Greenhouse Inoculation Assays: Mesocosm Experiments

Two independent 1-year-long mesocosm experiments were conducted as previously described (Bonilla et al., 2015) to perform disease assessment, bacterial survival and metagenomic analysis. The independent experiments were named “assay 1” (season 2017/18) and “assay 2” (season 2018/19). These assays started 120 days before R. necatrix was inoculated (which was considered day 0) and ended 110 days after pathogen inoculation (the experiments last 230 days in total; Figure 1).

Experimental design of the mesocosm assays. Two independent experiments were performed, treatments in each independent experiment are detailed in Table 2. Seventeen independent mesocosms were used for each of the treatments assayed (eleven mesocosms were inoculated with R. necatrix, and six mesocosms remained non-inoculated). (A) Schematic view of the mesocosms timeline. Experiments started 120 days (–120 days) prior to the R. necatrix inoculation (considered as day 0). One hundred and two 2 years-old avocado plants were sown in commercial soil. At this time started the treatments with the positive control, amending composted almond shells to the corresponding pots. At –50 days, the preventive treatment with PcPCL1606 and PcPCL1606-GFP were applied. 70 days after R. necatrix inoculation, the curative PcPCL1606 treatment was applied. Disease monitoring was performed just after R. necatrix inoculation. Sampling points for further analysis were stablished at T0 (previous to the preventive treatment), T1 (after preventive treatment), T2 (after R. necatrix inoculation), T3* (taken only during biocontrol in “assay 2”) and T4 (end of the experiment). (B) Aspect of the mesocosms experiment at –70, 30 and 110 days along the experiment.

Briefly, an experimental microplot platform that mimicked field conditions was designed and constructed for the plant assays at the IHSM-UMA-CSIC “La Mayora” (Algarrobo Costa, Spain, 36°45′37.74″ N – 4°02′26.28″ W). The greenhouse was built as an open structure with double roofing to allow air passage for improved ventilation, and the microplots (mesocosms of 35 liter plant pots) were planted in a white gravel bank to reduce oscillation of the soil temperature. Environmental conditions were monitored during each experiment by using a portable data logger, which recorded the air temperature and relative humidity.

In each independent assay, a total of 102 two-year-old commercial avocado seedling plants (cv. Topa-Topa) were independently transplanted to 35 liter pots filled with a blend (1:1) of solarized natural soil and peat and randomly placed into the experimental area. Each plant into its pot constitute an independent mesocosom. Seventeen independent avocado plants were used for each of the treatments assayed in these studies as listed in Table 2. For each independent treatment, eleven plants were inoculated with R. necatrix to study multitrophic interactions during biocontrol, and the remaining six non-inoculated plants were used as controls to study multitrophic interactions without the presence of the pathogen. Fungal inoculation was performed as previously described (Sztejnberg and Madar, 1980; Cazorla et al., 2006). Briefly, four holes per pot were made on the soil surface using a punch, and 16 g of wheat colonized with R. necatrix strain CH53 was distributed in the holes before filling them with the surrounding soil.

Main characteristics of the treatments used in the microcosm assay.

The first assay, “assay 1,” was designed to test the biocontrol of different treatments with formulated PcPCL1606 as a biologically active product against R. necatrix and to study the impact of the application of PcPCL1606 on natural microbial populations with R. necatrix inoculation. PcPCL1606 treatments were applied by irrigating a final cell concentration of 1.0 × 10¹⁰ cfu suspended in 200 ml of sterile water. Treatments were applied using watering to properly distribute the bacterial cells onto the whole pot surface. One of the bacterial treatments was a preventive application (PcPCL1606 preventive), consisting of a single application with a semi-commercial formulate of PcPCL1606 (PcPCL1606 preventive) performed 50 days before inoculation with R. necatrix. A second treatment was a curative application (PcPCL1606 curative), using the same semi-commercial formulate of PcPCL1606, which was added after symptoms appeared. The application was performed 70 days after inoculation with R. necatrix. A third treatment was included and consisted of a control treatment designed to compare the accuracy of the bacterial counts in different culture media. For this, the PcPCL1606-GFP derivative strain was used in the experiments, following the preventive protocol described above (Table 1 and Figure 1). A bacterial suspension of PcPCL1606-GFP (growing in liquid TPG medium for 24 h at 25°C and 180 rpm) reached a bacterial concentration of 1.4 × 10⁹ cfu/ml. A total of 10¹⁰ cfu per plant was applied in this treatment (as described above) and allowed specific bacterial counts from soil and rhizosphere samples. In a second assay (“assay 2”), only the preventive semi-commercial formulated PcPCL1606 treatment was repeated to confirm its biocontrol efficacy on the pathogen in the previous assay and to study the impact of PcPCL1606 application on the microbial communities during biocontrol (Table 1 and Figure 1).

In both assay 1 and assay 2, two control treatments were included. First, a positive control of biocontrol consisted of developing soil-induced suppressiveness to R. necatrix (Vida et al., 2016). For this, 19 liters of composted almond shells (ASO treatment) was placed on the top layer of 16 liters of soil and peat. This positive control treatment was initiated 150 days before R. necatrix inoculation to allow the soil to induce suppressiveness (Figure 1 and Table 1; Vida et al., 2016). The negative control consisted of a group of 17 avocado plants without bacterial treatments.