Reliable and early diagnosis of bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae using sensitive PCR techniques

Doddaraju, Pushpa; Kumar, Pavan; Gunnaiah, Raghavendra; Gowda, Abhishek A.; Lokesh, Veeresh; Pujer, Parvati; Manjunatha, Girigowda

doi:10.1038/s41598-019-46588-9

Cited by 34 publications

(20 citation statements)

References 23 publications

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“…Immediately afterwards, without further bacterial growth, we harvested leaf discs within inoculated areas using a cork borer. We ground the discs and used some of the lysate for Xe 85-10 CFU counting, and the remainder for DNA extraction and hamPCR targeting the V4 16S rDNA and the pepper GI gene (CaGI), and qPCR, targeting the xopQ gene for a Xe type III effector (Doddaraju et al, 2019), and the C. annuum UBI-3 gene for a ubiquitin-conjugating protein (Wan et al, 2011).…”

Section: Utility In Diverse Hosts and Crops With Large Genomesmentioning

confidence: 99%

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Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Lundberg

Ayutthaya

Strauß

et al. 2021

eLife

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The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

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Section: Utility In Diverse Hosts and Crops With Large Genomesmentioning

confidence: 99%

“…A primer set targeting the gene for the type III effector XopQ of pathogenic Xanthomonas was used to measure abundance of Xe 85-10 ( Doddaraju et al, 2019). For C. annuum, primers targeting the UBI-3 gene encoding a ubiquitin-conjugating protein were used (Wan et al, 2011).…”

Section: Quantitative Real Time Pcr On C Annuum Samplesmentioning

confidence: 99%

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Lundberg

Ayutthaya

Strauß

et al. 2021

eLife

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show abstract

“…Immediately afterwards, without further bacterial growth, we harvested leaf discs within inoculated areas using a cork borer. We ground the discs and used some of the lysate for Xe 85-10 CFU counting, and the remainder for DNA extraction and hamPCR targeting the V4 16S rDNA and the pepper GI gene ( CaGI ), and qPCR, targeting the xopQ gene for a Xe type III effector 54 , and the C. annuum UBI-3 gene for a ubiquitin-conjugating protein 55 . Sequencing the hamPCR libraries revealed that as the Xe 85-10 infiltration concentration increased, so did the resulting load of the ASV corresponding to Xe 85-10 ( Supplementary Figure 16 and 17a ).…”

Section: Utility In Diverse Hosts and Crops With Large Genomesmentioning

confidence: 99%

Host-associated microbe PCR (hamPCR): accessing new biology through convenient measurement of both microbial load and community composition

Lundberg

Ayutthaya

Strauß

et al. 2020

Preprint

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The microbial population size, or load, in a host is a fundamental metric of colonization by commensals or infection by pathogens. Sequence-based measurement of DNA amount contributed by host and microbes in a sample provides a simple way of measuring microbial load, and it also has the ability to estimate microbiome diversity. Unfortunately, it is also very costly, especially when host DNA greatly outweighs microbial DNA. We introduce a robust two-step PCR strategy to quantify absolute abundance of the host-associated microbiome and describe its composition in a single, cost-effective amplicon library. We demonstrate the accuracy and flexibility of this method across multiple amplicons and hosts, and further present a simple technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without a loss of information.

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“…Presently, the normalization of quantitative gene expression is carried out by several traditional reference genes, including 18S [7], Actin [8], GAPDH [9], and CYP [9]. However, in pomegranate reference genes such as 18S rRNA, Ribosomal protein S, named PgRPSΙΙ, and GAPDH has been used for gene normalization in various gene expression studies [10][11][12]. However, this may misinterpret the gene expression because all reference genes may not constantly express in different tissues under diverse environmental conditions.…”

Section: Introductionmentioning

confidence: 99%

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

2021

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Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis.In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

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Reliable and early diagnosis of bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae using sensitive PCR techniques

Cited by 34 publications

References 23 publications

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Host-associated microbe PCR (hamPCR): accessing new biology through convenient measurement of both microbial load and community composition

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

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