Identification and molecular characterization of Phyllosticta yuccae isolates causing Yucca leaf spot in Iran

INTRODUCTION

Yucca sp. (family Asparagaceae) is widely grown as an ornamental plant (Chase etal. 2009). Yucca plants are native to the hot and dry regions of the Americas and Caribbean and tolerate a wide range of conditions (Irish & Irish 2000, Dorling 2008). Many species of Yucca plants such as Yucca elephantipes, are commonly grown as houseplants, while some of them including Y. filamentosa, Y. flaccida, and Y. gloriosa are commonly grown outside in gardens (Irish & Irish 2000). This ornamental plant, which is cultivated as a perennial shrub and tree, provides a dramatic accent to landscape design (Chase etal. 2009).

Ornamental plants, like the other plant species, are affected by various fungal species causing leaf spot diseases. According to the surveys in Iran, different species of Alternaria, Ascochyta, Cercospora, Cladosporium, Colletotrichum, Coniothyrium, Ectophoma, Fusarium, Glomerella, Graphiola, Pestalotiopsis, Phoma, Phyllosticta, and Septoria have been reported as the causal agents of leaf spot on different ornamental plants (Azimi-Motem & Osipyan 2009, Ershad 2009, Bagherabadi et al. 2018, Hosseinnia & Mohammadi 2018, Larki et al. 2018, Bakhshi et al. 2019, Bakhshi & Zare 2020). Since the beauty of the ornamental plants is the major factor in their cultivation, any pathogenic problem could be serious from the cultivator's point of view. To that end, infected plants are never accepted in flower gardens and they are not profitable in commercial cultivations at all (Tilford 1932). Identification of leaf spot agents on ornamental plants can be helpful in plant disease management and lead to prevent their spread and progress of the disease. Therefore, the aim of this study was to identify and characterize the Phyllosticta sp. isolated from Yucca plants in Iran, using morphological and molecular characteristics and test them for pathogenicity.

MATERIALS AND METHODS 

Fungal isolation and morphological characterization

During 2017-2018, mound-lily Yucca (Yucca gloriosa) and Spineless Yucca (Y. elephantipes) plants showing typical leaf spot symptoms were collected from different areas in Fars, Bushehr, and Tehran provinces of Iran (Table 1). Small and excised portions of leaves with characteristic lesions were sterilized in 1% sodium hypochlorite, washed twice with sterile distilled water, dried using filter paper, and placed onto potato dextrose agar medium (PDA).

Table 1. Details and GenBank accession numbers of Phyllosticta strains included in this study.

T: Type strain     Strains isolated in this study are in boldface.

The fungal colonies grown from infected tissues were purified using single spore technique (Sinclair & Dhingra 1995). Cultural characteristics and morphological features of the isolates were determined on PDA, 2% malt extract agar (MEA; 20 g/L malt extract, 16 g/L agar), and oatmeal agar (OA; 60 g/L oatmeal, 16 g/L agar) media (Bissett 1986, Wikee et al. 2013).

DNA extraction, amplification, and phylogenetic analysis

BLAST search (which is available at https:// blast.ncbi.nlm.nih.gov/) was conducted to compare newly obtained sequences against NCBI database. All sequences used in this study are listed in Table 1. Sequences were aligned using CLUSTAL W program and concatenated following alphabetic order of the genes, ending in a sequence of 951 base pairs: nucleotides 1 to 223 foract, 224 to 718 for ITS, and 719 to 951 for tef1.

The best evolutionary model was determined using the Modeltest option from MEGA 6.06. Phylogenetic trees were constructed using the maximum likelihood algorithm (Tamura et al. 2013). Diplodia seriata (CMW25477) was chosen as an outgroup taxon. The bootstrap value was adjusted to 1,000 replications. The final tree was illustrated with infix pdf editor (https://www.iceni.com/infix.htm).

Pathogenicity test

The pathogenicity test was performed using the protocol described by Wikee et al. (2013) with three treatments as wounded leaves, unwounded leaves, and control. Before inoculation, attached and healthy leaves of two plant species (Y. gloriosa and Y. elephantipes) were surface sterilized using 70 % ethanol and washed three times with sterile, distilled water. The leaves were then dried with sterile tissue paper. Mycelial plug inocula (0.7 mm) were obtained from the edges of 15-day-old cultures and transferred to the wounded and unwounded leaves. For control treatments, leaves were inoculated with PDA plugs. All inoculated plants were then incubated in a moist chamber. Two weeks after inoculation, the fungal isolates on the inoculated leaves with leaf spot symptoms were re-isolated and cultured on PDA. The

morphological characteristics of the isolates were compared with those of the original isolates from the leaf spots to confirm Koch's postulates.

