Limitations in controlling white mold on common beans with Trichoderma spp. at the fall-winter season

1EPAMIG, Vila Gianetti 47, CEP 36570-000 Viçosa, MG, Brazil; 2EPAMIG, CP 176, CEP 37200-000 Lavras, MG, Brazil; 3Embrapa Meio Ambiente, CP 69, CEP 13820-000 Jaguariúna, SP, Brazil; 4Univ. Fed. de Viçosa, CEP 36570-000 Viçosa, MG, Brazil. E-mail: hudsont@epamig.br, rfvieira@epamig.br, mmorandi@cnpma.embrapa.br, millerlehner@gmail.com, renan.lima@ufv.br, jesc@ufv.br Autor para correspondência: Trazilbo José de Paula Júnior (trazilbo@epamig.br) Data de chegada: 24/04/2012. Aceito para publicação em: 29/08/2012. 1815

The spreading of white mold caused by Sclerotinia sclerotiorum (Lib.) de Bary have been favored by the environmental conditions of many irrigated areas cultivated with common beans (Phaseolus vulgaris L.) in Brazil, that usually present moderate temperatures (15-22 °C) during the fall-winter season (14). Most of these areas are infested with sclerotia leading frequently to expressive losses if disease is not adequately controlled. S. sclerotiorum can attack more than 400 plant species (3). Applications of fungicide are generally efficient to control the disease, but they increase the costs and have important ecological implications. Other strategies used to manage the disease include sowing of non-infested seeds, low plant population, upright cultivars, crop rotation with cereals, low irrigation frequency, and biological control (4, 14,16).
Trichoderma is one of the more intensively investigated biocontrol agents (7). Trichoderma spp. are particularly prevalent in humid environments, but they can be isolated from all climatic zones (8).
This antagonist associates with sclerotia of S. sclerotiorum and cause sclerotia to degrade or not germinate. The potential of the biological control with isolates of Trichoderma spp. has been reported for S. sclerotiorum in vitro (10). Applications of T. harzianum 1306 in Brazilian cerrado fields have successfully controlled the pathogen (4). The demand for Trichoderma products for the control of soilborne pathogens has increased significantly in Brazil (2, 12). However, applications of Trichoderma conidia to control white mold at moderate temperatures may be not efficient, since the antagonist is more adapted to temperatures above 25 °C (8). In a field trial with average temperature bellow 18 °C, Paula Júnior et al. (15) have not found antagonistic effects of T. harzianum against S. sclerotiorum. The purpose of this work was to test the application of Trichoderma isolates for white mold control at the fall-winter season.
A field experiment was carried out from May 5, 2008, in an experimental area of the Federal University of Viçosa, Minas Gerais, RESUMO Palavras-chave adicionais: Phaseolus vulgaris, Sclerotinia sclerotiorum, controle biológico, manejo integrado. Brazil (20°46'05" S, 42°52'10" W, elevation 662 m). The inoculum of S. sclerotiorum in this area is high and homogenously distributed. Treatments were arranged in a randomized complete block design with four replicates. An isolate of T. harzianum (LQC 88) successfully selected in vitro and in greenhouse for S. sclerotiorum control at 18-22 ºC (11) and four Trichoderma isolates from commercial products were tested (Table 1). In the case of the isolate LQC 88, two 5 mm diameter mycelial-agar disks of the growing fungus on Petri dishes with PDA were transferred to 200 mL-Erlenmeyers flasks containing rice grains. After incubation for 15 days at 25 °C for conidia production, the inoculum was ground on trays. All Trichoderma isolates were applied (1 x 10 9 conidia g -1 ) over the plants and soil through sprinkler irrigation water at 20 and 46 days after emergence (DAE). Another treatment was carried out with applications of the fungicide fluazinam (0.5 L ha -1 ) at 46 (pre-flowering stage) and 56 DAE by a backpack spray equipped with one cone nozzle delivering 667 L ha -1 of fungicide solution. These treatments were compared with water (untreated control).
