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Population density of Beauveria bassiana in soil under the action of fungicides and native microbial populations

Densidade populacional de Beauveria bassiana no solo sob a ação de fungicidas e de populações microbianas nativas

ABSTRACT.

This study investigated whether populations of naturally-occurring soil bacteria, fungi and actinomycetes influence the effect of fungicides on the survival and growth of Beauveria bassiana. The toxicity of methyl thiophanate, pyraclostrobin, mancozeb and copper oxychloride at the recommended doses was analyzed in culture medium and in soil inoculated with fungus at various time points after addition of fungicides. All fungicides completely inhibited the growth and sporulation of B. bassiana in the culture medium. The fungicides were less toxic in soil, emphasizing the action of the microbial populations, which interfered with the toxic effects of these products to the fungus. Actinomycetes had the greatest influence on the entomopathogen, inhibiting it or degrading the fungicides to contribute to the survival and growth of B. bassiana in soil. Native populations of fungi and bacteria had a smaller influence on the population density of B. bassiana and the action of fungicides towards entomopathogen. The toxic effect of the fungicides was greater when added to the soil one hour before or after inoculation than at 48h after inoculation.

Keywords:
biological control; entomopathogenic fungus; soil microorganisms; compatibility; toxicity

RESUMO.

Este estudo investigou se as populações nativas de bactérias, fungos e actinomicetos do solo influenciam a ação de fungicidas na sobrevivência e crescimento de Beauveria bassiana. A toxicidade de tiofanato-metil, piraclostrobina, mancozeb e oxicloreto de cobre usados nas doses recomendadas pelos fabricantes, foi analisada no meio de cultura e no solo inoculado com o fungo após diferentes períodos de adição dos fungicidas. Todos os fungicidas inibiram completamente o crescimento e esporulação de B. bassiana no meio de cultura. No solo, os fungicidas foram menos tóxicos, enfatizando-se a ação das populações microbianas que interferiram na toxicidade destes produtos para o fungo. Os actinomicetos mostraram a maior influência; inibiram B. bassiana ou degradaram os fungicidas, contribuindo para a sobrevivência e crescimento do fungo no solo. As populações nativas de fungos e bactérias tiveram menor influência sobre a densidade populacional de B. bassiana e na ação dos fungicidas para o entomopatógeno. A toxicidade dos fungicidas foi maior quando adicionados ao solo uma hora antes ou após a inoculação do fungo, do que 48h após.

Palavras-chave:
controle biológico; fungo entomopatogênico; micro-organismos do solo; compatibilidade; toxicidade

Introduction

Chemical pesticides can be a problem for integrated pest management (IPM) because they can get contact with non-target organisms in soil, such as entomopathogenic fungi (Mochi, Monteiro, & Barbosa, 2005Mochi, D. A., Monteiro, A. C., & Barbosa, J. C. (2005). Action of pesticides to Metarhizium anisopliae in soil. Neotropical Entomology, 34(6), 961-971.). The pesticides affect these organisms in various ways (Jaros-Su, Groden, & Zhang, 1999Jaros-Su, J., Groden, E., & Zhang, J. (1999). Effects of selected fungicides and the timing of fungicide application on Beauveria bassiana-induced mortality of the Colorado potato beetle (Coleoptera: Chrysomelidae). Biological Control, 15(3), 259-269.). Tests of the compatibility of pesticides and beneficial fungi are essential for developing successful IPM strategies (Jaros-Su et al., 1999). Most tests are conducted by cultivating the fungi in synthetic culture medium containing these products; however this method does not reflect the conditions in the soil where contact between the two control agents occurs (Mochi et al., 2005Mochi, D. A., Monteiro, A. C., & Barbosa, J. C. (2005). Action of pesticides to Metarhizium anisopliae in soil. Neotropical Entomology, 34(6), 961-971.).

The survival of entomopathogenic fungi in natural environments is influenced by biotic and abiotic factors. The main biotic factors are reservoirs of invertebrates and natural and artificial antagonists (McCoy, Quintela, & Faria, 2002McCoy, C., Quintela, E. D., & Faria, M. (2002). Environmental persistence of entomopathogenic fungi. In M. E. Baur, & J. R. Fuxa (Eds.), Factors affecting the survival of entomopathogens (p. 20-30). Lousiana, US: State University Agricultural Center, Southern Cooperative Series Bulletin 400.). Natural antagonists are the native microbial populations of the soil and are one of the most active factors in determining the persistence of fungi in these environments (Jaronski, 2010Jaronski, S. T. (2010). Ecological factors in the inundative use of fungal entomopathogens. Bio Control, 55(1), 129-145.).

