Biological control of white mold by Trichoderma harzianum in common bean under field conditions

1220 DD.C. Carvalho et al. Notas Científicas Biological control of white mold by Trichoderma harzianum in common bean under field conditions Daniel Diego Costa Carvalho(1), Alaerson Maia Geraldine(2), Murillo Lobo Junior(2) and Sueli Corrêa Marques de Mello(3) (1) Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Campus Universitário Darcy Ribeiro, Bloco E, s/no, Asa Norte, CEP 70910‑700 Brasília, DF, Brazil. E‑mail: daniel.carvalho@ueg.br (2)Embrapa Arroz e Feijão, Rodovia GO‑462, Km 12, Zona Rural, Caixa Postal 179, CEP 75375‑000 Santo Antônio de Goiás, GO, Brazil. E‑mail: alaerson.geraldine@ifgoiano.edu.br, murillo.lobo@embrapa.br (3)Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Avenida W5 Norte (Final), Caixa Postal 02372, CEP 70770‑917 Brasília, DF, Brazil. E‑mail: sueli.mello@embrapa.br Abstract – The objective of this work was to evaluate Trichoderma harzianum isolates for biological control of white mold in common bean (Phaseolus vulgaris). Five isolates were evaluated for biocontrol of white mold in 'Perola' common bean under field conditions, in the 2009 and 2010 crop seasons. A commercial isolate (1306) and a control treatment were included. Foliar applications at 2x109 conidia mL-1 were performed at 42 and 52 days after sowing (DAS), in 2009, and at 52 DAS in 2010. The CEN287, CEN316, and 1306 isolates decreased the number of Sclerotinia sclerotiorum apothecia per square meter in comparison to the control, in both crop seasons. CEN287, CEN316, and 1306 decreased white mold severity during the experimental period, when compared to the control. Index terms: Phaseolus vulgaris, Sclerotinia sclerotiorum, antagonists, hyperparasitism, soilborne pathogen. Controle biológico do mofo‑branco por Trichoderma harzianum em feijão em condições de campo Resumo – O objetivo deste trabalho foi avaliar isolados de Trichoderma harzianum para o controle biológico do mofo-branco em feijão (Phaseolus vulgaris). Cinco isolados foram avaliados para o biocontrole do mofo‑branco em feijão 'Pérola', em condições de campo, nos anos agrícolas 2009 e 2010. Um isolado comercial (1306) e um tratamento testemunha foram incluídos. Aplicações foliares a 2x109 conídios mL-1 foram realizadas aos 42 e 52 dias após a semeadura (DAS), em 2009, e aos 52 DAS em 2010. Os isolados CEN287, CEN316 e 1306 reduziram o número de apotécios por metro quadrado de Sclerotinia sclerotiorum, em comparação à testemunha, nos dois anos agrícolas. CEN287, CEN316 e 1306 reduziram a severidade do mofo‑branco no período experimental, quando comparados à testemunha. Termos para indexação: Phaseolus vulgaris, Sclerotinia sclerotiorum, antagonistas, hiperparasitismo, patógenos habitantes do solo. Fungicides have been used to manage white mold, but highly‑infested areas require many applications, which greatly increase production costs. Moreover, chemical fungicides do not always provide satisfactory control and may have adverse effects on nontarget organisms (Naseby et al., 2000). In this respect, biological control is advantageous over conventional pesticides, as it provides an alternative to reduce the soil inoculum potential, without harmful effects on the environment (Harman et al., 2004). Pesq. agropec. bras., Brasília, v.50, n.12, p.1220-1224, dez. 2015 DOI: 10.1590/S0100-204X2015001200012 Disease biocontrol promoted by Trichoderma, considering active components of natural soil, consists in a complex process that can occur through antibiosis, competition for nutrients, and mycoparasitism, among other mechanisms (Harman et al., 2004). As an additional advantage, some isolates of Trichoderma may also act as plant growth promoters (Carvalho et al., 2011). Different T. harzianum isolates have effectively reduced the incidence of white mold and other diseases in several economically important crops, Biological control of white mold by Trichoderma harzianum