First report of Apiognomonia errabunda on Quercus ilex in Algeria

Four sites were examined: Bordjem, Tougurt, Ain Ali and Chelaâlaâ (Fig. 1). Leaves of Q. ilex showing typical symptoms of anthracnose (Morelet 1989; Moricca and Ragazzi 2011) were sampled at each site (Fig. 2). Each twig was subdivided into apical or distal (AP) and non-apical (NAP) parts. Twigs were sampled from four cardinal directions (North, South, East and West). Leaves were stored in paper bags for transfer to the laboratory.

Figure 1

Figure 2

The severity of the symptoms on the leaves was determined by direct quantification of the necrotic areas. The leaves were glued on to A4 white paper and scanned at 700 dpi resolution (Epson Perfection V370, Seiko Epson Co.). The scan was performed against a background of white light to maximize the contrast of the object before processing images with ImageJ software (Abràmoff et al. 2004; Ferreira and Rasband 2012). The resulting images were calibrated in mm against a ruler placed in the background. Image files were saved in TIFF format and analysed in the RGB (Red-Green-Blue) scale. A reference line (20 mm) was drawn to standardize the dimensions to the ruler and the object, and the set scale menu activated to set the measurement unit. Using the colour thresholds extraction method, selected necrotic areas of the leaf blade were extracted and measured (mm²).

Disease severity index (DSI) was determined as the ratio between the percentage of necrotic leaf area (%) and the corresponding severity scale (Horsfall and Barratt 1945; Nutter et Esker 2006), as follows:

DSI = (Σ Xi.ni / R Nt) x 100, here Xi: disease severity (scale score), ni: leaf number with i severity, R: the highest score of the scale and Nt: the total number of examined leaves.

Since the sampling of symptomatic leaves was based on the orientation of the twigs and the position of the leaf (insertion point) on, severity was also considered in relation to these two parameters.

To isolate and identify the causative agent, leaves were surface-sterilized in 10% H₂O₂ for 1 5 m in followed by threefold rinsing in sterile distilled water (Ragazzi et al. 2001). After sterilization, the leaves were dried on sterile filter paper and aseptically cut into small square pieces (5 mm²) containing for each both necrotic and non-necrotic areas. Leaf pieces were placed on malt extract agar (MEA), as 5 pieces per Petri dish incubated at 24°C for one week, with daily observations to characterize the appearance of hyphae giving the fungal colonies. Mycelium was identified on morphological traits (Hughes 1953; Monod 1983; Ellis and Ellis 1997; Sogonov et al. 2008). Characterization of morphotypes was based on macroscopic features including colony shape and colour (Ragazzi et al. 2000; Lacap et al. 2003). Once identified, the fungal cultures are subcultured by transferring to water agar (WA).

For molecular characterization, discs taken from three-week colonies were transferred to glass tubes of 15 ml WA. Tubes were incubated at 24°C. After 15 days, the purity of emerging colonies was tested according to Smith and Onion (1994). Subsequently, the mycelium was scraped from the agar surface and crushed on a sterilized stainless steel support. The crushed materials were placed in previously autoclaved glass tubes, alternating with layers of microgranules of autoclaved silica gel (Smith and Onion 1994). Tubes were sealed and stored at 4°C. Three days after, the crushed mycelia were recovered and resuspended in glass Petri dishes containing potato dextrose agar (PDA), then incubated at 26°C for 15 days, before subculturing to Erlenmeyer flasks containing 100 ml of potato dextrose broth. The flasks were incubated with shaking at 250 rpm at 25°C for one week prior to DNA extraction.

DNA extraction was performed according to Lee and Taylor (1990) using Tris-HCl (50 mM), EDTA (50 mM), 3% SDS, 1% 2-mercaptoethanol, chloroform-TE-saturated phenol 1:1 (v/v), NaOAc (3M)/pH: 8.0; isopropanol, and 70% ethanol as lysis buffer. PCR was carried out according to White et al. (1990), using ITS1, 5.8S and ITS2 regions (White et al. 1990; O’Donnell et al. 2000).

The sequences of the used primers were 5’-TCCGTAGGTGAACCTGCGG-3’ (ITS1) and 5’-TCCTCCGCTTATTGATATGC-3’ (ITS4) (White et al. 1990; O’Donnell et al. 2000).