In agricultural ecosystems, pesticides reach the soil directly or indirectly through rainwater and interact with native microbial populations to inhibit the survival and growth of microorganism populations exposed to chemical products (Ampofo, Tetteh, & Belo, 2009Ampofo, J. A., Tetteh, W., & Bello, M. (2009). Impact of commonly used agrochemicals on bacterial diversity in cultivated soils. Indian Journal of Microbiology, 49(3), 223-229.) or suffering degradation (Arbeli & Fuentes, 2007Arbeli, Z., & Fuentes, C. L. (2007). Accelerated biodegradation of pesticides: an overview of the phenomenon, its basis and possible solutions, and a discussion on the tropical dimension. Crop Protection, 26(2), 1733-1746.). During microbial degradation, the chemical compound is used as a source of carbon by the soil microbial populations to fuel considerable population growth (Bhuyan, Sreedharan, Adhya, & Sethunathan, 1993Bhuyan, S., Sreedharan, B., Adhya, T. K., & Sethunathan, N. (1993). Enhanced biodegradation of g-hexachlorocyclohexane (g-HCH) in HCH (commerial) acclimatized flooded soil: factors affecting its development and persistence. Pesticide Science, 38(1), 49-55.). This, in turn, can influence the survival of soil microorganisms such as fungi through fungistasis (Lanza, Monteiro, & Malheiros, 2004Lanza, L. M., Monteiro, A. C., & Malheiros, E. B. (2004). População de Metarhizium anisopliae em diferentes tipos e graus de compactação do solo. Ciência Rural, 34(6), 1757-1762.). The microbial degradation of chemical products benefits soil fungal populations by transforming chemicals into less-toxic byproducts (Lopes, Batista, Batista, Mitidieri, Bataus, & Fernandes, 2010Lopes, F. M., Batista, K. A., Batista, G. L. A., Mitidieri, S., Bataus, L. A. M., & Fernandes, K. F. (2010). Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil. World Journal of Microbiology and Biotechnology, 26(7), 1155-1161.).

In addition to action against native microbial populations indirectly affect entomopathogenic soil fungi, fungicides can act directly on these natural enemies of pests by inhibiting their development and reproduction, with negative effects on the IPM (Malo, 1993Malo, A. R. (1993). Estudio sobre la compatibilidad del hongo Beauveria bassiana (Bals.) Vuill. con formulaciones comerciales de fungicidas e insecticidas. Revista Colombiana de Entomologia, 19(1), 151-158.). Due to its cosmopolitan distribution and frequent presence in nature, B. bassiana is one of the most important and abundant entomopathogenic fungi (Rehner, 2005Rehner, A. S. (2005). Ecology and evolution of fungal endophytes and their role against insects. In F. E. Vega, & M. Blackwell (Eds.), Insect-fungal associations: ecology and evolution (p. 74-96). Oxford, UK: Oxford University Press.). The effect of fungicides on fungi used for biological control has been poorly explored in soil mainly under the effect of microbial populations. Thus, this study investigated whether the populations of naturally-occurring soil bacteria, fungi and actinomycetes influence the action of fungicides applied in various ways on the survival and growth of B. bassiana.

Material and methods

Test fungal culture and fungicides

The B. bassiana isolate JAB 06 (GenBank - accession No. KX599544 - ITS1-5.8S-ITS2 region) from the collection of the Microbiology Laboratory of the Department of Plant Production in the Faculty of Agrarian and Veterinary Sciences, São Paulo State University, was used for all tests. The fungus was cultivated in a medium of potato, dextrose, and agar (PDA) and kept in an incubator at 27 ± 1ºC in the dark for 25 days.

Information on active ingredients, chemical groups, recommended dose of commercial product (c. p.), and spraying volume of the fungicides is shown in Table 1. The concentrations of products in spraying liquids were: a) thiophanate-methyl: 2.5 mg of c. p. mL-1; b) pyraclostrobin: 0.00015 mL of c. p. mL-1; c) mancozeb: 2 mg of c. p. mL-1; and d) copper oxychloride: 2.5 mg of c. p. mL-1.

Experiment with culture medium

The amount of fungicide (per milliliter of culture medium) needed to obtain a concentration equivalent to the liquid used for spraying was calculated and added to the PDA medium at temperatures ranging from 45 to 50°C to avoid possible alterations of the properties of the fungicide. After pouring the medium into Petri dishes, each dish was inoculated in the center by pricking with a platinum needle dipped in a suspension containing 107 conidia mL-1 obtained from 15-days-old colonies of the fungus.