such as tomato (Abdullah et al., 2008) and common bean (Geraldine et al., 2013; Carvalho et al., 2014). Brazilian soils contain a rich diversity of beneficial microorganisms. Among them, Trichoderma species have been targets for collection and screening, aiming at the discovery of efficient isolates for biological control (Carvalho et al., 2014). Five isolates (CEN287, CEN288, CEN289, CEN290, and CEN316) were previously selected in vitro against major common bean pathogens (Carvalho et al., 2011). Their effectiveness, previously observed in controlled‑environment and field studies (Carvalho et al., 2015), motivated further tests, also designed to determine their biocontrol amplitude in agroecosystems. The objective of this work was to evaluate Trichoderma harzianum isolates for biological control of white mold in common bean (Phaseolus vulgaris). The five isolates of T. harzianum used in the present study belong to the fungi collection for biological control of plant pathogens and weeds of Embrapa Recursos Genéticos e Biotecnologia, located in the municipality of Brasília, in Distrito Federal, Brazil. All Trichoderma isolates were originally obtained from the Cerrado biome. Cultures were stored in liquid nitrogen and recovered in potato dextrose agar medium. In addition, a commercial isolate of T. harzianum, 1306 Trichodermil, (Itaforte Bioprodutos, Itapetininga, SP, Brazil), recommended for the biocontrol of soilborne pathogens and for plant growth promotion, was used. Two field experiments were conducted in the same area, at Fazenda Palmital (16º26'04"S, 49º24'07"W, at an altitude of 735 m), within Embrapa Arroz e Feijão facilities, located in the municipality of Goianira, in the state of Goiás, Brazil, in the 2009 and 2010 crop seasons (July‑October), when the average air temperature was 21.2 and 21.5°C, respectively. The soil of the experimental area is classified as a Latossolo Vermelho ácrico (Rhodic Acrustox) (Santos et al., 2006) with clay texture, and presented the following characteristics: 6.6 pH; 3.24, 1.25, 0, and 3.35 cmolc dm-³ Ca, Mg, Al, and H+Al, respectively; 11.6, 111, 2.9, 3.6, 110, and 50 mg dm-³ P, K, Cu, Zn, Fe, and Mn, respectively; and 20 g dm-³ organic matter. The experimental area had been previously cropped for pasture and had no record of previous annual crop. A total of 2.5 L ha-1 glyphosate was applied, and the distribution of sclerotia in the experimental area was carried out soon after crop sowing, with an average of 1221 145 sclerotia per square meter in 2009 and 2010, in alignment with Huang et al. (2000). The experimental area was fertilized with N‑P2O5‑K2O (5-25-15 at 400 kg ha-1) and sown with 'Pérola' common bean (24 seeds per square meter). Plots composed by five 2.5‑m planting rows were arranged in a randomized complete block design, with four replicates. The spacing between rows was 0.5 m, whereas plots were spaced at 1.0 m. Outer guard rows (2.5 m) were sown with the same crop to protect the total experimental area (400 m2) and to support a disease‑conducive microclimate. The experiments were sprinkled irrigated, favoring proper common bean growth and apothecia development. Other cultural practices followed the recommendations of Barbosa & Gonzaga (2012). In order to produce T. harzianum inoculum, 5.0‑mm mycelial plugs of each T. harzianum isolate were transferred to 250‑mL flasks (six plugs per flask), containing 15 g parboiled rice, previously moistened (60% w/v) and autoclaved (121ºC for 40 min). Flasks were kept at 25ºC, under a 12‑hour photoperiod. After 7 days, spores were harvested with distilled water and filtered through sterile gauze, and their concentration was adjusted to 1×106 conidia mL-1 with a Neubauer chamber. In 2009, two T. harzianum applications of 1.5 L of the conidial suspension were performed per plot (6.25 m2, equivalent to 2.4×1012 conidia ha-1): the first at 5% bean flowering, at 42 days after sowing (DAS), and the second, 10 days after