EVALUATION OF TWO PROTOTYPES BASED ON Bacillus amyloliquefaciens FOR THE CONTROL OF Fusarium oxysporum IN CAPE GOOSEBERRY García Jennifer, Grijalba Erika, Díaz Andrés, Alarcón Erika, Gómez Martha CORPOICA (Colombian Corporation for Agricultural Research), Tibaitatá Research Center, Mosquera; Colombia. e-mail:
[email protected].
INTRODUCTION The disease known as vascular wilt caused by the phytopathogenic fungus Fusarium oxysporum can potentially cause losses between 80% and 100% in Cape Gooseberry, significantly affecting the yield and productivity of this crop (Fischer et al., 2014, Amézquita et al., 2010, Góngora et al., 2006). In this context, the use of beneficial microorganisms is an environmentally friendly alternative for the control of aggressive pathogens such as F. oxysporum, for which there is not an effective and safe chemical pesticide in the market. The bacterium Bacillus amyloliquefaciens has the capability to produce a wide range of biodegradable and low toxicity compounds with antifungal, antibacterial and plant growth promotion properties; it also has the ability to form spores that make it ideal for the development of biopesticides (Yanez-Mendizabal et al., 2012). In recent studies conducted in Corpoica, the strain of Bacillus amyloliquefaciens Bs006 showed its potential as biocontrol agent of F. oxysporum MAP5. Therefore, two formulation prototypes based on B. amyloliquefaciens were developed and its microbiological stability at different temperatures and biological activity against F. oxysporum MAP5 under controlled conditions were evaluated.
METHODOLOGY
RESULTS AND DISCUSSION Evaluation of stability and biocontrol activity of prototypes based on B. amyloliquefaciens
B. amyloliquefaciens strain Bs006 was obtained from the Germplasm Bank of Microorganisms of Corpoica and was maintained in Luria Bertani LB agar (yeast extract 5 g, peptone 10 g, NaCl 10 g, and agar 10 g in 1 L of water) at 4°C. The bacteria was grown for 48 h at 28°C with shaking (125 rev min-1) in a culture media previously standardized (Díaz et al., 2015). This culture was used as inoculum for fermentation process in a 13 liters STR bioreactor (Infors® Labfors 3), with a working volume of 8 liters and applying a pre-established cascade of agitation and aeration during 48 hours. Development of prototypes based on B. amyloliquefaciens Bs006
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10,5 Log10 (CUF/ml)
Microorganism, inoculum development and liquid fermentation
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P1. 0 m
P1. 1,5 m
P1. 3 m
P2. 0 m
Separated Biomass
Prototype P1
Fermentation broth
4°C
Prototype P1 Prototype P2
Response Variable:
25°C 35°C
Cell Viability (CFU/ml) to 0, 1.5, 3 months
The experimental unit was one Falcon® Centrifugate Tube (50 ml) by triplicate for each temperature, and a full randomized complete block design was applied. The response variable was analyzed by ANOVA procedure followed by means comparison (Tukey´s test, 95%) using program Statistix® v.8 Evaluation of B. amyloliquefaciens prototypes against F. oxysporum MAP5 in greenhouse Cape gooseberry plants (height ≈ 15 cm), were transplanted to nursery bags containing 700 grams of a mix of sterilized soil, peat and rice husk in proportion of 3:1:1. All treatments (Table 1) were inoculated with F. oxysporum MAP5 (1x104 microconidia/ g soil) before sowing the plants.
AUDPC
Stability evaluation of Prototypes based on B. amyloliquefaciens Bs006
STORAGE BY 3 MONTHS
C. 1,5 m
C. 3 m
Figure 1. Storage stability of formulation prototypes based on Bs006. P1: Prototype 1, P2: Prototype 2, C: Fermentation broth without formulation. Letters represent homogeneous groups obtained using Tukey analysis (P < 0.05) by each prototype
Prototype P2
Fermentation broth
C. 0 m
The P1 formulation prototype presented a presented 12% viability loss at 4°C at 25° and 35° C viability losses were less than 10%, in the P2 prototype viability losses at the three temperatures were lower than 6%. This shows that the used formulation auxiliaries conferred additional protection to the organism due to the unique nature of the components used in each formulation (Jayaraj et al., 2005). On the other hand, the control treatment (unformulated fermentation broth) showed losses of 11.7%, 12% and 5.5% at 4°C, 25°C and 35°C respectively at the end of 3 months of storage, which proves the inherent resilience of the spores of B. amyloliquefaciens to the formulation and storage processes (Kloepper, 1991 cited by Jayaraj et al., 2005). In general, prototypes and control were stable (
