Endosphere microbiome comparison between symptomatic and asymptomatic roots of Brassica napus infected with Plasmodiophora brassicae

Abstract

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

Fig 1. Symptoms of Brassica napus samples and PCR detection.

(a), Symptoms of B. napus samples collected from the same clubroot field; photos of asymptomatic (above) and symptomatic (underneath) samples. (b), Detection of P. brassicae in surface-peeled roots of RS1 and RS2 samples; Lane 7: sterile water; Lane 8: P. brassicae DNA; Lane M: DNA marker. (c), q-PCR detection of P. brassicae in surface-peeled roots of RS1 and RS2 samples; The B. napus actin gene served as the internal control for normalization.

Fig 2. Number of operational taxonomic units (OTUs) in different samples.

Venn diagrams were used to show the number of standardized OTUs in the three RS1 (asymptomatic) samples (RS1.1, RS1.2 and RS1.3) detected from 16S rRNA (a) and ITS (d) sequencing results, as well as the OTU numbers in the three RS2 (asymptomatic) samples (RS2.1, RS2.2 and RS2.3) detected from 16S rRNA (c) and ITS (f) sequencing results; The Venn diagrams were also used to express the number of OTUs present in at least two samples from each group between the RS1 (asymptomatic) samples (RS1 ≥2) and the RS2 (asymptomatic) samples (RS2 ≥2) detected from 16S rRNA (b) and ITS (e) sequencing results.

Fig 3

Relative microbial abundance from 16S rRNA sequencing results at the family level (a) and ITS sequencing results at the genus level (b). (a), Relative abundance of the top 15 bacterial families from the 16S rRNA sequencing results; (b), Relative abundance of the top 16 fungal genera from the ITS sequencing results.

Fig 4

Abundance of clusters from 16S rRNA with unique families (a) and shared families (b). The 16S rRNA sequencing results revealed (a) 21 unique families (16 families in the asymptomatic samples and five families in the symptomatic samples) and (b) 21 families that were common to both the asymptomatic and symptomatic samples. The clusters were based on the Z value. The Z value of one sample in a classification is equal to the value calculated from the difference between the relative abundance in the sample and the average relative abundance in all samples, divided by the standard deviation of all samples.

Fig 5

Abundance of clusters from ITS with unique families (a) and shared families (b). The ITS sequencing results revealed (a) 34 unique families (30 families in the asymptomatic samples and four families in the symptomatic samples) and (b) 37 families shared in both the asymptomatic and symptomatic samples. The clusters were based on the Z value. The Z value of one sample in a classification was equal to the value calculated for the difference between the relative abundance in the sample and the average relative abundance in all samples, divided by the standard deviation of all samples.