the first one (Huang et al., 2000). In 2010, T. harzianum was spread only once at 42 DAS, at the same dose as in 2009. In both experiments, a pre‑compression sprayer, model 417‑02 (Guarany Indústria e Comércio Ltda., Itu, SP, Brazil), with real tank volume of 3.8 L was used to spray the conidial suspensions. After the antagonist was applied, the experiments were irrigated to facilitate the spread of conidia in the soil. The number of apothecia present on the soil surface was estimated at the full‑bloom and first‑pod formation stages (62 DAS), in a 1.0‑m2 area in the center of each plot. White mold severity was evaluated at 72 DAS, at the pod‑filling stage. For this evaluation, two 1.0-m2 areas were randomly chosen within each plot. For severity evaluation, the rating scale described by Napoleão et al. (2005) was used. For statistical analysis, the mean point of each assigned grade was Pesq. agropec. bras., Brasília, v.50, n.12, p.1220-1224, dez. 2015 DOI: 10.1590/S0100-204X2015001200012 1222 DD.C. Carvalho et al. considered. Manual harvesting was performed at 97 DAS in the two central rows of each plot at 1.5‑m length. Besides grain yield (kg ha-1), the number of pods per plant, grains per pod, and mass of 100 grains (g) were determined. The results were subjected to the analysis of variance and to the Scott‑Knott test, at 5% probability, using the software Sisvar, version 5.3 (Ufla, Lavras, MG, Brazil). Soil infestation was successful, because it allowed the development of white mold in all plots, in both experiments, in 2009 and 2010. Differences were found among treatments. The CEN287, CEN316, and 1306 isolates reduced the pathogen inoculum density in both crop seasons, when compared to the control treatment not inoculated with T. harzianum (Table 1). The reduction in the average number of apothecia per square meter observed in the plots treated with these effective isolates was 46, 58, and 62% in 2009, and 73, 61, and 52% in 2010, respectively. CEN290 also reduced the number of apothecia in 2009, but did not present the same effect the following year, under higher inoculum pressure. Furthermore, only the CEN287, CEN316, and 1306 isolates proved to be effective biocontrol agents of white mold under field conditions in the two crop seasons, reducing disease severity by 77, 74, and 76% in 2009, and by 96, 80, and 84% in 2010, respectively, in comparison to the untreated control. Even with the increase in the general mean of white mold severity between 2009 and 2010, these three isolates maintained their efficiency in controlling the disease, as shown by the grouped means obtained by the Scott‑Knot test in both crop seasons. There were no differences between the isolates for yield and its components (Table 2). However, in the first experiment, in 2009, the mass of 100 grains of CEN287 was higher than that of the other isolates. This was the only significant difference observed in the means of yield and its components (Table 2). In addition, an increase in the general grain yield was verified between 2009 and 2010. Reductions in the number of apothecia per square meter and in the severity of white mold did not positively Table 1. Effect of Trichoderma harzianum on the number of apothecia per square meter and on white mold severity in 'Pérola' common bean (Phaseolus vulgaris) under field conditions, in the 2009 and 2010 crop seasons(1). Trichoderam harzianum isolate CEN287 CEN288 CEN289 CEN290 CEN316 1306 Control(3) Mean CV (%) Apothecia per square meter 2009 2010 6.7aA 10.5bA 17.5cA 8.5aA 5.2aA 4.7aA 12.5bA 9.39A 21.61 28.2aB 114.2bB 50.7aB 85.7bB 40.5aB 50.2aB 105.5bB 67.89B 27.06 Severity(2) (%) 2009 2010 6.7aA 11.6aA 27.5bA 6.8aA 7.5aA 6.7aA 28.5bA 13.62A 25.36 2.3aA 33.7bB 32.2bA 32.2bB 11.7aA 9.2aA 58.7cB 25.76B 29.60 Means followed by equal letters, lowercase in the columns and uppercase in the rows, do not differ by the Scott‑Knott test, at 5% probability. (2)Severity was evaluated according to the rating