First report of Graphium carbonarium associated with walnut dieback in Iran

INTRODUCTION

Walnut (Juglans regia, Juglandaceae) as one of the most important nut crops has long been of interest to humans to produce wood and fruit. According to FAO (2018), Iran with 150,000 ha and 405,000 tons of walnut, is the third-largest producer of walnut in the world.The genus Graphium sensu lato has been identified by usually well-developed dark synnemata, producing single-celled conidia in slimy masses. Graphium species have been isolated from soil, plant debris, woody substrate and galleries of bark beetles (Jacobs et al. 2003). Graphium carbonarium Paciura, Z.W. de Beer, X.D. Zhou & M.J. Wingf. was first identified associated with a Pissodes sp. on Salix babylonica (Paciura et al. 2010). This species has also been reported from Tsuga dumosa (Paciura et al. 2010), Larix olgensis (Liu et al. 2016) from China, Ricinus communis in China and Acacia auriculiformis trees in Vietnam (Lynch et al. 2016). In the current study, 10 isolates of a Graphium species were isolated from walnut trees showing decline symptoms in Yazd province. The objective of this study was to identify these isolates using morphological and molecular characteristics.

MATERIALS AND METHODS

Sampling and fungal isolation

During 2017 and 2018, field surveys were conducted on walnut orchards in Yazd province to study of fungal pathogens associated with trees showing decline symptoms. Samples were collected from branches of trees with canker, dieback and gumming symptoms. Fungal isolations were conducted from internal wood necrotic tissues. In the laboratory, small wood segments (5×5 mm) were cut from affected tissues and surface-sterilized in 0.5 % sodium hypochlorite for 2 min followed by two rinses in sterile distilled water and then placed on potato dextrose agar (PDA, Merck, Germany) amended with streptomycin sulphate. The cultures were incubated in the dark at 25°C. For further study, pure cultures were obtained from each isolate based on single spore method.

Morphological and molecular identification and phylogeny

The putative identities of isolates were based on morphology following methods of Paciura et al. (2010). In order to molecular identification of the isolates, the total genomic DNA was first extracted from the aerial mycelium using a CTAB method (Doyle & Doyle 1990). All DNA samples were incubated at −17°C until used for PCR amplification. The internal transcribed spacer 1 and 2 including the intervening 5.8S nrDNA gene (ITS-rDNA) and a partial sequence of the translation elongation factor 1-alpha (tef-1α) gene were amplified using primer sets ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone & Kohn 1999), respectively. PCR products were purified and sequenced by Macrogen (Madrid, Spain). All sequences were read and edited with Sequencher software v. 1.8 (Gene Codes Corporation, Ann Arbor, MI), and then run through the BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm. nih.gov/Blast.cgi) to determine their basic identity.

For phylogenetic analyses, individual loci sequences obtained in this study and those references retrieved from GenBank were aligned using default settings of Clustal W algorithm (Thompson et al. 1994) included within MEGAX software package (Kumar et al. 2018). The alignments were manually checked and improved where necessary. Phylogenetic analyses for each locus and concatenated datasets were based on Maximum Likelihood (ML) and Maximum Parsimony (MP). Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC). The robustness of the topology was evaluated by 1000 bootstrap replications (Felsenstein, 1985). All sequences were deposited in GenBank (Table 1).

RESULTS AND DISSCUSSION

Field surveys were conducted on walnut orchards in Yazd province. The most important external disease symptoms observed on walnut trees were gumming, branch cankers and dieback. Internal symptoms included central, irregular, watery and v-shaped necrosis, which were observed in cross sections of diseased branches (Fig. 1). Graphium isolates produced dark gray-olive colonies with white margin, aerial mycelium and abundant synnemata on PDA. Conidia aseptate, hyaline, curved cylindrical, aggregate in a hyaline mucilaginous mass at the apices of the synnemata. Based on morphological and cultural features, the fungal isolates identified tentatively as Graphium sp. (Paciura et al. 2010). Iranian isolates had synnematous anamorph in culture and sexual structures were never observed. Conidiophores organized in synnemata, that generally formed in groups and sometimes singly. Synnemata had 125-290 µm long, and 43–59 μm wide at the apex. Conidia aseptate, hyaline, curved, cylindrical with truncated bases, 3.5-6 µm × 1-3.5 µm (Fig. 2). The optimum growth temperature is 25–30°C. Colonies reaching a radius of 4.5-6 mm in 7 d and 9.5-10.5 mm in 14 d at 25 °C. Graphium carbonarium is most closely related to G. basitruncatum (Matsush.) Seifert & G. Okada and G. euwallaceae Twizeyim., S.C. Lynch & Eskalen. However, they also have morphological differences. Graphium carbonarium has larger synnemata and conidia than G. basitruncatum. The latter species is characterized by conidiophores (70-)72–131(-158) μm in length, conidiogenous apparatus (19-)24–45(-56) μm wide and conidia of 5–6×1–2 μm in size (Paciura et al. 2010). Conidia in G. euwallaceae are also shorter and slender compared to G. basitruncatum and G. carbonarium (Lynch et al. 2016). DNA sequence comparisons were conducted to confirm the identity of these isolates. The two individual phylogenetic analyses (ITS and tef-1α) resulted in similar tree topology (data not shown). Sequences of two Iranian isolates, 17 reference isolates of Graphium spp. (include of nine species) and Pseudallescheria boydii (Shear) McGinnis, A.A. Padhye & Ajello (as outgroup) were aligned. The combined alignment consisted of 1082 characters including gaps (ITS: 563 and tef-1α: 519). Of these, 626 were constant and 276 parsimony informative.

Fig. 1. Main branch canker and trunk disease symptoms found on walnut trees. (a-c) external disease symptoms, a) gumming, b) branch dieback, c) branch canker, (d-g) internal wood lesion types, d) central necrosis, e) v-shaped necrosis, f) irregular wood necrosis, g) watery necrosis.

Fig. 2. Graphium carbonarium. Colony on PDA after a) 14 days and b) 28 days, c) Conidia, d-g) Synnemata, Scale bar 40µm, C: 5µm.

Fig. 3. One of the most parsimonious trees for Graphium obtained from combined ITS-rDNA and tef1-α sequence data. ML/MP bootstrap support (1000 replicates) above 70 % are shown at the nodes. Pseudallescheria boydii (CBS 101.22) was used as outgroup and Iranian isolates (IRNPm47 and IRNPm48) obtained in this study and isolates of G. carbonarium retrieved from GenBank shown in bold type. Bar represents 50 changes. 

 Table 1. Origins, host and GenBank accession numbers of the Graphium strains used in phylogenetic analyses (Iranian isolates are shown in bold type).

^T Ex-type strains