INTRODUCTION

Several plant species in the Lamiaceae (Labiatae) and Asteraceae (Compositae) families are important ornamental, medicinal and aromatic plants which produce essential oils that are used in traditional and modern medicine. A large number of medicinal plants from the Lamiaceae and Asteraceae are grown in Kerman province, Iran. Kerman is the second-largest province of Iran with an area of 180,726 km² encompasses nearly 11 percent of the land area of the country with unique climatic conditions which has rangelands with a large variety of medicinal plants (Rajaei & Mohamadi 2012). In the recent years, much interests have been dedicated in medicinal plants and herbal derived products all over the world, including Iran.

The fungal genus Fusarium is notorious as a plant pathogen (Vujanovic et al. 2006, Bacon & Yates 2006) while several species are significant mycotoxigenic species or causal agents of human diseases. They are also found as saprophytes in air, water and soil, and as epiphytes or endophytes in living plants (Leslie & Summerell 2006, O’Donnell et al. 2010; Aoki et al. 2014). Members of the genus Fusarium are considered among ten globally most important genera of plant pathogenic fungi (Dean et al. 2012). The Fusarium oxysporum, is cosmopolitan fungi and several strains are known as sever threads agents for many crops. Furthermore, species of F. graminearum as well as F. proliferatum, F. verticillioides and F. sporotrichoides, produce mycotoxins on crops including wheat, maize, barley, rice and other foodstuffs (Mirocha et al. 1989, Bánáti et al. 2017).

Fusarium species such as Fusarium solani (Mart.) Sacc. (Green & Skotland 1993, Nasr Esfahani & Monazzah 2011) and F. oxysporum Schltdl. (Sattar & Husain 1980, Borges et al. 2018, Ortu et al. 2018) are reported as diseases agents on Lamiaceae species worldwide. Furthermore, F. oxysporum and F. solani are reported to be associated with several hosts from the Asteraceae family (Kourany et al. 1988, Koike 2011, Kim et al. 2016, Garibaldi et al. 2017, Matić et al. 2018). However, a comprehensive study of Fusarium species associated with medicinal species in the Lamiaceae and Asteraceae has not been investigated in Iran. To the best of our knowledge, no comprehensive information is available on fungal flora associated with these medicinal species in Iran. One reason is that these plant species are mainly distributed in less accessible areas of rangelands such as high elevations of mountains and rocky areas. Characterization of Fusarium species in association with these plant species is of great importance to understand their complex interactions among plant, fungus and habitat. Therefore, this study was designed to isolate and identify Fusarium species associated with selected medicinal species of the Lamiaceae and Asteraceae based on the morphological and molecular characteristics and thereafter, to study the pathogenecity of isolates on their hosts. Consequently, studies on pathogenicity of Fusarium spp. on these hosts are critical in order to develop management measures for this group of fungi wherever commercial plantations have been started.

MATERIALS AND METHODS

Sample collection

In 2017, a survey was conducted across the main medicinal plants reservoirs of northern Kerman province, Iran. The following species including Teucrium polium, Ziziphora tenuior, Thymus carmanicus, Mentha piperita and Ziziphora cliniopodiodes from Lamiaceae and Achillea wilhelmsii and Artemisia dracunculus from Asteraceae, were selected for samplings. Sixty-five samples were collected from the wild plants and the main cultivation sites in northern regions of Kerman, Iran. At each site, three asymptomatic plants as well as three plants showing yellowing or wilting and brown discoloration of vascular tissues were collected 10 m apart in the form of whole plant and transferred to the laboratory in paper bags. Three soil samples (100 g) were collected in proximity of plant roots from a depth of 0–15 cm using a trowel. Soil samples were sieved, air-dried, and stored at 4°C until they were assessed.

