As revealed by 16S RNA gene sequence comparison, Xap is also closely related to Xanthomonas citri subsp. citri, encodes 4,385 protein coding genes, 50 tRNA and 3 rRNA genes 11. Xap genome (4.94 Mb) is >99% identical to Xanthomonas axonopodis pv. Xap produces smooth, circular, light yellow, glistening mucoid, butyrous and convex colonies with entire margins. Xap is cultured in vitro on different synthetic media peptone yeast extract dextrose media, nutrient glucose agar and Luria-Bertani media. Xap is a gram negative, rod shaped bacterium that measures 0.4 to 0.75 × 1.0 to 3.0 μm with single polar flagellum 10. Bacterial blight is gaining international attention through its recent spread to other major growing areas of the world such as Pakistan 7, South Africa 8, and Turkey 9. Severe disease outbreaks can cause 60 to 80% yield losses 5. Fruits exhibit isolated water-soaked lesions followed by necrosis with small cracks, leading to splitting of the entire fruit. The initial water-soaked lesions appear only after 6 to 7 days of infection under favourable field conditions and develop into late necrotic blighting 6. Bacterial blight mostly affects above ground parts of pomegranate such as leaves, twigs, and fruits 5. punicae ( Xap) is a major constraint of pomegranate cultivation and production 4. Bacterial blight caused by Xanthomonas axonopodis pv. The annual pomegranate fruit export is approximately 35,000 tonnes 3. India is the largest producer of pomegranate in the world with an annual production of 2,442 thousand tonnes grown in 209 thousand hectares 2. Additionally, the long shelf life of pomegranate encourages huge demand in domestic and international markets. Pomegranate is an important fruit crop of subtropical and tropical regions of the world and is promoted as a functional food and nutraceutical source with health promoting benefits 1. Our data demonstrate that qPCR is more sensitive than other PCR methods along with being reliable for early diagnosis. qPCR detected bacterial blight in orchards that did not show any disease symptoms. However, conventional PCR-AGE detected pathogen at the onset of disease symptoms with a detection limit of 10 pg of bacterial DNA. PCR-CE and qPCR were capable of diagnosing bacterial blight 6 to 10 days before symptom appearance, with detection limits of 100 fg and 10 fg of bacterial DNA respectively. PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and real-time PCR (qPCR) were applied for the early and accurate diagnosis of bacterial blight in pomegranate. DNA-based disease diagnostics using polymerase chain reaction (PCR) are reliable, precise, accurate and quick. Symptoms based disease diagnostic methods are labor-intensive, time-consuming and may not detect disease on asymptomatic plants. Precise and early diagnosis of bacterial blight is crucial for active surveillance and effective management of the disease. Bacterial blight drastically reduces the yield and quality of fruits, which are critical for pomegranate production. punicae is a major disease of pomegranate.
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