The vegetative growth, sporulation, and germination of conidia were used as parameters to evaluate the performance of the fungus after treatment with a fungicide. The growth of the colonies was quantified by measuring, in millimeters, three equidistant diameters previously marked on the bottom outside of the Petri dish. The measurements were taken every three days from day three to fifteen after inoculation. Each dish corresponded to a repetition, and each treatment was replicated five times.

On day 15, sporulation was assessed by collecting three samples per dish (one each from the center, middle and edge of each colony) with the aid of a sterilized metallic 8-mm diameter ring. Three replicate colonies were used for sample collection. Each sample was individually transferred to a test tube containing 10 mL of a 1:1 sterilized solution of NaCl (0.89% w v-1) and Tween 80® (0.1% v v-1) and vigorously agitated in an electric tube agitator. Conidia were counted with an optical microscope using a Neubauer counting chamber, and the suspension was diluted when necessary.

Table 1
Fungicidesa used in this study with the entomopathogenic fungus Beauveria bassiana isolate JAB 06.

Germination was assessed by direct examination of conidia on a microscope slide. After demarcation of three areas, a sterile microscope slide was covered with a thin layer of PDA medium containing fungicides at the recommended doses. A drop (approximately 0.05 mL) of the fungal suspension with 105 conidia mL-1 was placed in the region of the culture medium corresponding to each of these areas. After incubation for 15h at 27 ± 1°C in the dark, the germination process was stopped with a drop of dye in each area. A dye stock solution was prepared by adding 1 g of methylene blue in 20 mL of lactic acid. The working dye was composed of 1 mL of the stock solution added to 29 mL of lactic acid. A total of 150 conidia were observed in each slide area to establish the percentage of germinated conidia. Three replicate slides were used for each treatment, and the replicates were inoculated with suspensions obtained from different 15 day-old colonies.

A single trial was organized using a completely randomized design. The treatments consisted of media containing fungicides and fungicide-free PDA medium was used as a control. Variance was assessed using the F-test, and mean values were compared using Tukey’s test (p ≤ 0.05). AgroEstat (Barbosa & Maldonado, 2010Barbosa, J. C., & Maldonado Jr., W. (2010). AgroEstat - Sistema para Análises Estatísticas de Ensaios Agronômicos, Versão 1.0.) software was used for statistical analysis.

To determine the toxic effect of the fungicides, we used the formula described by Rossi-Zalaf, Alves, Lopes, Siqueira Neto, and Tanzini (2008Rossi-Zalaf, L. S., Alves, S. B., Lopes, R. B., Silveira Neto, S., & Tanzini, M. R. (2008). Interação de microrganismos com outros agentes de controle de pragas e doenças. In S. B. Alves, & R. B. Lopes (Eds.), Controle microbiano de pragas na América Latina: avanços e desafios (p. 270-302). Piracicaba, SP: Fundação de Estudos Agrários Luiz de Queiroz .), including parameters for vegetative growth, sporulation, and germination:

BI = 47 (VG) + 43 (SP) + 10 (GER) ÷ 100

where: BI = is the Biological Index; VG = is the percentage of vegetative growth of the colony after 15 days, relative to the control; SP = is the percentage of sporulation after 15 days, relative to the control; and GER = is the percentage of germination of the conidia after 15 hours, relative to the control. No decimal places were used in the calculation of BI.

Using the obtained BI values we classified the fungicides according to their level of toxicity using the following scale: 0 to 41 = toxic, 42 to 66 = moderately toxic, and > 66 = compatible (Rossi-Zalaf et al., 2008Rossi-Zalaf, L. S., Alves, S. B., Lopes, R. B., Silveira Neto, S., & Tanzini, M. R. (2008). Interação de microrganismos com outros agentes de controle de pragas e doenças. In S. B. Alves, & R. B. Lopes (Eds.), Controle microbiano de pragas na América Latina: avanços e desafios (p. 270-302). Piracicaba, SP: Fundação de Estudos Agrários Luiz de Queiroz .).

Soil experiment

The toxic effect of the fungicides on B. bassiana was also evaluated in soil. A Typic Eutrustox soil composed of 53% clay, 18% silt, and 29% sand was collected at a depth of 0 to 20 cm from an environmental preservation area belonging to the Faculty of Agrarian and Veterinary Sciences. The soil was dried at room temperature, loosened, and sifted through a 1-mm mesh sieve. For each fungicide test, an assay was carried out, for which a new sample of soil was collected. The water saturation capacity of the soil was determined before each trial was conducted.

