IDENTIFICATION OF COLLETOTRICHUM ACUTATUM FROM YELLOW AND ANDEAN LUPIN SEEDS USING PCR ASSAY

Tomasz Kulik¹, Agnieszka Pszczółkowska¹, Jacek Olszewski¹, Gabriel Fordoński¹, Krystyna Płodzień¹, Ewa Sawicka-Sienkiewicz^2
1 Department of Diagnostics and Plant Pathophysiology, University of Warmia and Mazury in Olsztyn
² Department of Genetics, Plant Breeding and Seed Production, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

PCR assays were used to identify Colletotrichum acutatum and C. gloeosporioides out of 14 fungal cultures obtained from seed samples of selected yellow and Andean lupin cultivars. The results allowed for a classification of all fungal cultures to C. acutatum. No C. gloeosporioides culture was identified. In addition, the comparison of rDNA sequence fragment from one of the cultures tested with sequences available in the NCBI/GeneBank database confirmed that the species identified was C. acutatum. The BIO-PCR technique was also used to detect potential inoculum of both species in two lupin seed samples. The results of BIO-PCR assays showed that two lupin seed samples were infected with C. acutatum while no isolate of C. gloeosporioides was detected.

Key words: yellow lupin, Andean lupin, anthracnose, Colletotrichum acutatum, Colletotrichum gloeosporioides, BIO-PCR.

INTRODUCTION

Anthracnose of lupin was believed to be mainly caused by Glomerella cingulata (Stonem) Spauld et v. Schrenck, and its conidial stage, Colletotrichum gloeosporioides. In the recent years the disease has become widespread in all parts of the world where lupin is cultivated [4,9]. In Poland its occurrence was observed in 1995 [4].

Anthracnose lesions can form on all above-ground parts of the lupin plant. The most distinctive symptom is bending and twisting of stems with a lesion in the crook of the bend. This is particularly noticeable during flowering. Stem lesions are usually dark brown and elongated up to about two centimeters in length. A pale pinkish spore mass develops within lesions. The stem is often completely girdled by these lesions or weakened so that it breaks. Both the main stem and lateral branches can be affected and a close inspection often shows similar symptoms on leaf petioles [8]. Seed infection by Colletotrichum spp. is considered to be the primary source of infection [4].

Both C. acutatum and C. gloeosporioides are collective species, encompassing various groups of strains and biotypes. Traditional methods for discriminating between species of Colletotrichum rely on morphology and host preference. The use of morphological characteristics is complicated due to environmental influences on these characteristics and the existence of intermediate forms [2,7]. As an alternative, species-specific PCR primers derived from ITS1 rDNA sequence have been used to distinguish between C. acutatum and C. gloeosporioides [1,3,7].

Most reports attribute lupin anthracnose in Europe to C. gloeosporioides [4,9]. However, the application of species-specific PCR revealed that the main cause of lupin anthracnose in Australia is C. acutatum [12].

The objective of the present study was:

1. identification of 14 isolates of Colletotrichum spp. isolated from seeds of yellow lupin grown in the Warmia and Mazury region and Andean lupin from Lower Silesia to a species level using PCR technique with species-specific primers,

2. detection of C. acutatum and C. gloeosporioides from two lupin seed samples using BIO-PCR assays.

Scientific hypothesis assumed identification and detection of Colletotrichum spp. with species-specific PCR assays infecting lupin seeds.

MATERIAL AND METHODS

Origin of seed samples and fungal cultures

Yellow lupin seeds (Lupinus luteus L.) originated from Warmia and Mazury region (`Amulet´, `Juno´, `Markiz´, `Legat´ and `Polo´) while Andean lupin seed sample came from Lower Silesia region (Table 1).

One hundred seeds of each sample were washed, surface sterilized for 3 min in sodium hypochlorite (1% available chlorine), rinsed in sterile distilled water and then placed on PDA. The material was incubated at 22°C for 24 hours. The fungal cultures were classified to the genus Colletotrichium according to their morphology (appearance of a culture, conidia) examined under microscope, numbered and inoculated on separate Petri dishes. After these preparations, the material was used for DNA isolation.

DNA isolation

Fungal DNA was extracted by the protocol according to Parry & Nicholson [11] with slight modifications. Mycelium (0.20 g) was scraped from a colony growing on a PDA plate, frozen in liquid nitrogen and ground to a fine powder in a pestle and mortar. DNA was extracted in 20 mL CTAB buffer (CTAB 8 g, sarkosyl 10 g, sorbitol 25 g, NACl 47 g, EDTA 8 g, PVPP 10 g in 1 L H₂O) at 65°C, for 30 min, in 1.5 mL centrifuge tubes. An equal volume of chloroform/octanol (24:1) was added, mixed and centrifuged at 14000 rpm for 10 min. The aqueous phase was removed to a fresh tube and an equal volume of isopropanol was added followed by centrifugation as above to precipitate the DNA. The pellet was washed in 70% ethanol and dissolved in TE buffer (10 mM Tris- HCl pH 8, 0.1 mM EDTA).

