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Quantitative polymerase chain reaction (qPCR) is a commonly used technique for plant disease diagnosis, which generally requires DNA extraction from the plant samples. The efficiency of DNA extraction has a significant influence on the accuracy of qPCR. Thus, including a DNA extraction efficiency control is important in qPCR analyses. To develop and evaluate a DNA extraction efficiency control, a Stagonospora nodorum (syn. Phaeosphaeria nodorum , synonym and correct taxonomic name: Parastagonospora nodorum) strain named HUA-22 was created by transferring a green florescent protein (GFP) gene into the genome of the S. nodorum strain Sn15. HUA-22 showed a similar pathogenicity as Sn15 but carried a strong GFP activity. A qPCR primers/probe set named P-GFP, targeting the GFP sequence, was designed. Using P-GFP, qPCR analysis was conducted on DNA extracted from replicated samples of HUA-22 conidia, and confirmed that HUA-22 could be used to characterize the variation in replicated DNA extractions. Conidia of HUA-22 were used to spike soil samples inoculated with the canola clubroot pathogen Plasmodiophora brassicae , canola stem samples infected with the blackleg pathogens Leptosphaeria biglobosa and/or L. maculans and wheat/barley samples infected with Xanthomonas translucens pv. translucens (Xtt) or X. translucens pv. undulosa (Xtu). Duplex qPCR using P-GFP and a primers/probe set specific to P. brassicae , triplex qPCR using P-GFP and primers/probe sets specific to L. biglobosa and L. maculans , and triplex qPCR using P-GFP and primers/probe sets specific to Xtt and Xtu were conducted. The results indicated that HUA-22 could be used as a standard for DNA extraction efficiency in qPCR-based plant disease diagnosis. Adding HUA-22 conidia to plant or soil samples prior to DNA extraction, and subsequent use of the P-GFP detection control, provided an added control that could distinguish truly negative from false-negative qPCR results. • A GFP -labeled S. nodorum strain named HUA-22 was created by transformation. • GFP in HUA-22 can be used as a DNA control in qPCR diagnosis of plant diseases. • HUA-22 was tested in the diagnosis of plant diseases caused by five pathogens. [ABSTRACT FROM AUTHOR]

Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa , is a devastating cereal disease that is becoming more common in Iran. A cost-effective and realistic strategy to cope with BLS is planting of BLS-resistant cultivars. To discover BLS-resistant genotypes, 360 hexaploid wheat genotypes were selected and inoculated with a local strain of Xanthomonas translucens pv. undulosa in the field and greenhouse in two years (2019 & 2020). In the field experiments, disease reaction of wheat plants was assessed based on both percentage of disease incidence (PDI) and the percentage of disease severity (PDS). In the greenhouse experiment, disease reaction was evaluated as the disease severity (DS). The genotypes with PDS, PDI, and DS values ≤ 20% were considered BLS-resistant or semi-resistant. Genotypes with zero or very low PDS, PDI and DS during the first and second years were considered highly resistant to the disease. Despite resistant genotypes being more prevalent than susceptible genotypes in each year, genotype responses to BLS varied over the two years, but in both years, most genotypes showed a resistance response. Forty-six genotypes (12.8%) were consistently BLS-resistant across years in the field and greenhouse trials. In 2020, field BLS intensity ratings (PDI and PDS) were positively correlated with flag leaf width and days to maturity. In contrast, PDI and PDS were negatively correlated with plant height, peduncle length, and spike length. In 2019, no significant correlations were found between PDI, PDS, and phenotypic traits. Cluster heatmaps and principal component analysis (PCA) separated resistant and susceptible genotypes. In BLS-resistant genotypes, plant height, peduncle length, and spike length were significantly longer, but flag leaf width and days to maturity were shorter. This study identified new and diverse sources of resistance to BLS that could be used in wheat breeding efforts. • Three hundred and sixty wheat genotypes were screened for bacterial leaf streak response. • Field-disease and phenotypic values varied greatly among wheat genotypes. • Some disease-resistant wheat genotypes remained stable in greenhouse and fields. • A negative or positive correlation was found between disease and phenotypic traits. • Heat maps separated wheat genotypes by disease response and phenotypic traits. [ABSTRACT FROM AUTHOR]

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS) are highly destructive diseases that severely impact rice production when outbreaks occur, especially BLB. Timely identification of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) as the causative agents of these diseases is crucial to prevent and manage their spread. This study developed a detection method called loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD) to facilitate the surveillance of Xoo and Xoc in rice fields. LAMP-LFD using either Xoo80 or Xoc3863 to detect Xoo and Xoc , respectively was subjected to a heat block at 63 °C for 50 min. The LAMP products from both reactions were then hybridized with Xoo or Xoc specific probes in a single reaction and examined using a lateral pad with two tested lines. LAMP-LFD using Xoo80 or Xoc3863 demonstrated high specificity, yielding no false positives for other bacterial species. The sensitivity of LAMP-LFD using Xoo80 (with a detection limit of 2.5 × 104 cfu/ml) was ten-fold higher than Xoc3863 (with a detection limit of 2.5 × 105 cfu/ml). To evaluate the efficacy of this approach, 42 rice field samples were tested, resulting in the detection of nine and seven positives for Xoo and Xoc , respectively with two samples indicating the presence of both. LAMP-LFD using either Xoo80 or Xoc3863 showed significant promise as a simultaneous detection tool for Xoo and Xoc surveillance and management of rice disease outbreaks, ultimately reducing rice yield losses. [Display omitted] • LAMP-LFD exhibits specific detection for Xoo and Xoc in <90 mins. • LAMP-LFD enables dual detection of Xoo and Xoc in a single strip. • The limit of detection is as low as 0.2 ng/μl of gDNA and 2.5 × 105 cfu/ml of bacterial cell. • LAMP-LFD can be used for Xoo and Xoc detection in rice leaves samples. [ABSTRACT FROM AUTHOR]

Abstract: Molecular-based techniques are becoming desirable as tools for identification and detection of plant diseases. Amongst the Xanthomonas oryzae pathovars, there is a need to differentiate X. oryzae pv. oryzicola from X. oryzae pv. oryzae, as misidentification could lead to improper treatment of rice crops. Therefore, we developed a novel SYBR green real-time PCR assay to target a putative membrane protein gene of X. oryzae pv. oryzicola BLS256. The specificity of the primer set was tested using purified DNA from eleven X. oryzae pv. oryzicola strains, three X. oryzae pv. oryzae strains, and twenty-six other reference pathogenic bacteria. The primer set amplified a single band of expected size from genomic DNA obtained from X. oryzae pv. oryzicola strains, but not from X. oryzae pv. oryzae or from other bacterial strains. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA, or 0.1 × 10−5 OD600 unit of cells per reaction, respectively. This assay represents a novel approach to bacterial leaf streak diagnosis that will advance the field of rice disease research. [Copyright &y& Elsevier]

Abstract: Rxo1 cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxo1, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation. [Copyright &y& Elsevier]