We used 100-mm Petri dishes containing 80 g of non-autoclaved soil. To provide a gap for gas exchange, the space between the two halves of the dishes was increased by attaching two wooden sticks inside the upper plate. In an aseptic chamber, sterile distilled water was added to the soil until it reached 65% of its saturation capacity, and samples were left to rest for one hour for stabilization.

Fungicide was added in accordance with the volume and concentration of spraying liquid used in the field. Based on the manufacturer’s recommendation, we calculated the amount of liquid used for spraying (in milliliters) to be deposited per cm2 of soil, assuming a direct and homogeneous distribution of fungicide in the field. We calculated the soil surface area in the Petri dish and a proportional volume of liquid used for spraying to be applied in this area. A fungicidal solution with the same concentration of the liquid used for spraying in the field was prepared. The appropriate volume for the Petri dish area was carefully distributed (droplets of approximately 0.05 mL) on the soil surface with the aid of a micropipette, and effort was taken to cover the soil surface homogeneously. Fungicide was applied at the following time points: a) one hour before inoculation of fungus; b) one hour after inoculation of fungus; and c) 48 hours after of inoculation of fungus. Inoculation with a micropipette was performed by distributing 2 mL of fungal suspension containing 1.0 x 107 conidia mL-1 onto the entire surface of the soil in the most homogeneous manner possible.

Dishes with soil remained in the incubator at 27 ± 1ºC in the dark for 28 days. A tray with water was kept in the incubator to minimize water loss. Dishes were checked for water loss weekly, and sterile distilled water was added to replace water loss by evaporation. The control treatment consisted of soil inoculated with the fungal suspension without the addition of fungicide.

For evaluation of fungal survival, colony-forming units (CFUs) were counted after 0, 7, 14, 21, and 28 days of incubation. In each dish, a 1-g sample of damp soil was collected from 12 to 15 points on the surface of the soil and suspended in 9 mL of a 0.1% (v v-1) solution of Tween 80®. Serial dilutions were performed from the initial suspension, and these dilutions were used to seed 0.1 mL in Petri dishes that contained culture media to determine the populations of B. bassiana (Joussier & Catroux 1976Joussier, D., & Catroux, G. (1976). Mise au point d´un milieu de culture pour le denombrement de Beauveria tenella dans les sols. Entomophaga, 21(3), 223-225., with the exclusion of vegetable juice and oxygall), bacteria (Bunt & Rovira, 1955Bunt, J. S., & Rovira, A. D. (1955). Microbiological studies of some subantarctic soils. Journal of Soil Science, 6(1), 119-128.), fungi (Martin, 1950Martin, J. P. (1950). Use of acid, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Science, 69(3), 215-232.), and actinomycetes (Hsu & Lockwood, 1973Hsu, S., & Lockwood, L. (1973). Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Applied Environmental Microbiology, 29(3), 422-426.).

For each fungicide, a trial was performed using a completely randomized design with four repetitions per treatment. The data were subjected to an analysis of variance using an F-test to examine a) growth of each microorganism over the course of the evaluation period in a single treatment and b) growth of B. bassiana among the treatments. The averages were compared using a Tukey’s test at the 5% probability level. The experiment was analyzed according to a split-plot-in-time design, considering the effects of treatment (1 hour before inoculation, 1 hour after inoculation, and 48 hours after inoculation) and time period (0, 7, 14, 21, and 28 days of incubation) and the treatment versus time period interaction. The Pearson coefficients of correlation were determined at a 5% probability level to verify interactions between populations of bacteria, fungi, and actinomycetes with population of B. bassiana. AgroEstat software (Barbosa & Maldonado, 2010Barbosa, J. C., & Maldonado Jr., W. (2010). AgroEstat - Sistema para Análises Estatísticas de Ensaios Agronômicos, Versão 1.0.) was used for the statistical analyses.

Results and discussion

Experiment with culture medium

No vegetative growth or sporulation of B. bassiana on media containing the recommended doses of the tested fungicides was observed. With the exception of mancozeb, germination of conidia was not significantly different among the tested fungicides and the control. In spite of these results, all fungicides were rated as toxic to B. bassiana based on the calculated BI (Table 2). A similar result was obtained by Loureiro, Moino, Arnosti, and Souza (2002Loureiro, E. S., Moino Jr., A., Arnosti, A., & Souza, G. C. (2002). Efeito de produtos fitossanitários químicos utilizados em alface e crisântemo sobre fungos entomopatogênicos. Neotropical Entomology, 31(2), 263-269.) who found that fungicides containing thiophanate-methyl and mancozeb as active ingredients were also classified as very toxic to the CB 66 isolate of B. bassiana.

