The biocontrol bacterium Pseudomonas fluorescens Pf29Arp strain affects the pathogenesis‐related gene expression of the take‐all fungus Gaeumannomyces graminis var. tritici on wheat roots

Abstract

The main effects of antagonistic rhizobacteria on plant pathogenic fungi are antibiosis, fungistasis or an indirect constraint through the induction of a plant defence response. To explore different biocontrol mechanisms, an in vitro confrontation assay was conducted with the rhizobacterium Pseudomonas fluorescens Pf29Arp as a biocontrol agent of the fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In parallel with the assessment of disease extension, together with the bacterial and fungal root colonization rates, the transcript levels of candidate fungal pathogenicity and plant-induced genes were monitored during the 10-day infection process. The bacterial inoculation of wheat roots with the Pf29Arp strain reduced the development of Ggt-induced disease expressed as attack frequency and necrosis length. The growth rates of Ggt and Pf29Arp, monitored through quantitative polymerase chain reaction of DNA amounts with a part of the Ggt 18S rDNA gene and a specific Pf29Arp strain detection probe, respectively, increased throughout the interactions. Bacterial antagonism and colonization had no significant effect on root colonization by Ggt. The expression of fungal and plant genes was quantified in planta by quantitative reverse transcription-polymerase chain reaction during the interactions thanks to the design of specific primers and an innovative universal reference system. During the early stages of the tripartite interaction, several of the fungal genes assayed were down-regulated by Pf29Arp, including two laccases, a β-1,3-exoglucanase and a mitogen-activated protein kinase. The plant host glutathione-S-transferase gene was induced by Ggt alone and up-regulated by Pf29Arp bacteria in interaction with the pathogen. We conclude that Pf29Arp antagonism acts through the alteration of fungal pathogenesis and probably through the activation of host defences.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease

“…At 3 weeks post-inoculation, the disease severity in each plant was assessed as the percentage area of take-all lesions covering the root system (Daval et al ., 2011). The infection types (ITs) were categorized from 0 to 4 according to Bithell et al .…”

“…To investigate the take-all resistance of transgenic wheat lines further, Q-RT–PCR was used to assess the relative abundance of Ggt in transgenic wheat plants based on Ggt 18S rRNA (FJ771002; Daval et al ., 2011) in reference to wheat 18S rRNA (AY049040). The primer sequences for Ggt 18S rRNA and wheat 18S rRNA in Q-RT–PCR analyses are listed in Table 1.…”

“…The disease known as take-all, caused by the necrotrophic fungus Gaeumannomyces graminis var. tritici ( Ggt ), is one of the most destructive root diseases of wheat worldwide (Gutteridge et al ., 2003; Daval et al ., 2011). The disease begins by Ggt hyphae penetrating the cortical cells of the root and progresses upwards into the base of the stem, and even leads to premature death of the infected plant.…”

pH effect on strain-specific transcriptomes of the take-all fungus

“…The expression levels of 8 selected DEGs were determined by qRT-PCR to confirm the results of RNA-Seq analysis. Total RNA from Ggt mycelium was reverse transcribed with a set of two external RNA quality controls as previously described [8,23]. Briefly, for all the strains and the pH conditions, 750 ng of total RNA from fungi were mixed with known quantities of the two external controls.…”

“…Moreover, each specific amplicon was sequenced (Genoscreen, Lille, France) to confirm it corresponds the expected sequence. The expression levels of transcripts were normalized using external RNA controls [23,24] from three independent biological replicates, each with three technical PCR replicates. Data were analyzed using the ANOVA procedure of the R statistical analysis software.…”