Histopathology of rust infection in wheat and barley
Maree, Gerrie Johanna
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The rate at which new virulent rust races emerge continues to leave global cereal production vulnerable. Since genetic defence is the most effective method to combat rust pathogens and minimize crop losses, the continuous identification and incorporation of diverse, durable resistance sources in new varieties are imperative for sustainable cereal production. In addition to visual scoring, histological analysis is a valuable method to investigate resistance mechanisms whether conferred by a single gene or several quantitative trait loci (QTL). Earlier, assessments of wheat (Triticum aestivum L.) stripe rust [caused by Puccinia striiformis Westend. f. sp. tritici (Pst)] resistance in South Africa resulted in identification of the durable adult plant resistance (APR) gene Lr34/Yr18/Sr57 and QTL QYr.sgi-2B and QYr.sgi-4A in the cultivar Kariega. In the present study, stripe rust colonization in Kariega x Avocet S doubled haploid (DH) lines, carrying different gene and/or QTL combinations, was compared through fluorescence microscopy. The flag leaves of field-infected adult plants stained with fluorophore Uvitex 2B, attested to the increased resistance effects of multiple loci appearing in a single host genotype compared to individual resistance loci. Additionally, the importance of gene/QTL identity in specific combinations was highlighted, with significant variation displayed among DH lines in terms of colony length (μm), number of haustorial mother cells per colony and hypersensitivity indexes (P<0.05). To diversify the sources of stripe rust resistance in South African wheat germplasm, Palmiet (QYr.ufs-4B) was previously crossed with breeding line Yr16DH70, retaining contributing APR QTL QYr.ufs-2A, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D from Cappelle-Desprez. Evaluation of recombinant inbred lines under field conditions included Pst colony length visualised with fluorescence microscopy and accumulation of fungal biomass through RT-qPCR. Besides the confirmation of enhanced resistance of co-occurring resistance loci, the choice of QTL was accentuated in the varying levels of defence conditioned by different QTL combinations. Carriers of QYr.ufs-2A or QYr.ufs-2D accompanied by at least one other QTL exhibited higher resistance levels than single QTL and combinations not including either the 2A- or 2D-chromosome QTL, with significant variation found among lines across parameters (P<0.05). Barley (Hordeum vulgare), host to the wheat and rye attacking forms of the stem rust pathogen, Puccinia graminis (Pg), is considered inherently more resistant to Pg than wheat. To investigate whether this enhanced basal defence is associated with the early infection or colonization processes, adult plants of selected barley lines and wheat control entries were inoculated with Pg f. sp. tritici Erikss. and Henning pathotypes UVPgt54 and UVPgt60, and Pg f. sp. secalis Erikss. and Henning pathotype UVPgs01. Flag leaf sheaths on the last stem internode were sampled for analysis. Using scanning electron microscopy and epidermal stripping, no obvious differences in early infection structure development were observed between barley and wheat. Sub-stomatal vesicle appearance and production of haustorial mother cells were similar at 24 and 48 hours post-inoculation (hpi), respectively. Aborted Pg infection structures were regularly detected in, specifically, the rpg4/Rpg5 gene complex-containing barley entries, and may be an indication of early onset of defences in certain host genotypes. Parallel data was obtained at 120 hpi using the WGA-FITC probe to measure colony sizes (μm2) and RT-qPCR for assessment of accumulated fungal biomass. Significant variation occurred among entries (P<0.05) while the difference between rust pathotypes as well as the interaction between the two factors proved insignificant (P>0.05). The latent period between 120 and 240 hpi may hold some explanation, as a possibly steeper increase in accumulated fungal biomass in the susceptible wheat line did not reflect in opposing susceptible barley entries at 240 hpi.