Table 2
Percent germination of Beauveria bassiana conidia cultivated on media containing fungicides at the recommended doses, Biological Index (BI) and fungal toxicity rating of the fungicides.

The germination of conidia requires a source of carbon as an extracellular signal for translation to occur which then triggers germination (Osherov & May, 2000Osherov, N., & May, G. (2000). Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis. Genetics, 155(2), 647-656.). This process can explain the utilization of endogenous sources of carbon and nitrogen, without the use of exogenous sources present in the culture medium (Lefevbre, 1931Lefebvre, C. L. (1931). Preliminary observations on two species of Beauveria attacking the corn borer, Pyrausta nubilalis Hübner. Phytopathology, 21(12), 1115-1128.). Thus, the germination of B. bassiana conidia would have occurred before the fungus contacted the fungicides added to the medium, and thus the fungicides could not have inhibited conidia germination. However, for vegetative growth and sporulation hyphal extension must occur after germination. At this point, the fungus requires exogenous sources of carbon and nitrogen from the culture medium, and must therefore come into contact with fungicide which inhibits further growth.

These results are important for IPM because they show that fungicides, although toxic to B. bassiana, do not completely inhibit the germination of conidia, which is the event that starts penetration of a fungus into the host (Alves & Lecuona, 1998Alves, S. B., & Lecuona, R. E. (1998). Epizootiologia aplicada ao controle microbiano de insetos. In S. B. Alves (Ed.), Controle microbiano de insetos (p. 97-170). Piracicaba, SP: Fundação de Estudos Agrários Luiz de Queiroz.). In addition, the toxicity of fungicides in soil environment can be smaller due to the influence of native microbial populations.

Soil experiments

Thiophanate-methyl

In soil added one hour after inoculation with B. bassiana, we observed a linear and directly proportional correlation (r = 0.78532, p ≤ 0.01) between the growth of the entomopathogen population and that of other fungi (Table 3), suggesting that the growth of these populations may be linked (Figure 1A). Results obtained by Draganova, Donkova, and Georgieva (2008Draganova, S., Donkova, R., & Georgieva, D. (2008). Impact of strains of entomopathogenic fungi on some main groups of soil microrganisms. Journal of Plant Protection Research, 48(2), 169-179.) showed that lineage 412 of B. bassiana stimulated the growth of soil fungi 30-fold above the control value. Because these microorganisms are heterotrophic, there may be a synergistic interaction between these populations (Draganova et al., 2008Draganova, S., Donkova, R., & Georgieva, D. (2008). Impact of strains of entomopathogenic fungi on some main groups of soil microrganisms. Journal of Plant Protection Research, 48(2), 169-179.). This interaction is important for the degradation of organic soil composts because degradation takes place in stages and includes the action of many physiologically different microorganisms (Stamford, Rodrigues, Heck, & Andrade, 2005Stamford, N. P., Rodrigues, J. J. V., Heck, R. J., & Andrade, D. E. G. T. (2005). Microbiota dos solos tropicais. In S. J. Michereff, D. E. G. T. Andrade, & M. Menezes (Eds.), Ecologia e manejo de patógenos radiculares em solos tropicais (p. 61-92). Recife, PE: Imprensa Universitária, Universidade Federal Rural de Pernambuco.).

In joint degradation of organic composts, both populations benefit by taking advantage of nutrients and show similar growth patterns. In this treatment (Figure 1A), the correlation between populations of actinomycetes and B. bassiana was inversely proportional (r = -0.57798, p ≤ 0.05) indicating that the increase in the population of actinomycetes is related to the reduction in the population of the fungus (Table 3). The growth of actinomycetes, starting on the seventh day of evaluation, may generate a competitive relationship with B. bassiana for nutrients and space, resulting in inhibition of the fungus. Another explanation would be amensalism; actinomycetes may release microbiostatic compounds that cause fungistasis (Stamford et al., 2005Stamford, N. P., Rodrigues, J. J. V., Heck, R. J., & Andrade, D. E. G. T. (2005). Microbiota dos solos tropicais. In S. J. Michereff, D. E. G. T. Andrade, & M. Menezes (Eds.), Ecologia e manejo de patógenos radiculares em solos tropicais (p. 61-92). Recife, PE: Imprensa Universitária, Universidade Federal Rural de Pernambuco.). Autoclaved soils that are re-infested with actinomycetes have been shown to contain volatile fungistatic substances inhibit the germination of spores of Trichoderma viride Pers., Zygorhynchus vuileminii Namyslowski, and Gonatobotrys simplex Corda (Hora & Baker, 1972Hora, T. S., & Baker, R. (1972). Soil fungistasis: microflora producing a volatile inhibitor. Transactions of the British Mycological Socienty, 59(3), 491-500.).