RESULTS AND DISCUSSION

The disease symptoms including dark red-brown, irregular and circle necrotic spots, were observed on the Yucca leaves (Fig. 1 a). Dark brown spots commonly have subglobos pycnidia on the upper side of the leaf (Fig. 1 b). A total of 27 strains of Phyllosticta associated with Yucca leaf spots were collected from three different geographical locations in Iran (Table 1). Fifteenstrains isolated from infected Y. gloriosa and12 strains isolated from Y. elephantipes were morphologically identical. In all strains, conidiogenesis was blastic, and the conidiogenous cells were cylindrical, hyaline, and 5.7 to 9.5 × 2.8 to 6 μm. Conidia were one-celled, aseptate, hyaline, smooth-walled, coarsely granular, broadly ellipsoidal to subglobose or obovate, usually broadly rounded at both ends, and 7.5 to 14.6 × 6 to 9.5 μm. Conidia were also surrounded by a slime layer about 1 μm wide, usually with a hyaline, flexuous, narrowly conoidal or cylindrical, mucilaginous apical appendage that was 4 to 12 μm long (Fig. 1 c, d). These characteristics matched well with the description of Phyllosticta yuccae (Bissett 1986). The representative isolate (Ysh2) was deposited in the Culture Collection of the Iranian Research Institute of Plant Protection, Tehran, Iran (IRAN 4218C).

The morphological identification of the fungus was confirmed with molecular analysis and phylogenetic approach. The results of molecular identification based on three different loci and blasted against the available sequences in GenBankshowed that the strains belong to P. yuccae. 

Phylogenetic tree constructed with concatenated sequences of the act, ITS, and tef1showed that four strains isolated in Iran were clustered with P. yuccae strain (CBS 117136) with 2, 4, and 4 different nucleotides in the sequences of act (site: 160 and 182), ITS (Site: 252, 371, 394, and 456), and tef1(Site: 739, 807, 813, and 818), respectively (Fig. 2). Iranian strains were grouped close to P. rubella, but separated from P. rubella with 5, 5, and 8 different nucleotides in the sequences of act (Site: 69, 76, 88, 96, and 151), ITS (Site: 247, 319, 364, 394, and 578), and tef1(771, 772, 779, 812, 814, 856, 869, and 908), respectively.

The results of the pathogenicity test demonstrated that P. yuccae is the causal agent of leaf spots on Y. elephantipes and Y. gloriosa. Symptoms like dark brown and necrotic elliptical spots were observed on inoculated leaves in seven days after inoculation. The results showed symptoms similar to those occurred in naturally infected plants (Fig. 1 e, f). Species in the genus Phyllosticta have been reported as plant pathogens, endophytes, and saprobes (Baayen et al. 2002, van der Aa & Vanev 2002, Okane et al. 2003, Glienke et al. 2011). A wide range of economically important crops and ornamental plants are affected by many species of Phyllosticta, which lead to leaf spot symptoms and fruit diseases. For example, P. yuccae has previously reported as a causal agent of leaf spot disease on Y. filamentosa in Brazil (Glienke-Blanco et al. 2002, Silva & Pereira 2007, Silva et al. 2013).

Currently, more than 20 species of Phyllosticta, including P. hedericola, P. theae,and P. yuccae have been reported affecting different plant species such as Camellia japonica, Hedera helix, Magnolia grandiflora, Rosa sp., Schefflera sp., Syringa reticulate, and Y. elephantipes from Iran (Fatehi & Mirabolfathy 1994, Ershad 2009, Darsaraei et al. 2016, Esmaeilzadeh et al. 2020) It is for the first time that molecular identification (based on three loci) and the pathogenicity test of P. yuccae on Y. gloriosa have been studied in Iran.

Fig. 1. Leaf spot symptoms on leaves of wild Yucca plants (a-b); Phyllosticta pycnidia (c); conidia (d); non-inoculated plant (e); leaf spot symptoms on leaves two weeks after inoculation (f). — Scale bars = 20 μm.

Fig. 2. Concatenated maximum likelihood tree of act, ITS and tef1genes. Bootstrap values higher than 70% are shown as percentages of 1000 replicates. Diplodia seriata CMW 8232 was chosen as outgroup taxon. Red circles indicate the strains isolated in this study. T: Type strain.