Polymerase Chain Reaction

Primer pair: CaInt2 5´-GGGGAAGCCTCTCGCGG-3´ and ITS4 5´-TCCTCCGCTTATTGATATGC-3´ was used to identify C. acutatum, while primers CgInt 5´-GGCCTCCCGCCTCCGGGCGG-3´ and ITS4 (Brown et al., 1996) were used to identify inoculum of C. gloeosporioides. PCR amplifications were performed in a total volume of 25 μl, containing 10 to 100 ng of genomic DNA, 4 mM (NH₄)₂SO₄, pH 9.0, 10 mM Tris-HCl, 2 mM MgCl₂, 0.2 mM each of dATP, dTTP, dCTP and dGTP, 0.2 U MasterAmp Tfl DNA Polymerase (Epicentre Technologies) and 1 μM concetration of each primer. In a negative control DNA probe was replaced with 5 μl of H₂O instead of template DNA. Amplifications were conducted in Mastercycler gradient (Eppendorf) with an initial denaturation step (5 min at 95°C), followed by 30 cycles of denaturation (30 s at 95°C), annealing (30 s at 60°C), and extension (1.5 min at 72°C), and a final extension step (5 min at 72°C). Reactions were performed at least three times to check the consistency of the method. Aliquots (8 μl) of each PCR product were analyzed by electrophoresis in TBE buffer in 1.5% agarose gels stained with ethidium bromide.

Sequencing of the rDNA regionTo analyze rDNA region of Colletotrichum spp., fungal culture no. 13 was chosen as a representative. The ITS1 - 5.8S - ITS2 rDNA regions were amplified with the ITS1 and ITS4 primers [13]. The sequencing of the rDNA fragment was conducted on ABI PRISM 310 automated DNA sequencer with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) in both directions. The data were analyzed with the ABI PRISM DNA Sequencing Analysis Software. The sequences were analyzed using BLASTn [http://www.ncbi.nlm.nih.gov/BLAST/].

BIO-PCR assays

BIO-PCR assays were conducted on two lupin seed samples (`Juno´ and `Polo´, 2002) originated from Warmia and Mazury region. Mycelium for BIO-PCR assays was obtained by placing 200 seeds from each cultivar on moist filter paper. The seeds were incubated at room temperature for 48 hours. For DNA isolation, ten seeds with visible hyphae were selected for each cultivar. A single seed constituted a single sample in the material assayed.

RESULTS

Identification of Colletotrichum spp. using species-specific PCR assays

The PCR assay with primers CaInt2 and ITS4 (specific for C. acutatum) allowed for classification of all the 14 isolates to C. acutatum (Photo 1). The PCR products obtained with primers CaInt2 and ITS4 from all the C. acutatum cultures were approximately 500 bp, according to Brown et al. [3], Adaskaveg and Hartin [1] and Freeman et al. [5]. Other primers, CgInt and ITS4, specific for C. gloeosporides, were used as well. The relevant references imply that the size of the expected product is 450 bp [1,3,5]. No PCR product was obtained with these primers from all the 14 cultures tested (photo not shown).

Analysis of rDNA sequences

The results of the PCR assays were confirmed by the sequence comparison of rDNA fragment (386 bp) (Fig. 1) of fungal culture no. 13 with the sequences available in the NCBI/GeneBank database using BLASTn. The sequence comparison showed 91% homology to the C. acutatum isolate (GenBank accession number AF01292). Some errors may have occurred in the sequence as the results of the analysis have not been verified by any other institution.

BIO-PCR results

The inoculum of C. acutatum was detected from naturally infected lupin seed samples using BIO-PCR method. One isolate of C. acutatum was detected from cv. Juno (Photo 2), while five isolates of C. acutatum were detected from cv. Polo (Photo 3). No inoculum of C. gloeosporioides was detected.

DISCUSSION

It is obvious that molecular methods based on PCR used for the detection and identification of species of the genus Colletotrichum are more reliable than the conventional techniques, which are based on morphological characteristics (color and appearance of a culture, shape of conidia), growth rate and benomyl sensitivity [1,3,6]. Traditional identification is not always straightforward due to a high variability of fungal isolates caused by environmental factors [2,6,10,14].

The PCR technique enables the detection of the pathogen directly from plant tissue without prior isolation of microorganisms into selective media. The use of species-specific PCR assays as a diagnostic method allowed for a distinction between C. acutatum and C. gloeosporioides [1,3,7].

The use of PCR assays showed that the species responsible for lupin anthracnose worldwide is C. acutatum [12]. Our results also showed that anthracnose of lupins in Poland is caused by C. acutatum. We proved that BIO-PCR technique could be suitable for the identification of C. acutatum from lupin seeds. The BIO-PCR results showed that this technique could be applied as a valuable diagnostic tool for seed health testing.

CONCLUSIONS

1. The use of species-specific PCR assays in this study revealed that the species responsible for anthracnose of lupins in Poland is Colletotrichum acutatum.

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