A similar pattern of B. bassiana inhibition by actinomycetes was also observed in treatments (where) in which the fungicide was added to the soil one hour before (r = -0.49914, p ≤ 0.05) (Figure 1B) or 48h after (r = -0.51566, p ≤ 0.05) (Figure 1C) inoculation with the entomopathogen (Table 3). As described above, inhibition can be attributed to the release of compounds with inibitory effects by the population of actinomycete bacteria. Actinomycetes that release odors often described as earthy smell may be a productive source of inhibitory volatile compounds that are responsible for fungistasis (Hora & Baker, 1970Hora, T. S., & Baker, R. (1970). Volatile factor in soil fungistasis. Nature, 225(5237), 1071-1072.).

Table 3
Correlations between Beauveria bassian a population and populations of bacteria, fungi and actinomycetes obtained for treatments with different fungicides.

Figure 1
Interactions between Beauveria bassiana population and other naturally-occurring microbial populations in soil treated with fungicide containing the active ingredient thiophanate-methyl, applied A) 1 hour before inoculation with fungus, B) 1 hour after inoculation with fungus, C) 48 hours after inoculation with fungus. D is the control, to which no fungicide was added. Means (± standard deviation) with the same lower case letters for the incubation period of the same population were not significantly different (Tukey’s test, 5% probability). Statistical analysis performed and graphs plotted with data transformed in log (x+5).

¶In the control, the negative correlation (r = -0.55585, p ≤ 0.05) (Table 3) between populations of bacteria and B. bassiana suggests that the former inhibit the latter (Figure 1D). Bacteria are known to produce substances with fungistatic and even fungicidal effects. Zou, Mo, Gu, Zhou and Zang (2007Zou, C. S., Mo, M. H., Gu, Y. Q., Zhou, J. P., & Zang, K. Q. (2007). Possible contributions of volatile-producing bacteria to soil fungistasis. Soil Biological Biochemistry, 39(9), 2371-2379.) isolated 1,018 bacteria from soil and verified that among these, 328 produced volatile compounds with a fungistatic effect that inhibited the germination and mycelial growth of two species of fungi.

Population density averages for B. bassiana in different treatments indicated that the fungicide containing thiophanate-methyl had little toxic effect on this population. In the treatment without fungicide, there was greater stability of the fungal population, followed by the treatment where the fungicide was added to the soil 48 h after inoculation with B. bassiana (F = 258.55, p ≤ 0.01). The addition of fungicide 48h after inoculation probably provided sufficient time for the fungus to germinate and form hyphae before contacting the fungicide, increasing the chances of pathogen survival.

In treatments in which the product was added to soil one hour before or after inoculation with fungus, the population densities were statistically lower than control densities. The effect of thiophanate-methyl on the population of entomopathogen may be explained by the toxicity of its conversion product methyl-benzimidazol-2-yl carbamate, which inhibits fungi during cellular mitosis (Davidse, 1973Davidse, L. C. (1973). Antimitotic activity of methyl benzimidazol-2-yl carbamate (MBC) in Aspergillus nidulans. Pesticide Biochemistry and Physiology, 3(3), 317-325.). A progressive inhibition of the synthesis of DNA, RNA, and proteins causes flaws in normal mitosis or cytokinesis (Hammerschlag & Sisler, 1972Hammerschlag, R. S., & Sisler, H. D. (1972). Differential action of benomyl and methyl-2-benzimidazolecarbamate (MBC) in Saccharomyces pastorianus. Pesticide Biochemistry and Physiology, 2(1), 123-131.).

Pyraclostrobin

In soils where the inoculation of B. bassiana was performed one hour before (Figure 2B) or 48h after (Figure 2C) the addition of fungicide containing pyraclostrobin as an active ingredient, we observed a positive correlation between fungal growth and actinomycete populations (r = 0.58913 and r = 0.61853, respectively, p ≤ 0.01) (Table 3).

