Elaeagnus latifolia is an evergreen woody shrub, common in subtropical and temperate areas and belongs to family Elaeagnaceae. It is mostly grown in semi-wild conditions, fruits are locally known as Soh-Shang and are rich in vitamins A, C and E and contain several compounds which have anticancerous properties. It is considered as having great potential in this region because of its hardy nature and early bearing (Patel et al. 2008).

Rust of E. latifolia caused by Aecidium elaeagni-latifoliae is an emerging disease in this region. During a regular survey we came across hyperparasitised pustules of rust on the leaves of E. latifolia (Fig. 1). Microscopic examination was conducted using light and scanning electron microscopy. Molecular analysis was also done for confirmation. Light microscopy was conducted using an Olympus BX 53 microscope equipped with a digital camera DP 72, Olympus, and image analysis software-cellSens Standard 1.5, Olympus. Observations were made using 3 % potassium hydroxide as mounting medium. Hyperparsitised pustules were selected using a dissecting microscope and placed on double-sided cellotape then sputter-coated with gold under vacuum using Fine Coat Ion Sputter JFC–1100. Gold-coated samples were then placed on aluminium stubs for scanning electron microscopy (SEM) (JEOL JSM 6360, JEOL, Tokyo, Japan). A voucher specimen has been deposited in Ajrekar Mycological Herbarium (AMH- 9654). Microscopic analysis using light and scanning electron microscopy clearly indicated the affinity of the fungus with Simplicillium lanosoniveum. Phialides were solitary and conidial dimensions were 2.5 3.5 × 1 2 μm (Zare and Gams 2004). SEM also revealed, whole rust pustules were covered with the growth of the hyperparasite (Fig 2, 3, 4).

Fig. 1
figure 1

Parasitized and nonparasitized rust pustules on Elaeagnus latifolia

Full size image
Fig. 2
figure 2

Rust pustules covered with hyperparasite observed using SEM (Bar = 10 μm)

Full size image
Fig. 3
figure 3

Solitary phialides of Simplicillium lanosoniveum bearing conidia (Bar = 10 μm)

Full size image
Fig. 4
figure 4

Parasitized aeciospore of Aecidium elaeagni-latifoliae (Bar = 5 μm)

Full size image

Nested PCR was done using NL1 and LR5 for the first round then NL1 and LR3 for second round for amplification of large subunit (LSU) of nrDNA containing D1 and D2 domains (Vilgalys and Hester 1990). The ITS region comprising ITS1-5.8S-ITS2 was amplified using primers ITS1F and ITS4 (Gardes and Bruns 1993; White et al. 1990). PCR was done using initial denaturation for 5 min (94 °C), denaturation 30s (94 °C), annealing 40s (54-50 °C, touchdown with 1 °C decrement in every cycle), extension 1:10s or 50s (depending on the primer combination) (72 °C) and final extension 10 min (72 °C) (35 cycles). Sequencing was done using ITS1F and 4 for ITS region and NL1 and LR3 for LSU region. The sequences of ITS (KJ408447) and LSU (KJ135022) obtained in this study have been deposited in GenBank. Similarity checks were done at NCBI website. For further analysis, sequences of LSU and ITS of closely related sequences were downloaded from NCBI (Table 1, 2). Majority of the sequences included in the analysis were from Nonaka et al. (2013a) and Sung et al. (2001). Alignment was done using Clustal W implemented in MEGA 5.0 (Tamura et al. 2011 and references therein). Model selection was done using jModeltest (Posada 2008). Phylogenetic analysis was done using MrBayes v.3.1.2 (Huelsenbeck and Ronquist 2001). Parameters used were generations = 2,000,000, sampling frequency = 100, nst = 6, rates = gamma, no of chains set to 4 with two simultaneous runs and burnin = 5,000. Tracer was used for convergence analysis (Rambaut and Drummond 2007). FigTree was used for visualising the tree (Rambaut 2012).

Table 1 Large subunit sequences obtained from NCBI
Full size table
Table 2 ITS sequences obtained from NCBI
Full size table

Bayesian analysis with LSU sequences recovered the fungus in a well-supported (posterior probability = 1.0) clade with species of Simplicillium. (Fig. 5). The sequence of Verticillium dahliae (VDU17425) was used as root. Species determination was done by conducting phylogenetic analysis using ITS region and Verticillium chlamydosporium var. chlamydosporium (=Pochonia chlamydosporia var. chlamydosporia) (AJ292397) sequence was chosen as outgroup. Results clearly indicated that the fungus belongs to S. lanosonieveum since it clustered with S. lanosonieveum with posterior probability value of 1.0 (Fig. 6). For ITS region minimum evolution method as implemented in MEGA 5.0 (Tamura et al. 2011 and references therein) was also used and it also clustered our sequence with S. lanosoniveum with bootstrap support value of 98 %.

Fig. 5
figure 5

Phylogenetic analysis based on large subunit sequences

Full size image
Fig. 6
figure 6

Phylogenetic analysis based on ITS region

Full size image

Verticillium was regarded as heterogeneous group having phytopathogenic, fungicolous, entomopathogenic and saprophytic species. The genus was divided into sections Verticillium, Nigrescentia, Prostrata and Albo-erecta. Fungicolous and enomopathogenic species of Verticilillium were placed in the genera Simplicillium and Lecanicillium based on a systematic study by Zare and Gams (2004). A phylogenetic concordance method is used for species delimitation in these genera.

So, to our knowledge this is the first record of S. lanosoniveum on A. elaeagni-latifoliae causing rust of E. latifolia in India. Simplicillium lanosoniveum has a potential to be used for managing this rust as a sustainable and ecofriendly alternative.