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Stemphylium vesicarium causing Sansevieria trifasciata (viper’s bowstring hemp) leaf blight in Iran

  • Abdollah AhmadpourEmail author
  • Alireza Poursafar
Article
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Abstract

Leaves of Sansevieria trifasciata with leaf blight symptoms were collected from greenhouses of Miyandoab, West Azerbaijan province, Iran, during the summer of 2016. Leaf blight symptoms were large, elongated and gray to pale brown with brown margins. Three single spore isolates were obtained from diseased leaves and the pathogenicity of each isolate to plants was confirmed by inoculation tests based on Koch’s postulates. The pathogen was identified as Stemphylium vesicarium, based on both morphological characteristics and sequencing of the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. This is the first report of Stemphylium vesicarium causing leaf blight on Sansevieria trifasciata in Iran and the world.

Keywords

Pathogen identification GAPDH gene Morphology Pathogenicity 

Sansevieria trifasciata (viper’s bowstring hemp, Agavaceae) is commonly known as snake plant and/or mother-in-law’s tongue because of its leaf shape and margin sharpness. Sansevieria trifasciata is native to tropical Africa and was introduced to America, Asia, Australia, and the Pacific Islands as an ornamental and fiber crop (USDA-ARS 2017). In Iran, S. trifasciata is usually used as an ornamental plant in homes and offices. So far, several fungal species have been reported on Sansevieria spp. and some of them cause disease on leaves and roots (Farr and Rossman 2017).

During the summer of 2016, some leaves of S. trifasciata with leaf blight symptoms were collected from greenhouses of Miyandoab, West Azerbaijan province, Iran. Leaf blight symptoms were large, elongated and gray to pale brown with brown margins (Fig. 1a). A single spore produced on the surface-sterilized leaf was transferred to water-agar and then fungal colonies were cultured from hyphal tips on potato dextrose agar (PDA) culture medium. A subculture was transferred to Potato Carrot Agar (PCA) and incubated at 22–25 °C under cool/white fluorescent with 10/14 h light/dark photoperiod for 5–7 days (Simmons 1969). Macro- and micro-morphological features were recorded and compared with available literature. Three single-spore isolates of Stemphylium sp. made from lesions on symptomatic leaves were deposited in the fungal culture collection of Urmia University (CCUU) as CCUU1362, CCUU1363, and CCUU1364. Measurements and microphotographs were taken from slide mounts in lactophenol using an Olympus light microscope (model BH2). Based on morphological characteristics, the isolates were further identified as Stemphylium vesicarium (Simmons 1969). Colonies on PCA after seven days were ca 60 mm diam., olivaceous green to light brown with concentric zones of growth and sporulation. Sporulation abundant mostly from superficial hyphae and to a lesser extent from submerged hyphae (Fig. 2a). Hyphae pale brown, septate, branched and 5–7 μm wide. Conidiophores straight or curved, pale brown, septate, short to moderate, 20–75 × 5–7 μm in size. Conidiogenous cells swollen at the apex, dark brown, 6–8 μm wide and occasionally with 1–4 apical proliferation. Matured conidia dark brown, spherical to oblong, cylindrical to rectangular, rounded at the base and conical to angular at the apex, with a verrucose conidial wall, 6–7 transverse septa, 1–5 longitudinal septa and 1–4 oblique septa, 24–48 × 11–20 μm (Fig. 2b).
Fig. 1

Symptoms of leaf blight caused by Stemphylium vesicarium on Sansevieria trifasciata. a leaf blight symptoms on naturally infected S. trifasciata in greenhouse. b leaf blight symptoms on inoculated S. trifasciata under greenhouse conditions 10 days after inoculation

Fig. 2

Stemphylium vesicarium. a Colony on PCA; b Conidiphores and conidia. Bar (b) = 20 μm

Genomic DNA was extracted from the three isolates using the method of Liu et al. (2000). The Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified for each isolate with PCR using the primer pair gpd1/gpd2 (Berbee et al. 1999). Each PCR mixture contained 0.4 pM of each primer, 8 μL of a ready master mix (Taq 2X Master Mix Red 1.5 Mm, Amplicon Company, Denmark) and about 10 ng of template DNA in a final volume of 25 μL. A touchdown (TD) PCR method was used (Korbie and Mattick 2008) with an initial denaturation for 90 s at 95 °C and then, a cycle of 60 s denaturation at 95 °C, 60 s annealing at 62 °C and 60 s extension at 72 °C, followed by 10 cycles with a 62–57 °C annealing temperature (annealing temperature decreased 0.5 °C per cycle) and 25 cycles with a 57 °C annealing temperature and a final extension for 5 min at 72 °C. The PCR products were purified and sequenced by Macrogen Corporation (South Korea). The newly generated sequences of GAPDH in this study were deposited in GenBank (MG668997, MG914073 and MG914074).

