Early Detection and Quantification of Tilletia spp. Infections in Wheat

Oana-Alina Boiu-Sicuia^1,2, Constantin-Alexandru Aldea¹,
Camelia Diguță¹, Indira Galit³, Matilda Ciucă³

¹University of Agronomic Sciences and Veterinary Medicine of Bucharest,
Faculty of Biotechnologies
²Research-Development Institute for Plant Protection, Bucharest
³National Agricultural Research and Development Institute Fundulea

Keywords: wheat, Tilletia spp., common bunt, PCR, Real-Time PCR, early detection.

Abstract: Wheat bunt is caused by various Tilletia spp. infections. The pathogens are non-cultivable. This particularity restricts their study to in vivo experiments, which must be conducted on cereal plants. Since the symptoms defining the infection emerge only in the advanced stages of crop development, near the harvest time, evaluating disease management efficacy becomes difficult, significantly delaying the identification of effective control measurements. Developing and validating rapid molecular methods, focused on early detection of Tilletia spp. infections in young wheat plants, would considerably reduce the time required to evaluate the efficacy of preventive strategies.
The aim of this study was to develop and validate a laboratory testing system for early identification and quantification of Tilletia spp. in young wheat plants. A bunt-susceptible wheat variety, Capo, was used for experimental inoculation with Tilletia spp. spores and grown under two controlled laboratory cultivation systems. After seven weeks, surface disinfected young plants were subjected to fungal DNA extraction. The samples were quantified and used in conventional PCR reactions with genus-specific primers for Tilletia spp., as well as species-specific primers targeting T. caries and T. laevis. Real-Time PCR was also applied for early detection and quantification of T. caries bunt infections in wheat.
Among the two tested laboratory cultivation methods, only one enabled a successful bunt infection of wheat. To achieve this, the cultures were incubated for 4 weeks at 6ºC, followed by 3 weeks of growth under diurnal conditions, at 20ºC during daylight and 15ºC at night, favoring the infection, in laboratory conditions. Molecular biology analysis confirmed the infection. Using the genus-specific primers for Tilletia spp., an expected 361 bp amplification product was obtained, whereas with the species-specific primers, an expected 276 bp product was obtained for T. caries and a 660 bp product for T. laevis. Thus, conventional PCR was validated for early detection of wheat bunt, at BBCH 12 growth stage. The protocol was further validated for T. caries using Teal-Time PCR with SYBR Green, for semi-quantitative detection.
The study concludes that wheat bunt infections are temperature dependent. A successful experimental bunt contamination, under laboratory conditions, involves culture exposure at 6ºC for one month, prior to diurnal plant growth at 20ºC/15ºC (day/night). Using specific molecular markers for targeting the pathogen, allowed early detection of infected plants, using either conventional or semi-quantitative PCR techniques. The proposed testing and analysis protocol significantly reduces the time required to validate plant protection strategies.

Acknowledgement: This research is conducted under the frame of the Romanian sectorial project ADER 3.1.1./2023 “Research on the use of molecular markers for the creation and promotion of wheat varieties with genetic resistance to cryptogamic diseases” financed by Ministry of Agriculture and Rural Development.