Endospore-Forming Bacteria Present in a Commercial Stabilized Poultry Manure Determines the Fusarium Biocontrol and the Tomato Growth Promotion
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agronomy Article Endospore-Forming Bacteria Present in a Commercial Stabilized Poultry Manure Determines the Fusarium Biocontrol and the Tomato Growth Promotion German F. Sepúlveda Chavera 1,*, Mabel Arismendi Macuer 1,2 and Patricio Muñoz Torres 1,2 1 Laboratorio de Patología Vegetal y Bioproductos, Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Parcela 27, Colonia Juan Noé, Campus Azapa km 12 Valle de Azapa, Arica 1000000, Chile; [email protected] (M.A.M.); [email protected] (P.M.T.) 2 UC Davis Chile Life Sciences Innovation Center, Av. Andrés Bello 2299, Providencia, Santiago 1102, Chile * Correspondence: [email protected] Received: 7 September 2020; Accepted: 14 October 2020; Published: 23 October 2020 Abstract: Stabilized organic amendments (SOA) from poultry are used in agriculture to improve the conditions of the soil. SOAs favor the growth of the crops and reduces the effect of soil-borne plant-pathogens. However, in northern Chile, there are no studies to support this observation, nor have the mechanisms involved in the beneficial effects observed in the field been established. This work aims to establish whether the promotion of growth and control of soil fungi in tomato observed in the field as a result of commercial SOA application can be attributed to different endospore-forming bacteria (EFB). The effect of commercial SOA on nutrient availability was determined. EFB isolated from a commercial product, and the application of bacterial isolates were compared with the commercial formulation of SOA, for plant growth promotion (PGP) and biocontrol of Fusarium oxysporum fsp. radicis-lycopersici (FORL). The local tomato cultivar Poncho Negro was used given its sensitivity to different nutritional alterations and FORL. A series of measurements of growth parameters were carried out in plants submitted to different mixtures of SOA treatments. Isolates were identified by biochemical tests and sequencing of the 16S rRNA gene. Eleven EFB were isolated from SOA, and some tests were performed to determine the PGP and biocontrol of FORL activities of each isolate. Notably, isolates BAC22 (Bacillus megaterium), BAC21, and BAC23 (B. amyloliquefaciens/velezencis) were associated with PGP, highlighting the ability to produce indole-3-acetic acid, a trait that in many cases is key to explaining the effects of Bacillus spp. Keywords: PGP bacteria; poultry manure; biocontrol; Fusarium oxysporum fsp. radicis-lycopersici 1. Introduction The soil of the Azapa Valley (Arica, Chile) is naturally poor in organic matter [1], and the cultivation of vegetables requires incorporating organic amendments in large quantities to achieve acceptable production levels. The stabilized organic amendments (SOA), generated from spent beds of the poultry industry, are mainly used in tomato production, and its incorporation allows the improvement of the biological conditions of the soil. The application of SOA, derivated from the poultry industry, favors the growth and mitigation of the effect of soil-borne plant pathogens, such as Fusarium oxysporum in tomato crops. However, there are no studies that support this observation, nor have the mechanisms involved in the effects observed in the field been established. Different authors have shown that the use of manure as an amendment can replace the use of synthetic fertilizers, providing nutrients, improving soil structure, water retention capacity, porosity, as well as increasing the beneficial microbial population [2–6]. The microbial population present or induced as a result of the application of manure is largely responsible for the effects attributed to the Agronomy 2020, 10, 1636; doi:10.3390/agronomy10111636 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1636 2 of 14 use of organic amendments [7], through an enhancement of nutrient availability, including carbon, nitrogen, and phosphorus, and improved soil pH, increasing microbial biomass, species richness and promoting the proliferation of certain species, which participate in the decomposition of complex organic matters and the promotion of plant growth [8]. The commercial SOA used in the Azapa Valley is produced through a standardized aerobic process that aims to eliminate phytopathogens, reduce odors, and decrease or eliminate potential putrefaction [9]. This process is maintained for more than two months, making sure that temperatures above 70 ◦C are produced for at least 30 days, decreasing the particle size considerably, and the C/N ratio drops below 35, allowing the marketing of this commercial SOA. This process favors the persistence of endospore-forming bacteria, which could play a key role in the beneficial effects on tomato plants by the application of SOA to the soil. Among the endospore-forming bacteria that act as promoters of plant growth or plant growth-promoting rhizobacteria or PGPR [10], members of Bacillus genus are the most widely