Alternaria telliensis, a new causal agent of cabbage leaf spot disease in Iran

INTRODUCTION

Cabbage or headed cabbage (Brassica oleracea var. capitata), a member of Brassicaceae family, is an economically important leafy biennial vegetable crop grown worldwide as an annual crop for head harvest. Cabbages are used fresh in salads, as pickles, stir-fried and a fermented product (Wouters et al. 2013, Šamec et al. 2017) with antioxidant, antibacterial, anti-inflammatory, anti-obesity, anti-cancer, gastrointestinal and health-promoting effects (Podȿedek et al. 2006, Singh et al. 2006, Rokayya et al. 2013, Park et al. 2014, Lee et al. 2018). In 2017, the global production of cabbages and other related brassicas was 71.2 million tonnes (FAO 2018). In Iran, cabbage is cultivated on 6979 ha with a total annual production of 306781 tonnes (FAO 2018). Tehran province is one of the main cabbages-growing areas in Iran (Koocheki et al. 2013).

Cabbages and other cruciferous crops are severely affected by different pathogens, causing economic losses in yield and crop quality. Leaf spot diseases caused by Alternaria spp. are the most prevalent and destructive diseases of these plants worldwide as well as in Iran (Humpherson-Jones 1992, Nowicki et al. 2012, Kumar et al. 2014, Rahimloo & Ghosta 2015). These diseases are characterized by small, dark specks that enlarge into relatively large, circular, brown to black lesions with concentric rings and are sometimes surrounded by chlorotic halos (Koike et al. 2007, Siciliano et al. 2017). Historically, Alternaria brassicicola and A. brassicae have been considered as the main causative agents of dark leaf spot disease in cruciferous plants (Maude & Humpherson-Jones 1980, Rotem 1994, Pedras et al. 2009, Michereff et al. 2012). Seeds, seedlings and pods are also damaged by these pathogens (Köhl et al. 2010). In a monographic study on Alternaria diseases of crucifers, four species i.e., A. alternata, A. brassicae, A. brassicicola and A. japonica (as A. raphani) were reported to cause heavy losses in the crops (Verma & Saharan 1994). All four Alternaria species have been reported to be seed-borne and were detected from all parts of infected seeds (Saharan et al. 2016).

In a study on Alternaria species associated with cabbage leaf spot disease in Urmia, Iran, eight species were identified based only on morphological characteristics (Rahimloo & Ghosta 2015). In the most recent study on the incidence and diversity of Alternaria species associated with leaf spot disease of Brassica napus, 12 species were identified indicating high diversity of Alternaria species associated with the disease (Al-Lami et al. 2019). In the present study, a set of Alternaria isolates was isolated from cabbage plants representing leaf spot symptoms. Based on the combination of morphological characteristics and molecular data obtained from four genomic loci (ITS-rDNA, glyceraldehyde-3- phosphate dehydrogenase, the second-largest subunit of RNA Polymerase II and translation elongation factor 1-alpha), the isolates grouped well in Alternaria section Japonicae in close affinity with A. telliensis N. Bessadat, Ayad & P. Simoneau. Pathogenicity tests confirmed the pathogenic nature of the isolates on cabbage leaves. To our knowledge, this is the first occurrence of A. telliensis and its pathogenicity on cabbage plants in Iran.

MATERIALS AND METHODS

Sample collection and fungal isolation

Cabbage (Brassica oleracea var. capitata) leaves with necrotic leaf spot symptoms were collected randomly from different cabbage growing fields in Damavand city, Tehran province, Iran during the late summer and fall of 2017. Leaf samples were placed in new separate paper bags, transferred to the laboratory and processed immediately. Symptomatic leaf samples were washed gently under running tap water and surface disinfested by submerging in 70% ethanol for 2 min and washed again with sterile distilled water. Small pieces at the interfaces of healthy and necrotic areas were excised and plated directly onto potato dextrose agar (PDA, Merck, 39 g/L) medium. Also, some of the leaves were kept in moist chambers. The samples were examined at 48 h intervals for fungal growth, and the growing fungi were transferred onto new PDA Petri dishes. Purification of the fungi was done based on the single spore method. Pure cultures were grown on potato carrot agar slants (PCA, 20g carrot, 20g white potato, 20g agar in 1L distilled water) supplemented with twice-autoclaved pieces of filter papers and kept at 4°C.

