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2.2.9..1 Maize seed sterilization

Maize seeds were surface sterilized using the procedure of Martinez & Wang (2009) with some modifications. Approximately 200 g of seeds were placed in a 500 ml beaker along with a stir bar. Then ethanol (80%) was added and the beaker covered with an aluminium foil and placed on a stir plate and stirred at medium speed. After 3 min the ethanol was decanted, and seeds were washed with sterile water. After the first wash, HClO4 (50%) was added to the beaker containing the seeds and covered with the paper foil and stirred at medium speed. After 15 min the seeds were washed for four times with sterile water. The procedure with HClO4 (50%) and subsequent rinses with sterile water was repeated again. Finally, seeds were dried using sterile paper towels and filtered air in the laminar flow cabinet. All maize seed surface sterilization procedures were carried out in a laminar flow.

2.2.9..2 Root exudate collection

Root exudates were obtained following the methodology described by Wyrebek, Huber, Sasan, & Bidochka (2011) with minor modifications. Approximately 40 surface-sterilized maize seeds were transferred to an Erlenmeyer flask (250 ml) containing 100 ml of sterile water. Flask were closed with sterile cotton plugs to avoid contamination while allowing gas transfer. The flasks containing the seeds were maintained on an orbital shaker at 140 rpm at room temperature. Once 90% of the seeds were physiologically germinated (protrusion of the radicle), samples were kept on the shaker at 25°C for an

filtration through a series of filters to reduce clogging. First exudates were filtered through a Whatman cellulose filter (grade 2, 8 µm), then through 0.8 µm Millipore filter and finally 0.2 µm Millipore filter (Figure 2.1). Composition of roots exudates in sugars, organic acids and amino acids were determined by HPLC (Shimadzu).

Figure 2.1 Root exudates collection. Sterilized maize seed was incubated in distilled water at room temperature in an orbital shaker platform at 140 rpm for 8 days

2.2.9..3 Spectrophotometric determination of fungal growth in RE and in PDB

The fungal growth was determined using the spectrophotometric methodology described by Meletiadis, Meis, Mouton, & Verweij (2001) with some modifications. For all the isolates tested the concentration of the conidial suspension was obtained in two stages. First, the suspension was adjusted to 1 x 105 _{conidia/ml in 0.01% Triton X-100, and then to 1 x 10}4 _{conidia/ml using the medium} where fungal growth would be tested, PDB or roots exudates. From this conidial suspension (1 x 104 conidia/mL) in 50% RE or PDB, 220 µl were taken and transferred to a well of a 96-well plate. Each isolate was tested in duplicate per plate. The spectrophotometer was set at 25°C, 200 RPM and at 405 nm. The plate was subjected to 10 s of orbital agitation before the optical density (OD) was measured every 15 min.

2.2.9..4 Kinetic parameters determination

In order to compare the growth curves for each isolate in both nutrient media, a number of kinetic parameters were calculated based on the changes of the OD over time with the Skanlt Sofware 3.2.0.36 RE for Multiskan GO (Multiskan GO 1.00.40) and based on the obtained growth curve of each isolate in each medium. The kinetic parameters determined were: the highest OD (Maximum), the average growth rate (Rate, s-1_{), the integral (slope), the OD change as OD}

max– ODmin (Change), the Maximum rate (Max. rate min-1_{) and the time at which the maximum growth rate was reached (T MaxR, h.).} Additionally, from each curve the exponential growth was determined and equations that describe

kinetic microbial growth (Widdel, 2010) were calculated: the specific growth rate (µ min-1_{); the} duplication time or generation time (Td, h-1_{) and the time when the lag phase ends (Tlagf, h}-1_).

2.2.10Costelytra giveni bioassays with entomopathogenic fungal isolates

Bioassays were conducted using the method of Glare (1994) with some modifications. Second and 3rd instar larvae of C. giveni, were field collected and kept individually in sections of a 24 well-plate with a small piece of carrot at 18 ± 2°C in the dark. After 72 hrs, larvae were checked for feeding and natural mortality. Only healthy, feeding larvae were selected for bioassays. Conidia were harvested with 3 – 5 mL of a solution of 0.01% Triton X-100 added to 15 days old fungal colonies grown in half strength PDA at 20 ± 2 °C in light:dark conditions (12:12 h). Conidial suspensions were quantified with an improved Neubauer chamber and adjusted to 105_{, 10}7 _{and 10}9 _{conidia/mL. Soil was semi-sterilized at} 90°C for 48 hrs and then 10 grams of soil was added to Falcon tubes of 50 mL. To adjust the final soil moisture, each Falcon tube received 1 mL of 0.01% Triton X-100 and 1 mL of the corresponding conidial suspension (20% w/w) or 2 mL of 0.01% Triton X-100 and 1 mL of the conidial suspension (30% w/w). After soil and conidial suspension were mixed, one larva of C. giveni was added and the tube was loosely closed to allow gas exchange (Figure 2.2).

Figure 2.2 Costelytra giveni bioassays with entomopathogenic fungi. Soil was semi-sterilized, and 10 grams of soil were added to Falcon tubes of 50 mL. Each tube received 1 mL of the corresponding conidial suspension and moisture was adjusted to 20% w/w or 30% w/w. One larva of C. giveni was added, tube was closed loose to allow gas exchange and incubated at 20 ± 2 °C. Every 5 days larvae were checked for survival, mortality or fungal infection.

Larvae were kept for at least 40 days at 20 ± 2 °C and were checked every five days for mortality and fungal infection (mycosis). Dead larvae were kept in the Falcon tubes with the lid closed tight to keep

sporulation. For each bioassay, tubes were arranged in a randomized block design with four blocks. In each block, there were seven isolates at each conidial concentration (105_{, 10}7 _{and 10}9_{) plus an} untreated control (CS). The controls M. anisopliae A1080 and CS were replicated three times in each block. The bioassay was carried out three times. In order to combine data over the three bioassays, treatment means were input into an analysis of variance which treated bioassays as “blocks”, with treatments having a seven (isolates) by three (conidial concentrations) factorial structure. Untreated controls were omitted from this statistical modelling. The three conidial concentrations (105_{, 10}7 _and 109_{) were also further modelled using linear and quadratic polynomial contrasts (assuming equal} spacing on a log scale). Comparison of isolates employed an unprotected least significant difference (LSD) procedure (Saville, 2015).