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

*Biotechnological Applications of Biomass*

maize defense response to *A. flavus*.

**2. Materials and methods**

**2.1 Cultures of fungi**

**2.2 Maize cultivars**

regions (http://drylandseed.com).

**2.3 Media preparation and reagents**

extraction.

providing information on the bioecology [7, 8].

tion and identification of aflatoxigenic *Aspergillus* spp. [12, 13].

the potential use of qPCR for detection and quantification of fungal pathogens and for probable selection of resistant plant cultivars in breeding lines. This also helps in understanding the processes involved in infection in a host-pathogen system and

Mayer et al. [9] and Jurado et al. [10] used single copy mycotoxin biosynthetic genes to develop PCR assays for detecting mycotoxigenic fungi. Assay sensitivity increased when ITS1 and ITS2 spacer regions were included as, these regions have sufficient variability to enable discrimination of closely related species in the genus *Aspergillus* [11]. Subsequently, these regions have been successfully used for detec-

The objective of this study was to develop a sensitive, specific qPCR assay for quantifying *A. flavus* biomass in infected maize tissues. The assay was used to measure, the sensitivity of two dry land African maize lines grown in Kenya KDV1 and GAF4, when infected with an aflatoxigenic isolate (KSM014). Similar studies have been done with other fungal species, but this study is the first where the biomass of *A. flavus* from infected maize was detected and quantified with qPCR. This approach also could be used to discriminate between inbred maize lines that are sensitive or resistant to specific *A. flavus* strains and to help understand the mechanism of the

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

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

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

**512**

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 adjusted to 1 × 106 conidia ml−1 using a hemocytometer was used to inoculate the 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 nitrogen prior to DNA/RNA extraction and stored at −80°C.