Figure 2
Interactions between Beauveria bassiana population and other naturally-occurring microbial populations in soil treated with fungicide containing the active ingredient pyraclostrobin, applied A) 1 hour before inoculation with fungus, B) 1 hour after inoculation with fungus, C) 48 hours after inoculation with fungus. D is the control, to which no fungicide was added. Means (± standard deviation) with the same lower case letters for the incubation period of the same population were not significantly different (Tukey’s test, 5% probability). Statistical analysis was performed and graphs plotted with data transformed in log (x+5).

¶The fungicide may (have been) be used as a source of energy by the actinomycetes, which are the most active microorganisms in the degradation of complex soil composts (Goodfellow, 1983Goodfellow, M. (1983). Ecology of actinomycetes. Annual Review of Microbiology, 37(1), 189-216.); this would lead to an increase in their population density. The degradation of the product likely led to conversion of the active ingredient into a less-toxic byproduct and favored the growth of the entomopathogen.

The population density of B. bassiana was significantly lower (F = 5.79, p ≤ 0.05) in treatments in which pyraclostrobin was added to soil one hour before or one hour after inoculation with fungus. In the control and the treatment in which the fungicide was applied to soil 48 h after fungal inoculation, the population densities were higher. This result suggests the existence of an effect of both the product and of the treatments on the entomopathogen. Inoculation of B. bassiana in the soil one hour before and one hour after the addition of the fungicide did not allow sufficient time for the fungus to germinate, resulting in inhibition. Pyraclostrobin completely controlled the

growth of the fungus Colletotrichum lindemuthianum (Sacc. and Magn.), showing a fungistatic effect (Sartorato, 2006Sartorato, A. (2006). Sensibilidade “in vitro” de isolados Colletotrichum lindemuthianum a fungicidas. Pesquisa Agropecuária Tropical, 36(3), 211-213.), in accordance with the results of this study. However, in the treatment in which the fungicide was applied 48 h after inoculation of the fungus, the toxic action was diminished because there was time for germination prior to contact with the chemical, reducing inhibition.

Mancozeb

In the experiment conducted with the fungicide mancozeb, the correlation between the populations of B. bassiana and the actinomycetes was inversely proportional (r = -0.48714, p ≤ 0.05) (Table 3) in the treatment where the product was added to soil one hour before the fungus. This indicates that B. bassiana may have been slightly inhibited by actinomycetes, although statistical analysis of the growth of both populations (Figure 3B) did not detect this phenomenon. For the control (Figure 3D), however, there was no significant correlation between the two populations (Table 3),

Figure 3
Interactions between Beauveria bassiana population and other naturally-occurring microbial populations in soil treated with fungicide containing the active ingredient mancozeb, applied A) 1 hour before inoculation with fungus, B) 1 hour after inoculation with fungus, C) 48 hours after inoculation with fungus. D is the control, to which no fungicide was added. Means (± standard deviation) with the same lower case letters for the incubation period of the same population were not significantly different (Tukey’s test, 5% probability). Statistical analysis performed and graphs plotted with data transformed in log (x+5).

¶suggesting that inhibition of the population of B. bassiana may be a consequence of degradation of the fungicide by actinomycetes. In an experiment performed with several species of Streptomyces, 80% of the tested species inhibited fungal growth through the production of antibiotics, while the rest of the tested species prevented the fungi from acquiring nutrients (Broadbent, Baker & Waterworth, 1971Broadbent, P., Baker, K. F., & Waterworth, Y. (1971). Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Australian Journal of Biology Science, 24(4), 925-944.).

A difference in the toxicity to B. bassiana was observed when experiments with mancozeb in culture medium and soil were compared. In the culture medium experiment, the fungicide was classified as toxic to the fungus, while in the soil this toxicity was not observed, as the population of B. bassiana in the control did not differ significantly from those in the presence of fungicide (F = 1.27NS). This may be due to the influence of soil on the toxicity of chemical products to the microorganisms. The fungicides that had completely toxic effects on B. bassiana in the culture medium did not show the same effects when applied in the field (Clark, Casagrande & Wallace, 1982Clark, R. A., Casagrande, R. A., & Wallace, D. B. (1982). Influence of pesticides on Beauveria bassiana, a pathogen of the Colorado potato beetle. Environmental Entomology, 11(1), 67-70.).