studied, being more than a half of the bioformulations of agricultural application produced from strains of Bacillus genus [11]. There are two main reasons for the success of this group of bacteria as bioformulates for agricultural use: (1) they can promote plant growth and phytopathogen control [12,13]; and (2) they possess the capacity to produce endospores and have a high degree of tolerance to different environmental challenges, including temperature, desiccation, ultraviolet light, among others [14,15]. In this work, we propose to establish whether the promotion of growth and control of soil fungus Fusarium in tomato observed in the field is a result of the application of commercial SOA to soil and could be attributed to the presence of different species of Bacillus genus and other endospore-forming bacteria. For this purpose, the effect of the application of commercial SOA on nutrient availability was determined. Subsequently, endospore-forming bacteria were isolated from the product, and finally, the application of bacterial isolates was compared with the commercial formulation of SOA, in the plant growth promotion and biocontrol of Fusarium oxysporum fsp. radicis-lycopersici (FORL). 2. Materials and Method 2.1. Plant Material The local cultivar Poncho Negro was used, given its sensitivity to different nutritional alterations [16] and FORL [17]. The seeds were sown and grown for 20 days in the germination tray of 240 cavities in sterile peat before being used in the experiments. 2.2. FORL Strain Growth Conditions and Preparation The strain of FORL F62 used in this work was isolated from a tomato crop in the Azapa Valley. This strain was identified and characterized by Sepulveda et al. [18] and maintained in the Phytopathogens Collection of the Plant Pathology Laboratory (University of Tarapacá). To ensure and re-activate its pathogenicity before being used for the experiments, it was inoculated into tomato plants as described below: for the infection of FORL in tomato, the fungus was grown for 7 days in Petri dishes with solid PDA (15% agar-agar). The mycelium was taken and planted in a flask with liquid PDA supplemented with 3% agar-agar and incubated in an orbital shaker incubator Gyromax SK727 (Amerex instrument, Concord, CA, USA) for 72 h at 150 rpm and 26 ◦C. A suspension of conidia adjusted to 1 106 conidia/mL was used to infect tomato plants. × 2.3. Nutrients in Percolation and Tomato Growth with Different Doses of SOA Poncho Negro seedlings were taken after 20 days of growth and placed in 250 mL plastic containers with 200 mL of different SOA/perlite mixtures: control corresponding to 200 mL of perlite (SOA 0% w/v); SOA 5% w/v, 10 g SOA 190 mL perlite; SOA 10% w/v, 20 g SOA/180 mL perlite; SOA 20% w/v, 40 g SOA/160 mL perlite; SOA 40% w/v, 80 g SOA/120 mL perlite; SOA 80% w/v, 160 g SOA/40 mL perlite. Aiming to know the effect of the different doses of SOA on the availability of nutrients, pH values, and Agronomy 2020, 10, 1636 3 of 14 electrical conductivity (EC), after placing the plants in each of the respective treatments, holes were made in each plastic container to receive the percolation for 12 days. Table1 shows the nutrients and the method used for each parameter analyzed. Table 1. Organic matter, nitrogen content, acidity and electrical conductivity (EC) in stabilized organic amendment (SOA) samples. Parameter Unit Observation * EC 1:2.5 mS/cm 22.10 Acidity pH 8.01 NO3− mg/kg 246.00 Org. Mat. % 32.65 * Each value represents the average of three repetitions. 2.4. Growth Evaluation of Tomato Plants After 12 days, a series of measurements of growth parameters were carried out in the plants submitted to the different mixtures of SOA treatments. Plant height was measured from the neck of the plant to the last shoot, using a measuring tape. Leaf and root weight were determined using an analytical balance Adam NBL 84e (Adam equipment, Kingstone, UK). The number of leaves and length of leaves was registered. Additionally, the thickness of stems was measured using a digital caliper (Liaoning Mec Group Ltd., Liaoning, China). 2.5. Isolation of Endospore-Forming Bacteria (EFB) from SOA Isolation of EFB from SOA was performed using the thermal shock method described by Al-Humam [19] with some modifications, 10 g of SOA was placed in 90 mL of sterile distilled water and agitated at 150 rpm for 1 h, and subsequently placed in a thermal bath at 80 ◦C for 30 min. After this step, the solution was maintained without agitation at room temperature for 2 h to sediment soil particles. Samples of 100 µL were taken from three different batches to perform three serial dilutions in Petri dishes containing solid Lysogeny Broth (LB) medium (ATCC medium 1065) and kept at 25 1 C. ± ◦ The isolated colonies with different morphologies were reseeded in plates with LB to obtain pure cultures of each isolate. 2.6. Biochemical Tests and Molecular Identification Isolates were identified at the genus level using Gram staining, a catalase test, motility, starch hydrolysis, citrate utilization, endospore staining, and a hemolysis test [20].