**3. Results and discussion**

### **3.1 Gene specificity and qPCR assays**

To our knowledge, a qPCR assay for the detection and quantification of *A. flavus* biomass using extracted fungal DNA from control or infected maize tissues has not been previously reported. Since this is the first report, our discussion will be in comparison with reports for *Fusarium* spp. and related fungi where this assay is more commonly used.

In this study, the qPCR assay was developed to specifically detect and quantify *A. flavus* gDNA in maize tissues. Primers were designed, and their specificity was confirmed by testing against control and infected tissues (**Figure 1**). The fungal biomass in the co-infected shoots differed from the fungal biomass in the roots according to 1-way ANOVA analysis and TMCT test (P < 0.05).

Amplification of the MEP gene (203 bp) was used to detect maize DNA, while amplification of *β*-tubulin (118 bp) and *Ef1ɑ* (102 bp) were used to detect *A. flavus* DNA (**Table 1**; **Figure 1**). The specificity of the primer pairs was determined by conventional PCR (**Figure 1**) after *A. flavus* KSM014 infection of GAF4 and KDV1 maize lines. *A. flavus* DNA extracted from infected maize plant tissues, for both lines, gave an amplification product for both *β*-tubulin (118 bp) and *Ef1a* (102 bp) (**Figure 1**). However, there was amplification product for *Ef1a* than there was for to *β*-tubulin (**Figure 1**), especially in the roots. The *MEP* gene (203 bp) was amplified in both control and infected maize plants for both lines (**Figure 1**). *MEP* amplification was plant specific and *β*-Tub and *Ef1ɑ* were fungal specific. Based on these results, *β*-Tub is a better marker for detecting *A. flavus* in infected maize tissues than was *Ef1ɑ* (**Figure 1**) and was used for fungal biomass determination.

#### **Figure 1.**

*Gel images of the quantitative polymerase chain reaction amplicon sizes for maize maker gene (*MEP*) and*  A. flavus *maker genes (*Ef1ɑ, β-*tub) assessed on 2% agarose/EtBr gel run at 80 v for 45 min. M. 100 bp ladder; 1. NTC; 2. Pooled samples (maize gDNA and pure fungal gDNA); 3. GAF4 (control roots); 4. GAF4 (infected roots) 5. GAF4 (control shoots); 6. GAF4 (infected shoots); 7. KDV1 (control shoots); 8. KDV1 (infected shoots); 9. KDV1 (control roots); 10. KDV1 (infected roots); 11. KSM014 (positive control).*

**517**

**Figure 2.**

*plants.*

(**Figure 3**).

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

and Homa bay appeared more resistant to the infection (**Figure 2**).

and control plant tissues as revealed by the qPCR assay (**Figure 3**).

due to putative plant defense mechanisms [21].

*Aspergillus flavus* KSM014 infection of both maize lines resulted in changes in maize phenotype with the KDV1 showing more severe symptoms that GAF4 (**Figure 2**). After 3–14 days post infection, the infected kernels for both maize lines showed stunted growth compared to control kernels (**Figure 2**). Additionally, the shoots and roots exhibited minimal growth with the *A. flavus* fungi colonizing the kernels and this could possibly explain the reason for stunted growth or germination. The phenotypic observations suggest that KDV1 maize line grown in Makeuni is more susceptible to fungal infection (*A. flavus*), whereas GAF4, grown in Kisumu

The observed phenotypic characteristics were further supported by the detection and quantification of fungal biomass load in gDNA extracted from infected

Insignificant difference was observed in biomass of fungi between infected plant tissues for the GAF4 and the control maize line (**Figure 3a**). In contrast, significant differences in biomass of fungi for the KDV1 maize line was exhibited upon infection (p < 0.05) for both the shoot and root tissue (**Figure 3b**). Fungal gDNA level was observed to be lower in the infected GAF4 maize line tissues compared to KDV1 suggesting that GAF4 was more resistant to *A. flavus* KSM014 infection than KDV1

The fungal biomass of *Alternaria dauci* was observed to be equivalent in two carrot cultivars between 1 and 15 days of post-inoculation, whereas it was found to be four-fold higher in the more susceptible cultivar between 21 and 25 days postinoculation [21]. This suggests that fungal pathogens may colonize both susceptible and resistant cultivars in a similar manner during the first stages of the interaction, but fungal development is subsequently restricted in the partially resistant cultivar

It must be noted that we measured fungal biomass 14 days after infection when

symptoms of the infection was phenotypically visible. However, other fungal biomass studies have shown that specific fungi could be identified even before the development of the symptoms. The presence of *Colletotrichum acutatum* by qPCR in

*The GAF4 and KDV1 maize lines after 14 days of growth with and without* Aspergillus flavus *KSM014 infection. The red sticker shows infected maize plants while the white stickers are the control, uninfected maize* 

**3.2 Colonization of plant tissues by** *Aspergillus flavus*

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