Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Abstract

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers-ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.

Quantification of disease progression of Alternaria spp. on potato using real-time PCR

“…Since its development in the 1990s, real-time PCR has emerged as a reliable and sensitive method for detecting and quantifying phytopathogenic and antagonistic fungi (Schena et al 2004), and represents a highly sensitive and specific technique for the detection and quantification of nucleic acids (Tylor et al 2001). Protocols for the qualitative recognition of Alternaria species in sunflowers and foodstuff have recently been established (Pavón et al 2012;Udayashankar et al 2012). Quantitative detection of A. alternata in tomato has been developed by Schuhegger et al (2006).…”

Rapid, specific and sensitive molecular detection assay for Alternaria helianthi that causes leaf blight disease in sunflower

“…The ITS regions of ribosomal RNA often contains pathogen specific conserved sequences that enable designing of genera and species consensus primers (White et al 1990;Xue et al 1992). Also the high copy numbers of rRNA genes in a fungal genome serve as an (Jasalavich et al 1995;Kusaba and Tsuge 1995;Pavlina et al 2002;Udayashankar et al 2012). In the present instance, primers specificto-conserved as well as the unique sequences in the ITS2 region of rRNA of A. helianthi were used to develop the assay.…”

“…Healthy sunflower seeds were artificially infected by the method described by Udayashankar et al (2012) and observed under the stereo microscope for the presence of mycelial and conidial growth of A. helianthi after 8 days of incubation at 22±2°C under alternating 12 h periods of darkness and near ultraviolet (NUV) light.…”

“…Alternaria leaf blight reduces the average flower size, number of seeds per plant, seed yield per plant, seed weight and percentage filling of seed (Balasubramanyam and Kolte 1980). In India up to 90 % yield loss and 34 % oil loss have been documented due to this disease (Udayashankar et al 2012;Balasubramanyam and Kolte 1980). In addition, the pathogen was also reported to influence seed germination and seedling vigour drastically (Chander 2003).…”

“…Similarity in the conidial morphology of other Alternaria species associated with sunflower also increases the inaccuracy. PCRbased assays (Udayashankar et al 2012) available for detection of A. helianthi from infected plants and seeds lacks sensitivity and relies on the use of internal amplification control, to rule out the role of PCR inhibitors for false-negative results. Similarly, the conventional PCR based detection of A. helianthi could fail to detect pathogen from seed at low levels of infection.…”