 57.24 ab 46.50 b
ACB-69 at 10% ACB-69 (10%) + molasses (5%) Chemical control ACB-69 (5%) ACB-69 (5%) + molasses (5%) Control
42.31 ba 35.85 b 56.60 b 61.78 ab 57.75 ab 80.71 a
67.00 a 66.00 a 63.00 a 37.00 b 32.00 bc 26.00 c
a
Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). b ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease.
different flower growth stages previously described by Agustí et al. (2002): (1) closed visible flowers, (green bud), (FGB); (2) elongating flower petals; sepal covering corolla (white bud) (FWB); (3) most flowers with petals forming a hollow ball (HB); and (4) open flowers (FO). These treatments were compared to the (5) standard fungicide treatment (mancozeb + fomaxadone (Midas® ) and Derosal® ) used by the farm and to the (6) control treatment corresponding to the plants which received water applications. A total of nine treatments were tested, considering the applications: (7) in all flowering stages; and in the combinations of flower growth stages: (8) FGB + FWB and (9) FGB + FWB + HB, as described above. The inoculum production, applications and evaluations followed the same procedures as in the previous tests. All the treatments were applied from 20/09/2008 for each development stage, and the next applications were made from 7 after the first application, for each development stage. An experimental randomized block design was used with nine treatments and four repetitions. Each experimental plot was composed of 10 plants and the evaluations were realized in a sample of four branches per plant from the two central plants in the plot. 3. Results and discussion 3.1. Effect of the B. subtilis concentration to control C. acutatum under field conditions (2007/2008 growth season) The data related to how the B. subtilis concentration affects the control of C. acutatum under field conditions was evaluated according to the percentage of PFD symptomatic flowers and average number of effective fruits, as seen in Table 2. The results reveal that the disease occurred with great intensity and that the treatments affected the disease incidence. When the data referring to the percentage of symptomatic flowers was analyzed, it was verified that the only treatments that statistically differed from the control treatment (without disease control) were those which applied the bacteria at 5% concentration with either weekly or every 15 days applications or when the Bacillus (at 10%) was applied one week prior to the other treatments (prior to the “green bud bloom” stage). It was shown that when the plants were treated weekly with ACB-69 (at 5%) the quantity of symptomatic flowers was reduced by 47% while the chemical treatment showed a control efficiency of only 28.7% when compared to the control treatment data. The data presented by the average number of effective fruits showed that the best treatments were obtained when the ACB-69 isolate (at 10%) was applied one week prior to the other treatments and when the same treatment was applied weekly. These treatments did not differ from chemical control. The frequency of
a Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). b ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease.
application seems to cause more effects in the disease control and a single application before at the initial flowering stage provided good results. As for the applied methodologies evaluated, the percentage of symptomatic flowers used by Timmer and Zitko (1996), directly evaluates the effects of the disease, while the ANEF method proposed by de Goes (1995), apart from the direct effects of the disease also includes the number of fruit set. Considering the above, the results presented in this study reported that the treatment composted of an additional application favored the plant giving it protection to the pathogen attacks and consequently providing a greater number of fruit set. Kupper et al. (2003) reported that while developing the experiment using seven B. subtilis isolates and tree isolates from different Trichoderma species with cell concentrations of 1 × 107 cells per ml during the blossom period to control C. acutatum, the ACB-69 isolate differed from the control, statistically equating to the benomyl, causing a lower percentage of symptomatic flowers and a greater number of effective fruits. When this study, which was performed using a manual sprayer, is compared to the work of these authors, a greater audacity in terms of test installation is revealed, in this study was emphasized the possibility of the biocontrol agent to be applied to citrus plants on a commercial scale and with an air assisted spray. Several studies are found in literature proving the production of toxic metabolites with B. subtilis (Huang and Chang, 1975; Baker et al., 1983; Mckeen et al., 1986; Arras and Arru, 1997), which can inhibit the germination of spores or the growth of many fungi (Cubeta et al., 1985; Bettiol and Kimati, 1989, 1990; Kupper et al., 2003) which could affect the Bacillus’s own cells when in greater concentrations on the action site of the bio-fungicide broth. In this context, the higher concentration of B. subtilis in the broth did not reduce the biologic product efficiency. This was proven in the results obtained by the average number of effective fruits (Table 2), where the weekly application or the application that took place one week prior to the indicated period for the control of the disease, proved itself efficient in controlling C. acutatum. 3.2. Effect of carbon additions in the spraying of B. subtilis to control C. acutatum (2008/2009 growth season) The data presented in Table 3 shows that treatments where a sugar source was added during the application of a biological control agent did not statistically differ from the treatments where only the bacteria at a 10% concentration was applied. This was seen when evaluating the percentage of diseased flowers showing an efficiency of 48–56% in relation to the control. When the average number of effective fruits was evaluated, the addition of molasses to the Bacillus (at 10%) application also
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Table 4 Effect of timing of application of Bacillus subtilis on the development flowers, in the percentage of symptomatic flowers infected with Colletotrichum acutatum and the average number of effective fruits (ANEF) in ‘Valência’ orange plants, Botucatu, São Paulo State, Brazil, during 2008/2009 season. Treatments
Percentage of flowers with C. acutatum symptoms
ANEFc
Chemical control All flowering stagesd Hollow ball (HB) Green bud (FGB) FGB + FWB Open flower (FO) FGB + FWB + HB White bud (FWB) Control
10.54 ca , b 17.10 abc 20.43 ab 15.84 abc 15.06 abc 14.45 bc 15.94 abc 15.85 abc 21.73 a
86.00 ab 75.00 ab 66.75 bc 59.00 bcd 57.75 cd 51.25 cd 49.25 d 42.00 d 48.00 d
a