Amplification cycles were carried out in a ProFlex^TMPCR System (Applied Biosystems), with initial denaturation at 94°C (15 min) followed by three repeated phases (35 times): denaturation at 94°C for 2 min; annealing at 55°C for 1 min and elongation at 72°C for 3 min. A final elongation step of 72°C for 5 min was included. PCR products were examined on a 1% agarose gel in Tris-borate-EDTA buffer. DNA purification was carried out using the QiaQuick Gel Extraction kit (Qiagen, Germany). The product was stored at –20°C before sequencing.

Sequencing of the amplified fragments was performed in an ABI-Prism 373A DNA sequencer (Applied Biosystems) with enzymatic lysis and neo-amplification using the Big Dye Xterminator purification Kit (Applied Biosystems). Sequences were aligned using Bioedit Sequence Alignment Editor 7.0.0. (Hall 1999) and compared with those available in GenBank using BLAST search (http://blast.ncbi.nih.gov)

For phylogenetic analyses, multiple sequence alignment was performed including the genomic sequences of fungal isolates from the present study, compared with related mycotaxa in the Gnomoniaceae and Diaporthales using the ClustalW algorithm. The phylogenetic dendrogram was elaborated using MEGA 7.0 (Kumar et al. 2016) implying a Neighbour-joining statistical method. Evolutionary distances were computed using the Jukes-Cantor model (Yarza et al. 2017). The test adopted for phylogenetic analysis was the Bootstrap method with 1000 replicates. Statistical analyses were performed using XLSTAT 2014.5.03 (Addinsoft-Microsoft).

Digital image analyses of the symptomatic leaves of Q. ilex allowed characterizing the severity of anthracnose (Tab. 1).

Anthracnose severity varied according to the site. The most intense severity was recorded in the leaves sampled from Tougurt (34.52%), followed by Chelaâlaâ and Bordjem, with 21.96% and 21.85’, respectively, while the leaves sampled from Ain Ali were recorded with severity of 13.66% (P < 0.0001) (Tab. 1). Severity index and the Horsfall-Barratt (HB) scale also differed according to the site. The most important severity index was recorded for Tougurt (0.13), unlike Ain Ali with a minimum scale of 0.03 (Tab. 1).

Severity was greater in the south-facing twigs for all the sites (Tab. 2), while minimum values were recorded for the west-facing twigs (Tab. 2).

Regarding leaf position on a twig, we have found that anthracnose severity was more prominent apical leaves compared to non-apical leaves except for those sampled from Ain Ali (Tab. 3).

The causal agent of anthracnose was isolated in all leaf samples in the anamorphic form and identified under species level as Apiognomonia errabunda. 35 isolates distributed over five morphotypes were characterized from 330 leaf pieces (Tab. 4, Fig 3).

Figure 3

All the fungal colonies have a circular shape. After 15 days of growth at 25°C on MEA, colony diameters in Tougurt isolates averaged 43.57 mm; those of Bordjem have an average diameter of 40.95 mm. For Chelaâlaâ isolates, mean colony diameter was 42.28 mm and 43.19 mm for Ain Ali isolates (Tab. 4).

Colonies’ colour was usually light brown on both sides of the Petri dish, brownish-grey on the upper side and light grey on the underside, or light brown on the upper side and light-brown on the upper side with grey concentric circles on the lower side (Tab. 4). Conidia were ellipsoid, one-celled, 3.30–3.58 μm x 7.50–8.86 μm. Acervuli could not be detected, except in some isolates where they were spherical and grouped in clusters of 2 to 4.

The identification of A. errabunda species by molecular characterization was confirmed by comparing ITS sequences against GenBank accessions (Tab. 4). The database search yielded two strains: MH111403 with approximately 613 bp and MG594496 with approximately 595 bp. The two sequences were deposited at GenBank. The MH111403 had ITS similarity of 99% with sequences of A. errabunda isolated from Quercus pubescens in Italy (GQ452265, GQ452259). The phylogenetic tree showed isolates MH111403 and MG594496 together in a single clad (Fig. 4).

Figure 4