Morphological characterization

Morphological characteristics were examined according to Simmons (2007). Briefly, recovered isolates were cultured on PCA medium and incubated at 24 ± 1 °C with 8/16 h light/dark photoperiod under cool white fluorescent light without humidity control. Sporulation pattern was observed after 5–7 days at 64× magnification using a stereo-microscope. Microscopic slides were prepared using transparent clear tape and 25% lactic acid solution as mounting fluid (Schubert et al. 2007). Micro-morphological characteristics including dimensions, septation, color and surface ornamentation were recorded for 50 conidiophores and conidia. Colony characteristics were observed on PCA, PDA, hay decoction agar (HA) and V8 juice agar (V8-A) culture media after 7 days (Simmons 2007). The living cultures of examined isolates were deposited in the fungal culture collection of Agricultural Biotechnology Research Institute of Iran (ABRIICC) and additional isolates were kept in the fungal culture collection of Urmia University (FCCUU).

DNA extraction, amplification and sequencing

Freshly harvested mycelium from 5-day-old cultures grown in potato dextrose broth (PDB) was used for DNA isolation. Total genomic DNA was extracted using the Exgene™ Cell SV mini kit (GeneAll Biotechnology Co., Korea) following the manufacturer’s instruction. Polymerase chain reaction amplification of ITS-rDNA region and parts of TEF1-α, RPB2 and GAPDH gene regions were performed using primer pairs ITS1/ITS4 (White et al. 1990) for ITS-rDNA region, EF1-728F/EF1-986R (Carbone and Kohn 1999) for TEF1-α, fRPB2-5F/fRPB2-7cR (Liu et al. 1999; Sung et al. 2007) for RPB2 and gpd1/gpd2 (Berbee et al. 1999) for GAPDH. The thermal conditions for PCR amplification and the reaction mixtures were the same as Hashemlou et al. (2020). PCR products were cleaned up and sequenced by Microsynth DNA company (Balgach, Switzerland).

Phylogenetic analysis

Multiple sequence alignments were produced using the type or ex-type strains sequences retrieved from GenBank according to Woudenberg et al. (2013, 2015), Lawrence et al. (2014), Deng et al. (2018), Poursafar et al. (2018, 2019) and Bessadat et al. (2020) (Table 1), and aligned with MAFFT online service (https://mafft.cbrc.jp/alignment/server/index.html). Concatenated sequence dataset (ITS-rDNA + GAPDH + RPB2 + TEF1-α) was produced and proofed in Mesquite v. 2.74 (Maddison and Maddison 2010). Maximum likelihood analysis (ML) was performed on a concatenated dataset in RAxML-HPC BlackBox v. 8.2.12 (Stamatakis, 2014) through the CIPRES Science Gateway v 3.3 (Miller et al. 2010) using the GTRGAMMA+I as substitution model. The resultant phylogenetic tree was observed in FigTree v. 1.4.4 (Rambaut 2019). Sequences of Stemphylium vesicarium (formerly as S. herbarum) CBS 191.86 and S. botryosum strain CBS 714.68 served as the outgroup taxa.

Pathogenicity test

The healthy plants of white cabbage cv. Glory of Enkhuizen without any leaf symptoms were chosen for fungal inoculation. Cabbage leaves were firstly cleaned with moist cotton balls, then were sprayed with 70% ethanol and cleaned again with sterile distilled water. Mycelial plugs (5 mm diameter) containing fungal spores were taken from the edges of actively growing colonies (5-day-old) on PDA plates and placed upside down on cabbage leaves, both with and without small wounding caused by sterile needle. In the controls, only sterile agar plugs (without fungal mycelia or spores) were used. In addition, pathogenicity tests were done using the spore suspension (10⁶ spores/mL) prepared from PCA cultures. The inoculated plants were covered with plastic bags for 48 h in order to maintain high humidity. Disease symptoms were evaluated 10 days after inoculation. Four replicates were used for each isolate and all the experiments were repeated once. Re-isolation of the inoculated fungi was made from the necrotic lesions formed on the inoculated leaves after surface disinfestation and morphologically compared to the inoculated isolates to fulfill Koch’s postulates.

Table 1. Strains used for phylogenetic analysis in this study. Newly generated sequences are shown in bold.

RESULTS

Morphological characterization

Twenty-one Alternaria isolates with similar morphological characteristics were recovered from collected samples from different fields in Damavand city, Tehran province. Based on morphological characteristics, they were identified as A. telliensis.

