**2. Materials and methods**

## **2.1 Cultures of fungi**

The aflatoxigenic *A. flavus* KSM014 isolate was cultivated and maintained as described previously [14] and thereafter stored as spore suspension in 15% glycerol for short term storage at −20°C or for long term storage at −80°C prior to DNA/RNA extraction.

#### **2.2 Maize cultivars**

GAF4 maize lines and KDV1 varieties were obtained from Kenya Agricultural and Livestock Research Organization (KALRO), Kenya. The selection of the varieties are focused mainly on their drought tolerance and the agro-ecological zones in which they were grown. Striga tolerant variety (GAF4) is produced by KALRO Kibos, Kisumu County. GAF4 is cultivated in Homa Bay, Kisumu and Busia counties [15]. The Kenya Dryland Varieties 1 is an open pollinated hybrid recommended for medium to low altitude areas. KDV1 is drought tolerant, matures early and produce flowers after germination between 45 and 52 days. It is mainly cultivated in Homa Bay and Makueni regions (http://drylandseed.com).

### **2.3 Media preparation and reagents**

Phytagel, Nicotinic acid, Glycine, Thiamine hydrochloride, Murashige and Skoog medium (MS), Potassium hydroxide, Pyridoxine hydrochloride and Myoinositol were from Sigma-Aldrich (USA). MS vitamins; 5 g myo-inositol, 500 mg Thiamine-HCl, 500 mg pyridoxine-HCl, 250 mg nicotinic acid and 100 mg glycine were filter sterilized after preparation in distilled water and thereafter stored at −20°C according to the instructions of the manufacturer's (Sigma-Aldrich, USA). The modified MS media was briefly prepared, 2.15 g MS salt was dissolved in sterile H2O, thereafter, 10 ml MS vit. added and pH 5.7 adjusted using 1 M KOH and volume further adjusted to 1 l using sterile H2O. 5 g of phytagel was added to MS media

**513**

**2.6 Designing of primers**

*Fungal Biomass Load and* Aspergillus flavus *in a Controlled Environment*

**2.4 Seed sterilization and** *Aspergillus flavus* **infection**

nitrogen prior to DNA/RNA extraction and stored at −80°C.

**2.5 The extraction of gDNA from maize tissues and** *Aspergillus flavus*

Technologies, USA). DNA was diluted to 10 ng/μl for further analysis.

Sets of 3 primers (**Table 1**); elongation factor 1 alpha (*Ef1ɑ*), *β*-tubulin, and membrane protein (*MEP*) were used in the current research. *MEP* and *Ef1ɑ* were

A 100 mg of each of the following samples: control healthy maize tissues, infected and *A. flavus* KSM014 mycelia was used to extract DNA following the method of Möller et al. [16] with some modification. Briefly, 100 mM Tris pH 8.0, 2% SDS, modified TES buffer, 2% (w/v) polyvinylpyrrolidone (PVP) and 10 mM EDTA was prepared. 5 μl RNase (10 mg/ml) and 450 μl of TES buffer was added to microtube (2 ml) containing the tissues and thereafter, homogenized by vortexing for 15 min or with a microtube pestle. Twenty microlitres of Proteinase K (1 μg/μl) was added and vortexed for 1 min, thereafter, incubated for 1 h at 60°C. Seventy microliters of 10% CTAB (0.1 vol.) and 160 μl of 5 M NaCl (0.3 vol. was added and incubated at 65°C for 10 min). Seven hundred and fifty microliters of chloroform/ isoamyl-alcohol (24:1) was added, vortexed for 5 min, incubated on ice for 30 min and centrifuged at 14,000 rpm for 10 min. The aqueous phase was transferred cautiously onto a new microtube (2 ml) and 300–350 μl isopropanol (0.55 vol.) added and left to stand for 30 min at RT after mixing gently for 30 s. The mixture was centrifuged for 10 min at 14,000 rpm. Supernatant was cast-off, the pellets rinsed with chilled 700 μl of 70% ethanol twice and centrifuged again at 14,000 rpm for 2 min after mixing gently. The pellets were air dried and dissolved in 40 μl TE buffer (10 mM Tris-Cl pH 8, 1 mM EDTA pH 8) or nuclease free water after discarding the ethanol. The integrity of DNA was assessed on a 1% agarose/EtBr gel and the concentration quantified on a Nano-DropTM 1000 spectrophotometer (Nano Drop

and heated in microwave to dissolve the salts. Fifty milliliters of the media was dispensed into tissue culture bottles, autoclaved and thereafter, cooled in the level 2 biosafety cabinet for approx. 1 h prior to inoculations as previously described [14].

The seeds were sterilized in a biosafety cabinet, level 2 [Contained Air Solutions (CAS) BioMAT2, UK]. Twenty milliliters of 95–100% ethanol was used for sterilization of viable seeds for 1 min and briefly shaken for 15 s. The alcohol was replaced with 20 ml of sodium hypochlorite (2.5%). After 15 min of reaction at room temperature, the mixture was shaken for 30 s and thereafter, the liquid discarded. 30 ml of sterile H2O was used 5× to wash the seeds with intermittent shaking after every wash. 50 ml of sterile H2O was added and left to stand for 1 hr at rmt. The H2O was replaced with 20 ml of 2% Tween 20 and shaken for 30 s. Conidia suspensions

seeds. The seeds in the tubes were para filmed after sealing and kept for 30 min in a shaking incubator at 30°C. Controls were treated with sterile H2O instead of spores of conidia and thereafter, incubated following the same conditions. The seeds were left to dry in Petri dishes after inoculations overlaid with filter paper overnight (Whatman No. 1). The seeds were germinated in a plant growth chamber, Conviron (Winnipeg, Manitoba, Canada) set at 28°C after subsequent inoculations onto tissue culture bottles. The germination and growth were observed for a 14-day period, tissues of the plant (roots and shoots) were separately harvested, flash frozen in liquid

conidia ml−1 using a hemocytometer was used to inoculate the

*DOI: http://dx.doi.org/10.5772/intechopen.93307*

adjusted to 1 × 106

and heated in microwave to dissolve the salts. Fifty milliliters of the media was dispensed into tissue culture bottles, autoclaved and thereafter, cooled in the level 2 biosafety cabinet for approx. 1 h prior to inoculations as previously described [14].
