First report of Graphium carbonarium associated with walnut dieback in Iran

Document Type : Short Article

Authors

Department of Plant Protection, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

Abstract

Walnut tree is one of the most important nut crops in Iran. Dieback and decline of walnut trees are some of the factors limiting the cultivation and sustainability of this crop. During 2017 and 2018, field surveys were conducted on walnut orchards in Yazd province to study of fungal pathogens associated with diseased trees. Wood samples were collected from diseased branches showing canker, dieback and gumming symptoms. In the laboratory, affected branches were cut transversally and infected wood tissues were cut into small pieces. Wood pieces were plated on a potato-dextrose-agar (PDA) after surface sterilization. In this study, 10 isolates of a fungus were obtained from affected trees. Based on morphological and molecular (for two selected Iranian isolates based on ITS-rDNA and tef1-α gene sequences) characteristics, all isolates were identified as Graphium carbonarium. Based on literature reviews, this is the first report of this speciesassociated with necrotic wood of walnut trees in the world.

Keywords


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).

Isolates

Host

Origion

Genbank Accession Number

Species

Code

ITS

tef1-α

Graphium adansoniae

CMW30618T

Adasonia digitata

South Africa

KM592371

KM592363

 

CMW30620

A. digitata

South Africa

GQ200613

HM630597

Graphium basitruncatum

JCM9300

Forest soil

Solomon Islands

AB038427

KJ131248

Graphium carbonarium

CMW12420T

Salix babylonica / Pissodes sp.

China

FJ434979

HM630603

 

CMW12418

S. babylonica / Pissodes sp.

China

FJ434980

HM630602

 

IRNPm47

Juglans regia

Iran

MT605368

MT625161

 

IRNPm48

J. regia

Iran

MT605369

MT625162

Graphium euwallaceae

UCR 2980 T

Acasia sp.

Vietnam

KM592371

KM592363

 

UCRFD97

Acasia floribunda

California

KF540225

KF534806

Graphium fimbriisporum

CMW5605T

Picea abies

France

AY148177

HM630590

 

CMW5606

P. abies

Austeria

AY148180

HM630591

Graphium larics

CMW5601T

Larix deddua

Austeria

AY148162

HM630588

 

CMW5603

L. deddua

Austeria

AY148182

HM630589

Graphium madagascariense

CMW30628T

Adasonia rubrostipa

Austeria

HM630606

HM630595

 

CMW30629

A. rubrostipa

Austeria

HM630607

HM630594

Graphium penicillioides

CMW5292T

Populus nigra

Czech Republic

HQ335310

HM630600

 

CMW5295

P. nigra

Czech Republic

HQ335311

HM630601

Graphium pseudormiticum

CMW503T

Pinus sp.

South Africa

AY148186

HM630586

Pseudallescheria boydii

CBS 101.22

Homo sapiens

USA

AM887718

EF151369

T Ex-type strains

 

Maximum parsimony analysis resulted in three equally most parsimonious trees (TL=645, CI=0.817; RI=0.912, RC=0.745). MP tree of the respective datasets is presented as Fig. 3, with bootstrap results from the ML and MP trees. Based on MP analyses, the Iranian isolates clustered with the reference isolates of Graphium carbonarium. In this study, G. carbonarium collected from walnut trees in Yazd province. This species was first identified and described by Paciura et al. (2010) associated with a Pissodes sp. on Salix babylonica. Phylogenetic analyses reveal that G. carbonarium is distinct but closely related to Graphium euwallaceae and Graphium basitruncatum. This species has also been reported from Tsuga dumosa (Paciura et al. 2010) and Larix olgensis (Liu et al. 2016) from China. In 2016, G. carbonarium was isolated from Ricinus communis in China and Acacia auriculiformis trees in Vietnam (Lynch et al. 2016). Graphium basitruncatum was first described from forest soil in the Solomon Islands as Stilbum basitruncatum Matsush. (Matsushima 1971). This species has been isolated from a patient with leukemia in Canada, confirming that this species can act as an opportunistic human pathogen (Deepali et al. 2007). In a study conducted by Lynch et al. (2016), G. euwallaceae was reported as a pathogen of avocado and box elder (Lynch et al. 2016). Based on the literature review, the present study has shown that the walnut trees can also be considered as a new host for this species in the world.

 

ACKNOWLEDGEMENTS

 

This work was done at the Shahid Bahonar University of Kerman. The authors acknowledge the editorial board of Mycologia Iranica Journal as well as the respected reviewers for their helpful comments for improving the paper.

 

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