Phylogenetic analysis

PCR amplifications produced band sizes approximately 480 bp for ITS-rDNA, 531 bp for GAPDH, 909 bp for RPB2 and 224 bp for TEF-1α. Sequence combination of four loci for a total of 41 fungal strains including ingroup and outgroup taxa contained 1919-characters. The best scoring RaxML tree with the final ML optimization likelihood value of -11504.616823 (ln) is selected to demonstrate the phylogenetic relationships among the studied strains (Fig. 1). The results of the phylogenetic analysis revealed that the two studied Iranian Alternaria strains were closely related to the members of Alternaria section Japonicae with the nodal bootstrap support of 100% and clustered well in the subclade along with the strains of A. telliensis (Fig. 1), confirmed morphological identification.

Alternaria telliensis N. Bessadat, D. Ayad & P. Simoneau, Phytotaxa 440 (2): 92. 2020

Colonies on PCA reached 60 mm diam after 7 days, greenish olivaceous at the center and olivaceous grey at margins, with four well-defined concentric rings of growth and sporulation; 64 mm diam on PDA, smoke gray to whitish-grey; 54 mm diam on HA, hazel to brown and 65 mm diam on V-8 agar, greyish sepia to olivaceous. White aerial hyphae are present at the colony center of all media (Fig. 2a–c). Sporulation abundant after 4 days on PCA, HA and V8-A. On PCA, conidiophores arise directly from hyphae growing on agar surface or from aerial hyphae, mostly simple with an apical conidiogenous locus, sometimes with 2–3 geniculations, 20–90 × 3.5–5 µm; conidia mostly solitary, sometimes in chains of 2–3(–4) spores. Juvenile conidia are ovoid to ellipsoid, pale brown to brown, distinctly constricted at transverse septa, beakless, with 1–3 transverse septa and 0–1(–2) longitudinal septa, 17–40 × 10–17.5 µm. Mature conidia are long ovoid to cylindrical, brown to dark brown, outer wall punctate to verrucose, distinctly constricted at the transverse septa, 3–4(–5) transverse septa, 1–3 oblique septa and 1–3 longitudinal septa in the broadest transverse divisions, 40–65 × 18–43 µm, septa darker black-brown, 3–5 µm wide. In some of the conidia, body cells are enlarged and deformed the spore body. The apical secondary conidiophores that initiate chain formation are as single cells, slightly paler than that of the spore body, 5–8 × 5 µm, or a well-differentiated apical outgrowth up to ca 17–48 × 5 µm. A distinct feature of the studied isolates is the transformation of all cells of some of the conidiophores into chlamydospore-like structures, 8.5–12.5 × 5.5–10 µm (Fig. 3a–z). The sexual form was not formed.

Specimens examined. IRAN, Tehran province, Damavand City, on cabbage leaf (Brassica oleracea var. capitata) with leaf spot symptoms, Oct. 6, 2017. A. Poursafar, ABRIICC 10148, GenBank accession numbers : MK660798, ITS ; MK660796, GAPDH ; MK660800, RPB2 ; MK660802, TEF1-α ; Sep. 25, 2017. A. Poursafar, ABRIICC 10150, GenBank accession numbers : MK660799, ITS ; MK660797, GAPDH ; MK660801, RPB2 ; MK660803, TEF1-α.

Note. Alternaria telliensis is phylogenetically closely related to A. japonica and A. nepalensis in the Alternaria section Japonicae, but in a well-supported and distinct subclade. Alternaria japonica can easily be differentiated from A. telliensis by the production of distinctive chains of dark, thick-walled and often ornamented cells (micro-chlamydospores) in surface and subsurface hyphae and conidia with a smooth outer wall (Simmons 2007, Bessadat et al. 2020). Also, A. nepalensis can be differentiated from A. telliensis based on conidia with a smooth outer wall and with no formation of chlamydospores (Simmons 2007, Bessadat et al. 2020).

Pathogenicity test

All Alternaria isolates were used in pathogenicity experiments, and they produced conspicuous lesions on white cabbage leaves similar to those of naturally occurred under field conditions (Fig. 4a–e). No symptoms were formed on the control plants (Fig. 4f). Re-isolation of the inoculated fungus confirmed Koch’s postulates.

Fig. 1. Phylogenetic tree generated from maximum likelihood (ML) analysis based on the combined dataset of ITS, TEF1-α, GAPDH and RPB2 sequences of 39 Alternaria strains. The RAxML maximum likelihood bootstrap values (>50%) are given at the nodes. The tree was rooted to Stemphylium vesicarium strain CBS 191.86 and S. botryosum strain CBS 714.68. The bar indicates the number of substitutions per position. T: Ex-type strain. R: Representative strain.

Fig. 2. Alternaria telliensis (ABRIICC 10148). Colony after 7 d on: a. PCA; b. PDA; c. V8-A; d. HA.