Homology searches were performed using the NCBI BLAST search. BLAST searches showed that the sequence data were identical to those of S. vesicarium, S. herbarum, and S. alfalfae from GenBank. The GAPDH sequence comparison showed 99% identity (565 out of 567 identical base pairs) to S. vesicarium type strain CBS 138138 (GenBank accession KU850735) (Woudenberg et al. 2017) and 100% identity (501 out of 501 identical base pairs) to EPS45 strain (JX075155) (Puig et al. 2015). Phylogenetic analysis based on the maximum likelihood (ML) method of selected GAPDH sequences of Stemphylium spp. from GenBank (Woudenberg et al. 2017) was conducted using MEGA6.0 software (Tamura et al. 2013). ML analysis based on GAPDH sequences indicated that the three isolates from the present study grouped in a S. vesicarium clade, supported by bootstrap value of 75% (Fig. 3). According to morphological and phylogenetic species hypothesis, the isolates were identified as S. vesicarium.
Fig. 3

Maximum likelihood (ML) tree based on of the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences of Stemphylium spp. retrieved from GenBank. The numbers above the nodes are the bootstrap values obtained from 1000 replicates

Sequence data of GAPDH in S. vesicarium isolates were identical to those of S. alfalfae (KU850735) and S. herbarum (AF443884). Systematic studies have shown that S. alfalfae, S. brassicicola, S. cremanthodii, S. herbarum, S. mali, S. sedicola, S. tomatonis and S. vesicarium were conspecific (Inderbitzin et al. 2009; Woudenberg et al. 2017). Woudenberg et al. (2017) revised species of Stemphylium based on three loci ITS, GAPDH, and cmdA and synonymized these seven species under S. vesicarium.

A pathogenicity test was conducted with S. trifasciata plants that were sown in plastic pots containing sterilized soil in the greenhouse. Three isolates of S. vesicarium were cultured on PDA at 25 °C for 10 days. The conidial suspensions of S. vesicarium were adjusted to 105 conidia/ml using a hemacytometer (Köhl et al. 2009). Healthy leaves were inoculated by spraying a conidial suspension (105 conidia/ml) of the fungus. At the same time, some leaves were scratched with a sterile needle and the conidial suspension sprayed onto these wounded parts. The pathogenicity tests were conducted using three replicates of each isolate and three plants for each replicate. Inoculated plants were placed in the greenhouse at 20–25 °C and 75–85% RH along with control plants (three plants) that had been sprayed with sterile, distilled water. Plants were covered with plastic bags for 48 h to maintain high humidity. Inoculated plants were inspected daily for disease symptom development for 20 days after inoculation. Leaf lesions were visible on all healthy and wounded leaves of inoculated plants after 10 days (Fig. 1b), but symptoms were not observed on control plants. The spots were pale brown with brown margins (Fig. 1b). The fungus was re-isolated from leaf lesions on inoculated plants and morphologically identified, but not from leaves of control plants, confirming Koch’s postulates.

Stemphylium vesicarium was first described from onion plants by Simmons (1969). This species is known as a plant pathogenic fungus and causes leaf spot on a wide variety of plant species. According to Farr and Rossman (2017), it has been associated with more than 20 plant species worldwide. Stemphylium vesicarium is an economically important pathogen of agricultural crops. The fungus causes brown spot disease on leaves and fruits of pear (Köhl et al. 2009), leaf blight on onion or garlic (Simmons 1969) and leaf/purple spot on asparagus (Graf et al. 2016). Stemphylium vesicarium has previously been reported as leaf blight on broad bean (Sheikh et al. 2015), onion (Ershad 2009), Helianthus annuus (Arzanlou et al. 2012), and black (sooty) head mold of wheat and barley (Poursafar et al. 2016) in Iran. Recently, Kee et al. (2018) reported S. lycopersici as the cause of leaf spot on S. trifasciata in Malaysia and estimated disease severity and incidence at 25 and 30% (total of 30 plants), respectively. The lesions were circular brown spots that gradually enlarged, forming oval to irregular chlorotic lesions with reddish-brown borders on the leaves (Kee et al. 2018). Symptoms caused by S. lycopersici on S. trifasciata in Malaysia were similar to symptoms caused by S. vesicarium in the present study. However, morphological and molecular analysis showed that our isolates belonged to S. vesicarium (Fig. 3). To our knowledge, this is the first record of leaf blight disease on S. trifasciata caused by S. vesicarium in Iran and the world (Farr and Rossman 2017). Further studies are needed to determine the distribution and to assess the economic impacts of this disease in Iran.

Notes

Acknowledgements

The authors would like to thank of Iran’s National Elite Foundation (Grant No: 15/80035) and Urmia University for financial support.

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Copyright information

© Australasian Plant Pathology Society Inc. 2018

Authors and Affiliations

  1. 1.Higher Education Center Shahid Bakeri Miyandoab and Member of Iran’s National Elite FoundationUrmia UniversityUrmiaIran
  2. 2.Department of Plant ProtectionUniversity College of Agriculture and Natural Resources, University of TehranKarajIran

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