scale described by Napoleão et al. (2005). (3)Without application of Trichoderma harzianum. (1) Table 2. Effect of Trichoderma harzianum application, for biocontrol of white mold, on grain yield of 'Pérola' common bean (Phaseolus vulgaris) and its components, under field conditions, in the 2009 and 2010 crop seasons(1). Trichoderma harzianum isolate CEN287 CEN288 CEN289 CEN290 CEN316 1306 Control(2) Mean CV (%) Pods (number per plant) 2009 2010 10.7aA 10.5aA 9.8aA 12.2aA 8.7aA 11.3aA 9.1aA 8.5aA 12.0aA 11.3aA 11.9aA 11.3aA 8.4aA 9.4aA 10.1A 10.7A 19.33 29.36 Grains (number per pod) 2009 2010 5.4aA 5.0aA 5.1aA 5.1aA 5.1aA 5.0aA 5.2aA 5.1aA 5.2aA 5.1aA 5.5aA 5.0aA 5.2aA 5.3aA 5.2A 5.1A 7.00 10.11 Mass of 100 grains (g) 2009 2010 29.7aA 27.7aA 26.5bA 25.7aA 26.0bA 27.5aA 26.0bA 27.1aA 25.5bA 26.5aA 26.6bA 26.6aA 26.9bA 25.1aA 26.8A 26.6A 5.12 6.29 Grain yield (kg ha-1) 2009 2010 2,162aA 3,217aB 1,820aA 2,285aA 1,950aA 2,700aA 2,056aA 2,744aA 1,944aA 2,426aA 1,929aA 3,471aB 1,990aA 2,555aA 1,979A 2,771B 23.59 22.07 (1) Means followed by equal letters, lowercase in the columns and uppercase in the rows, do not differ by the Scott‑Knott test, at 5% probability. (2)Without application of Trichoderma harzianum. Pesq. agropec. bras., Brasília, v.50, n.12, p.1220‑1224, dez. 2015 DOI: 10.1590/S0100‑204X2015001200012 Biological control of white mold by Trichoderma harzianum affect grain yield. This can be explained by the fact that 'Pérola' common bean, a known susceptible cultivar to white mold (Napoleão et al., 2005; Geraldine et al., 2013), shows indeterminate growth linked to yield compensation after biotic or abiotic stress (Kelly et al., 1998). In this case, an extended flowering period may originate a new set of pods, out of reach of decayed apothecia, and compensate at least partial yield losses from the disease. Although severity of white mold was not enough to cause changes in grain yield, disease biocontrol with Trichoderma species can inhibit the formation of new sclerotia in the area (Abdullah et al., 2008). Pathogen inoculation in 2009, together with new sclerotia formed in the first experiment, and a new artificial infestation in 2010 resulted in increased inoculum density, from 9.4 to 67.9 apothecia per square meter in the experimental area, from one crop season to the other (Table 1). However, the antagonist did not survive for 2 years and had to be inoculated again. The increasing soil infestation with sclerotia is in alignment with reports on buildup of S. sclerotiorum inocula over time (Duncan et al., 2006); despite the increasing disease pressure, the effective antagonists CEN287, CEN316, and 1306 were still able to reduce the pathogen inoculum density in both experiments, showing their advantages for biocontrol (Table 1). The grouping of CEN287, CEN288, CEN290, CEN316, and 1306 regarding white mold severity in 2009 was considered a consequence of the lower number of apothecia per square meter in the first crop season. CEN288 was statistically similar to these isolates in terms of white mold severity in 2009, but presented a statistically higher average number of apothecia per square meter at 62 DAS, except when compared to CEN289 and the control. In the following experiment, only the CEN287 and CEN316 isolates were effective in showing stable results in both crop seasons, with their biocontrol capacity unchanged in 2010 with just one application and under increased disease pressure. This result is in agreement with Zeng et al. (2012), who highlighted that bioagents were more effective when disease pressure was high. CEN287 and CEN316 were sprayed as conidial suspension in water, devoid of the technological formulations of 1306, i.e., emulsifiable concentrate suspension. However, all three isolates proved to be effective for management of Fusarium wilt of common 1223 bean in field conditions (Carvalho et al., 2014, 2015; Guimarães et al., 2014). 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