Data transformed to: ar sen sqrt (x + 0.5). Means followed by the same letter are not significantly different to according to the Duncan’s test (P ≤ 0.05). c ANEF = (A/(A + B)) × 100, where A is the number of fruit sets and B is the number of persistent calyces and/or number of yellowish fruits due to the disease. d Flowering stages: visible flower still closed and green bud (FGB); flower petals elongating, sepal involving corolla and white bud (FWB); most flowers with petals forming a hollow bud (FHB) and Flowers open (FO). b
showed to be indifferent. With the evaluation methodology used, the best treatments were: ACB-69 applications at a 10% concentration, ACB-69 (10%) + molasses (5%) and the chemical control, with percentages of the average number of effective fruits varying from 63 to 67%, not recording statistical differences between the cited treatments. The positive results of B. subtilis reducing the percentage of symptomatic flowers and enabling a greater number of effective fruits, suggest that ACB-69 is a potential agent in the biological control of postbloom fruit drop and that the addition of a carbon source during application was completely innocuous to the antagonism of the bacteria (Table 3). 3.3. Biological control agent applications and growth stages of flowers (2008/2009 growth season) Experiments have proven that even under controlled conditions during pulverization, the desired control level is not always obtained. Among some of the possible factors that could influence the effectiveness of treatments are the applications of biological products, the time of pulverization and the respective flowering stage. In relation to the data in Table 4, it was shown that the best time to apply the B. subtilis ACB-69 isolate to control the disease was during the open flower stage, presenting approximately a 34% reduction of symptomatic flowers in relation to the without disease control treatment. This treatment did not differ from the chemical control which presented a control efficiency of approximately 52% in relation to the without disease control treatment. According to Kupper et al. (2003) antibiosis seems to be a major mechanism involved in the interaction between B. subtilis, especially the ACB-69 isolate and C. acutatum. Previous work related that ACB-69 isolate was one of the most effective in producing anti-fungal substances and in quantities sufficient to inhibit the germination of C. acutatum (Kupper et al., 2009). Consequently, the high activity of this isolate seems related principally to the preventive action of infection by C. acutatum considering to be pathogen spore germination an important stage of the disease cycle. In this context, when the disease control is evaluated using the method of diseased flower percentages (Timmer and Zitko, 1996), it is assumed that the best time to apply a bacterial application is during the open flower stage as shown in this research. Considering that the open flower stage is the stage in which most of the pathogen sporulation occurs, and since the environmental
conditions are favorable, this is also the stage where the flower is most susceptible to fungus attacks. According to Zulfiqar et al. (1996), after the initial infection of C. actutatum in citrus flowers, the petals are rapidly colonized and the first symptoms appear in less than 48 h, therefore the antagonist action of B. subtilis by antibiosis is essential in this phase in order to suppress the disease under natural conditions. The efficiency of the ACB-69 isolate in comparison to the standard fungicide treatment, once again confirmed the potential that was previously verified in vitro, in detached flowers (Kupper and Gimenes-Fernandes, 2002; Kupper et al., 2003) and under field conditions (Kupper et al., 2003). When the average number of effective fruits was evaluated, it was verified that the best results were obtained with the chemical product application and with the bacterial application during all flowering stages, having an average fruit set quantity of 86% and 75%, respectively. Several factors should be taken into consideration in an attempt to explain the necessity of applying B. subtilis during all flowering stages. First, in respect to the etiologic behavior of the pathogen, one of the survival forms of C. acutatum is by appresorias (Agostini et al., 1992; Zulfiqar et al., 1996), which germinate and produce hyphae and conidia on the surface of the leaves when in the presence of humidity and petal extracts (Timmer et al., 1994), such conditions were present during all trials; second, a long period of citrus plants blooming in the Botucatu area, exposing them to a longer period of susceptibility when compared with other regions and, third, the action mechanism used by the bacteria, the antibiosis, as previously mentioned which requires an immediate action in the presence of the phytopathogen. The long period of exposure of the flowers associated with a relatively short infection period and long periods of rain during the flowering stage, make it more difficult to plan and execute pulverizations with chemical products causing great difficulties to control the pathogen, which under favorable environmental conditions reproduce themselves intensely with consequent symptom expressions. Under these circumstances the disease manifests itself exponentially which does not permit delays in control programs even when adequate fungicides are used (de Goes et al., 2008). Considering that the bacteria was obtained in the same habitat and therefore adapted to the same conditions of the phytopathogen, the chances of establishing and exercising its antagonist activities are greater due to the above circumstances cited. On the other hand, the results obtained from this experiment diverge from the work with chemical products which are cited in literature. According to de Goes et al. (2008), the fungicides carbendazim and folpet were efficient in controlling postbloom fruit drop when they were applied in ‘green bud bloom’ and ‘hollow ball’ stages under field conditions; as for Roberto and Borges (2001), the best control was obtained with benomyl, when sweet orange plants were pulverized in the predominant ‘white round’ blossom stage until the ‘hollow ball’ stage in precocious flowering. The results obtained from this experiment also differ from others applications with B. subtilis (ACB-69) performed in previous seasons (Kupper et al., 2003, 2009). According to these authors, the best control was obtained with ACB-69, when sweet orange plants were pulverized in green bloom and hollow ball stages under field conditions. The extended bloom period in the Botucatu area was favorable for pathogen development as open flowers are more susceptible to the pathogens. Under these circumstances, and especially under elevated inoculums pressure and favorable environmental conditions, the application of biological product for flower protection during all bloom period was essential. Perhaps these reasons could explain the divergences found in this work, when compared with results from other authors.
K.C. Kupper et al. / Scientia Horticulturae 134 (2012) 139–143
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