Rapid in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

Abstract

As the incidence of fungicide resistance in plant pathogens continues to increase, control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9–100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

Globalizing plant health

“…Molecular methods of diagnosis are improving at an impressive pace (Bueno-Sancho et al, 2017;Dodhia et al, 2021;Hu et al, 2019;Hubbard et al, 2015;Knight et al, 2021;Radhakrishnan et al, 2019;Zulak et al, 2018), rendering a global sampling of wheat disease levels both technically and financially feasible. The COVID-19 pandemic has had the effect of distributing modern high-throughput molecular diagnostic capabilities to almost every country of the world, including all 60 that grow wheat at a significant scale.…”

Complex System Analysis of Blumeria graminis f.sp. tritici Isolates Collected in Central Hebei Province, China

“…In China, the steady growth of grain production is the basis of food security. As one of the three major grain varieties, wheat accounts for about 5% of China's total grain output [1,2]. At the same time, wheat is the main food source for residents in northern China and plays an important role in its dietary structure, so the fluctuation of wheat production has a great impact on national food security [3][4][5].…”

“…tritici starts from the 1980s, and the degree of harm and extension become enlarged increasingly [16][17][18][19]. e average annual incidence area of powdery mildew in major wheat areas in China is more than 6 million hm 2 . In 2019, the cumulative occurrence area of wheat powdery mildew in China was close to 8.7 million hm 2 , and the average diseased leaf rate in individual regions was even as high as 100% [20][21][22].…”

“…e average annual incidence area of powdery mildew in major wheat areas in China is more than 6 million hm 2 . In 2019, the cumulative occurrence area of wheat powdery mildew in China was close to 8.7 million hm 2 , and the average diseased leaf rate in individual regions was even as high as 100% [20][21][22]. In recent years, the damage of wheat powdery mildew continues to expand, showing a trend of gradual aggravation from south to north, which poses a serious threat to the safe production of wheat in China, but the proportion of resistant wheat varieties is low, and there is a lack of effective resistance genes [23][24][25][26].…”

Sensibilidad y resistencia a fungicidas de <i>Cercospora kikuchii</i>, agente causal del tizón de la hoja y mancha púrpura de la semilla de la soja

“…El modo o mecanismo de resistencia puede ser analizado en diferentes etapas tales como molecular, genética, bioquímica, fisiológica, y a nivel de población (Gisi et al, 2000;Rusell, 2003;Ishii, 2006;Hollomon e Ishii, 2015). Actualmente se están desarrollado diferentes "kits de campo" que facilitan la determinación de la resistencia in situ, ingresando la información en una base de datos coordinada vía internet, permitiendo el monitoreo oportuno de los perfiles de resistencia específicos para cada ubicación geográfica (Schnabel et al, 2012;Khater et al, 2017;Hu et al, 2019;Radhakrishnan et al, 2019;Mechan et al, 2020;Dodhia et al, 2021). Por lo tanto, el monitoreo en el tiempo de la sensibilidad de patógenos a fungicidas es de máxima importancia para poder maximizar la vida útil y la eficiencia de uso de los i.a.…”