Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus

Abstract

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

A Species-Specific PCR Based Assay for Rapid Detection of Mango Anthracnose Pathogen Colletotrichum gloeosporioides Penz. and Sacc

“…The identification of fungal pathogens based on polymerase chain reaction (PCR) using species-specific primers is now widely used, especially for economically important plant pathogens such as quarantine listed fungi or those that are difficult to isolate or cause symptomless [6,7]. The internal transcribed spacer regions (ITS1 and ITS4) within the nuclear ribosomal gene clusters are particularly attractive loci for the design of PCR-based detection assays since they are readily accessible using universal primers [8] typically present in high copy number increasing PCR sensitivity and often exhibiting sufficient inter-specific sequence divergence for the designing of species-specific primers [9].…”

Black Scab of Jojoba (Simmondsia chinensis) in Australia Caused by a Putative New Pathotype of Elsinoë australis

“…Several authors (12,14) have reported using the technique of Whiteside (25) to produce conidia of Sphaceloma spp. ; however, we were not able to induce sporulation using this technique.…”

“…The species-specific primers of Hyun et al (12) were used to further characterize the isolates of Elsinoë from jojoba. Primers Eaut1 and 5 along with EaNat1 were utilized.…”

“…In all, 17 isolates of Elsinoë from jojoba were examined for genetic diversity using random amplified polymorphic DNA (RAPD) primers OPX-8, OPX-12, and OPX-17 identified by Hyun et al (12), and compared with 6 Elsinoë isolates from citrus (E. australis strains Ea-2, Ea-4, VRG-BRT, 70212, FZ-STM-CL-1.4, and 70041) (13). Reactions (20 µl) consisted of 1× buffer (Bioline), 0.2 mM each dNTP, 2.5 mM MgCl 2 , 0.5 µM each primer, 1 unit of BioTaq DNA polymerase (Bioline), and 5 ng of template DNA.…”

Reaction of tangerines genotypes to Elsinoe fawcettii under natural infection conditions

“…It attacks a wide variety of citrus species and cultivars, resulting in scab disease on leaves, twigs, and fruits , Hyun et al 2007, Goes and Reis 2009.…”