**2.3 Screening of clone colonies of collected RWA samples for determination of potential biotypes**

The biotype of each RWA clone was determined by screening its feeding damage on 11 previously established plant resistant sources containing designated resistance genes *Dn*1 to *Dn*9 and *Dnx* and *Dny* (**Table 1**). Infestations of RWASA1 cause susceptible damage symptoms on wheat entries containing the *Dn2* and *Dn*3 gene (**Table 1**). RWASA2 cause susceptible damage symptoms on wheat entries containing *Dn*1, *Dn2*, *Dn*3, *Dn8* and *Dn*9 resistance genes (**Table 1**). RWASA3 is distinguished from RWASA2 by its added virulence to *Dn*4 and RWASA4 is distinguished from RWASA3 by its added virulence to *Dn5* (**Table 1**). RWASA5 is distinguished from RWASA4 by its added virulence to *Dn*6 and *Dn*x (**Table 1**).

Ten seeds of each plant entry were planted in a seedling tray filled with sterilized sand in a randomized complete block design with four replications for each biotype

#### **Figure 1.**

*Sampling sites for Russian wheat aphid (RWA) in the Western cape (winter rainfall area), South Africa from 2010 to 2019.*

#### **Figure 2.**

*Sampling sites for Russian wheat aphid (RWA) in the Free State (summer rainfall area), South Africa from 2010 to 2019.*

determination. Plant entries were randomly assigned to rows and were separated by border rows planted with RWA susceptible Tugela. Plants were kept in glasshouse cubicles at night/day temperatures of 16 °C/22 °C, natural light. Immediately after planting, the seedling trays were placed in gauze (315micron) cages to avoid contamination by secondary aphids. Plants were infested at the two-leaf stage with collected RWA clone colonies. Plants were rated with a ten-point damage rating scale, which included leaf chlorosis and leaf rolling [29]. A score from 1–4 describes leaf chlorosis, 5–6 striping on the leaves and 7–10 rolling. Once the susceptible wheat Tugela showed susceptible damage symptoms, all plants were rated. RWA biotypes were classified

*Russian Wheat Aphid Distribution in Wheat Production Areas: Consequences of Management… DOI: http://dx.doi.org/10.5772/intechopen.96375*


**Table 1.**

*Comparison of plant reaction of the five* Diuraphis noxia *biotypes identified in South Africa.*

by using damage ratings for each plant entry where the plant was considered resistant (R) if the damage rating was 1–6.5 and susceptible (S) if the damage rating was above 6.5–10. Each clone was given a biotype designation based on the differential virulence profile to the *Dn*1 to *Dn9* and Dnx and Dny resistance genes (**Table 1**).

Biotype (clones) groups across all plant differentials were analyzed using a two-way (clone, plant entry) analysis of variance (ANOVA). Mean damage rate entries with significant (P < 0.05) clone-by-plant interactions were separated by Fisher's protected least significant difference (LSD) test at the 5% level (SAS Institute 2003).
