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Optimized inoculation procedures are an important consideration in achieving repeatable plant infection when working with biotrophic rust fungi. Several plant pathology laboratories specializing in rust research employ a system where the collection and application of fungal spores are accomplished using an exchangeable gelatin capsule. Urediniospores are collected from erumpent pustules on plant surfaces into a capsule fitted to a cyclone collector controlled by a vacuum pump. By adding light mineral oil to the same capsule, the spore suspension is then sprayed onto plants by means of a dedicated atomizer (inoculator) connected to an air pressure source. Although devices are not commercially available, modern day technologies provide an opportunity to efficiently design and manufacture collectors and inoculators. Using a process called Additive Manufacturing (AM), also known as “3D printing,” the bodies of a collector and inoculator were digitally designed and then laser-sintered in nylon. Depending on availability, copper or aluminum tubes were fitted to the bodies of both devices afterward to either facilitate directed collection of spores from rust pustules on plant surfaces or act as a siphon tube to deliver the spore suspension contained in the capsule. No statistical differences were found between AM and metal inoculators for spray delivery time or spore deposition per unit area. In replicated collection and inoculation tests of wheat seedlings with urediniospore bulks or single pustule collections of Puccinia triticina and P. graminis f. sp. tritici , the causal organisms of leaf rust and stem rust, consistent and satisfactory infection levels were achieved. Immersing used devices in acetone for 60 s followed by a 2 h heat treatment at 75°C produced no contaminant infection in follow-up tests.

The bread wheat cultivar Kariega has maintained its stripe rust resistance since the first detection of Puccinia striiformis f. sp. tritici (Pst) in South Africa during 1996. Doubled haploid mapping population (MP) lines derived from a Kariega 9 Avocet S cross, carrying consistently detected adult plant resistance (APR) quantitative trait loci (QTLs)/gene combinations, were phenotyped at macroscopic and microscopic levels. Field data obtained over four seasons revealed that MP lines carrying a combination of any two of the APR loci QYr.sgi-2B.1, QYr.sgi-4A.1 or Yr18 displayed low coefficients of infection. Lines MP 45 and MP 65, carrying all three gene regions, showed leaf area infected and host reaction type ratings similar to Kariega. The microphenotype of lines was studied in flag leaves sampled from field plots during two seasons using fluorescence microscopy. Pst colony length, number of haustorial mother cells per colony and hypersensitivity index supported the phenotypic data. All three microscopy variables attested to low levels of disease in lines containing multiple stripe rust resistance loci. Lines MP 51 and MP 223 with a single QYr.sgi-2B.1 and Yr18, respectively, also showed adequate resistance, in contrast to lines carrying only QYr.sgi-4A.1 which showed significantly more disease symptoms. Host cell necrosis and lignification were revealed as mechanisms of resistance in some lines.

Depending on the pathogenicity of the stripe rust fungus Puccinia striiformis f. sp. tritici, the nature of resistance in the wheat host plant, and the environment, a broad range of disease phenotypes can be expressed. Therefore, the phenotyping of partial adult plant stripe rust resistance requires reliable and repeatable procedures, especially under controlled conditions. In this study, the development of a flag leaf point inoculation method, which resulted in a 100% initial infection rate, is reported. Flag leaf inoculations were achieved by placing 6-mm antibiotic test paper discs, dipped into a urediniospore and water suspension and covered with water-proof plastic tape, on the adaxial side of leaves. Results from independent trials allowed for the statistical comparison of stripe rust lesion expansion rate in wheat entries that differ in resistance. The technique is inexpensive, reliable, and applicable to routine screening for adult plant response type, quantitative comparison of stripe rust progress, environmental influences, and pathogenicity of different isolates.