The toxic effect of a chemical product on a soil microorganism in a pure culture is different from that produced on the same organism in its natural habitat. In the pure culture, the relationship is only between the product and the organism, while in the field, the relationship is between the chemical product and the organism as protected by the characteristics of the soil (Kreutzer, 1963Kreutzer, W. A. (1963). Selective toxicicity of chemicals to soil microorganisms. Annual Review of Phytopathology, 1(1), 101-126.).

Copper oxychloride

In treatments in which copper oxychloride was added to the soil one hour before (Figure 4B) and 48h after inoculation of fungus (Figure 4C), as well as in the control (Figure 4D), the coefficients of correlation between populations of bacteria and B. bassiana were positive (r = 0.69148, r = 0.82772, and r = 0.58644, respectively, p ≤ 0.01) (Table 3).

Figure 4
Interactions between Beauveria bassiana population and other naturally-occurring microbial populations in soil treated with fungicide containing the active ingredient copper oxychloride, applied A) 1 hour before inoculation with fungus, B) 1 hour after inoculation with fungus, C) 48 hours after inoculation with fungus. D is the control, to which no fungicide was added. Means (± standard deviation) with the same lower case letters for the incubation period of the same population were not significantly different (Tukey’s test, 5% probability). Statistical analysis performed and graphs plotted with data transformed in log (x+5).

¶This result suggests that the development of bacterial populations in soil does not restrict the growth of B. bassiana and may even stimulate it, as observed in the control, where the highest population density of the fungus occurred (Figure 4D). Reports indicate that this stimulus can occur due to hormesis, a phenomenon whereby bacteria secrete fungistatic substances that, if released in small quantities, can stimulate the genes of other microorganisms and initiate their growth (Mlot, 2009Mlot, C. (2009). Antibiotics in nature: beyond biological warfare. Science, 324(5935), 1637-1639.). Hormesis is a complex process that can have significant implications for organisms that are beneficial for pest management (Duke, 2014Duke, S. O. (2014). Hormesis with pesticides. Pest Management Science, 70(5), 689. ).

In the treatment in wich the addition of the fungicide to soil was carried out one hour prior to inoculation of the entomopathogenic fungus (Figure 4B), actinomycetes inhibited the population of B. bassiana (r = -0.46717, p ≤ 0.05) (Table 3). Actinomycetes are known to secrete substances that inhibit other microorganisms, such as antibiotics that can inhibit the growth and germination of fungi (Schippers, Bakker, & Bakker, 1987Schippers, B., Bakker, A. W., & Bakker, P. A. H. M. (1987). Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Annual Review of Phytopathology, 25(1), 339-358.). This secretion may have caused the negative correlation between the two populations.

The populations of fungi and B. bassiana had similar growth pattern, with a positive correlation between the growth of both populations in all treatments (r = 0.84943, r = 0.91818, r = 0.72213, and r = 0.95867, p ≤ 0.01) (Table 3). Considering the treated groups, this finding may stem from a lack of selectivity of the fungicide, which inhibited both populations. The actinomycetes grew in all soil, but this did not result in decreased fungicide toxicity for B. bassiana in the soil, as shown by the small reduction in entomopathogen population in the majority of the treatments.

Copper oxychloride had a toxic effect on B. bassiana in the soil, while in the control, the population density of the fungus was greater than in the other treatments (F = 52, p ≤ 0.01). However, inoculation of the fungus in the soil prior or after fungicide application did not influence the toxic effect.

Conclusion

All fungicides evaluated in this study showed a toxic effect to B. bassiana in culture medium, though some variation among compounds was observed. The naturally-occurring soil microbial populations interfered with the effect of the compounds, reducing the toxicity of these fungicides to the fungus. The actinomycetes played an important role by reducing toxicity through degradation of fungicides or inhibition of B. bassiana by fungistasis. The population of fungi may inhibit the population of B. bassiana through competition for nutrients or through release of toxic metabolites. However, in some cases, the population density of both groups had a similar pattern of increase or decrease. The population of bacteria had little effect. The toxic effect of fungicides was influenced by time point of application: the effect was greater when the addition occurred one hour before or after inoculation of the fungus in the soil than 48h after inoculation.

Acknowledgements

The authors are grateful to the National Council for Scientific and Technologic Development in Brazil for financial support of the first author’s master’s degree studies and to Dr. Mariah Valente Baggio and Dr. Andressa de Souza for molecular identification of the fungus.

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Publication Dates

  • Publication in this collection
    Oct 2017

History

  • Received
    21 July 2016
  • Accepted
    14 Oct 2016
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