Irrigation was provided as needed to promote good seedling emergence and establishment, and a rate of approximately 40 mm of water per week thereafter, as generally used in the region. The common bean cultivar BRSMG Majestoso (prostate plants, type II/III, carioca class) was sown in rows spaced 0.5 m apart. Plots of seven 3.5 mlong rows were sown on May 2008. Density of 10 plants per meter was adjusted by thinning two weeks after sowing. All plots received a basal fertilization: 24 kg ha -1 of N, 37 kg ha -1 of P, and 40 kg ha -1 of K. Ammonium sulfate application (200 kg ha -1 ) as side dressing was performed 20 DAE, together with foliage application of molybdenum (80 g ha -1 ) as sodium molybdate. Weeds were controlled by hand hoeing and with a commercial mixture of the herbicides fomesafen (250 g ha -1 ) and fluazifop-p-butyl (200 g ha -1 ). Insects were controlled, when needed, with monocrotophos (400 mL ha -1 ). The fungicide azoxystrobin (60 g ha -1 ) was applied once before flowering to protect beans against foliar diseases.
An area of 1.2 m 2 (one internal row without 0.3 m at each end) in the plots was harvested separately at 90 DAE for white mold evaluation. Disease incidence was calculated as the percentage of plants with symptoms. Plants were rated for disease severity index (DSI) by means of a "quarter scale" (6), where 0 = no disease present, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, and 4 = 76% to 100% of the plant with white mold symptoms. DSI was calculated on a percentage basis: DSI (%) = (scores of all plants)/[4 x (total number of plants)] x 100. Yield data were estimate based on mass of seeds with 12% moisture (w/w) harvested in 3.6 m 2 (included the 1.2 m 2 area harvested for disease evaluation). Data were subjected to variance analysis and means of either the Trichoderma or the fluazinam treatments were compared to the untreated control by Dunnett's test (P < 0.05).
In general, high intensity of white mold was observed, with disease incidence about 100% in all treatments (Table 1). Only the applications of fluazinam reduced the disease severity (26.3%), compared to the untreated control. There was no effect of Trichoderma spp. and fluazinam applications on bean yield. Results of field experiments in the same experimental area from 1997 to 2006 indicated an average yield increase of 35.6% with fluazinam application, with average disease incidence and severity of 58.0% and 31.8%, respectively (13). These results confirm that in 2008 the disease intensity was higher than the expected for this area, which can be explained by the climate conditions favorable to the white mold spread, especially high relative humidity and low temperatures (Fig.1).
In a study carried out at the 2004 fall-winter season in the same field, the attempts in controlling white mold on common beans with T. harzianum also failed (15). The high disease pressure in the field in 2004 and in this experiment may have influenced the efficacy of the antagonist, although the Trichoderma applications have been done according to manufactory's instructions in both trials. Low temperatures during the fall-winter crop may better explain the lack of effectiveness of the biological control of white mold on common beans with Trichoderma spp. in the Zona da Mata region in the State of Minas Gerais. Even the isolate LQC 88, which was selected at a suboptimal temperature, did not reduce the disease severity. Trichoderma is more adapted to temperatures higher than 25 °C (8). In this study, the average temperatures were 16.7 °C in June, 15.4 °C in July, and 18.4 °C in August (Fig. 1). The average of maximum temperatures was slight higher than 25 °C only in August. Isolates of Trichoderma spp. have been found in the soil (10) as well as associated to sclerotia of S. sclerotiorum (1) in Brazilian cerrado areas, where applications of the antagonist have reduced the severity of white mold in the field (4). In these areas average temperatures at the fallwinter season are higher than in the Zona da Mata region. Therefore, best results are expected for the antagonists in the cerrado areas, since the average temperature in these areas occurs outside the best temperature range for the pathogen (9). Interactions among bean plants, S. sclerotiorum and Trichoderma are also influenced by the relative  humidity, since the activities of the antagonist may be also increased by high humidity (5). However, our results suggest that low temperatures are determinant for non recommending Trichoderma applications for control of white mold on common beans in the Zona da Mata region and other Brazilian regions where beans are cultivated as fall-winter crop, since this condition favor more the pathogen than the antagonist. Further studies in these regions could focus the biological control with antagonists more adapted to moderate temperatures, as Conyothirium minitans Campbell (14).