Fusarium Isolation and storage conditions

The roots, crown and stem tissues were rinsed under running tap water for 30 minutes to remove excess soil particles and spores of fast-growing contaminant fungi present on tissues surface, surface-disinfected with 70% ethanol for 10 s, rinsed in sterile distilled water (SDW) for 10 s, then submerged for 2 min in 1% NaOCl, rinsed three times in SDW and dried on sterile filter paper. Tissues were cut into the 5-10 mm-long pieces, plated onto PDA (Potato Dextrose Agar) plates supplemented with antibiotic (0.2 g. L^-1 streptomycin sulfate). Fusarium species were isolated from soil using dilution method. The amount of 1.0 mL of 10⁻² to 10⁻⁴ fold diluted suspensions was inoculated into a modified Nash/Snyder medium (Nash & Snyder 1962, Burgess et al. 1994). Plates were incubated for 7 days at 25^°C and inspected for Fusarium-like colonies. Pure cultures were established using single spore techniques (Summerell et al. 2003) and stored on PDA slants at 4^°C. All the isolates were deposited in the KGUT Fungal Culture Collection at Kerman Graduate University of Advanced Technology, Kerman, Iran, stored in 15% glycerol at -80^°C.

Morphological characterization

Fusarium cultures were identified based on the approaches described by Nelson et al. (1983), Burgess et al. (1994), and Leslie & Summerell (2006). Isolates were cultured on SNA (Synthetic Nutrient-poor Agar) and CLA (Carnation Leaf Agar) and incubated at 22-25^°C in darkness. Microscopic characteristics were observed after 7–14 days of inoculation. Colony morphology (pigmentation and colony growth rate) was assessed on PDA (Leslie & Summerell 2006). Each strain was grown at 15, 20, 25, 30, 35^°C in darkness in triplicates. Colony diameter was measured at intervals of 24 h over a period of 96 h. Microphotographs of fungal features were taken from agar plates and microscopic slides using a Dino-eye microscope camera USB lens (The Microscope Store, LLC., USA). Thirty measurements were taken of the relevant parameters of the conidiophores and conidia.

DNA Extraction, PCR, Sequencing

Genomic DNA was extracted using a CTAB extraction procedure (Doyle & Doyle 1987, Zhang et al. 2010). Approximately, 100 mg of fresh mycelia was scraped off the PDA plate of each isolate and homogenized using liquid nitrogen. Afterward, cells were lysed using CTAB solution and the DNA was extracted using DNG^TM-plus DNA extraction solution (Sinaclon, Iran) according to the manufacturer’s instructions.

A standard polymerase chain reaction (PCR) protocol was used to amplify a ~700 bp portion of the TEF1-α gene (Translation Elongation Factor 1α) region using primers ef1 (forward primer; 5'-ATGGG TAAGGA(A/G)GACAAGAC-3') and ef2 (reverse primer; 5'-GGA(G/A)GTACCAGT(G/C)AT CATGT T-3') (O’Donnell et al. 1998, Geiser et al. 2004). Amplifications were performed in a Biometra TAdvanced Thermal Cycler (Biometra, Göttingen, Germany) with an initial denaturation of 5 min at 95^°C followed by 35 cycles of 30 s denaturation at 95^°C, 30 s annealing at 54^°C, 60 s extension at 72^°C and a final extension of 5 min at 72^°C. The quantity and quality of PCR products were evaluated on 1% agarose gels and for some of the PCR amplicons, product purification from the gel was conducted using the Kardan gel extraction kit (Kardan co, Iran). The PCR products sequencing was performed by Macrogen (Macrogen Inc., Seoul, South Korea).

Molecular identification and Phylogenetic analysis

The obtained sequences were manually edited by Geneious version 7 (Biomatters, USA). The sequencing results were compared with those in MLST Fusarium database and NCBI/Genebank database using a basic local alignment search tool (BLAST) (Altschul et al. 1990). All the sequences generated in this study were deposited in GenBank and the accession numbers were obtained (Table 1). Furthermore, the number of 31 DNA sequences was retrieved from GenBank according to the reliable published papers and included in the phylogenetic analyses. The sequences were aligned using Geneious version 7 (Biomatters, USA). Analyses of phylogenetic relatedness among the obtained Fusarium isolates and identification of the isolates to the species level were performed using PAUP* 4.0a133 (Swofford 2002) for parsimony and MrBayes v3.2.2 (Ronquist et al. 2012) for Bayesian analyses. All the alignment gaps were treated as missing data. Fusarium merismoides Corda was used as outgroup. The best-fit model (HKY+I+G) was selected using JModeltest v.2.1.4 (Posada 2008) and the Bayesian information criterion (BIC) ratio test.

Pathogenicity tests

The pathogenicity tests of selected Fusarium species being isolated from Lamiaceae and Asteraceae plant hosts were performed to satisfy Koch’s postulates. The pathogenicity tests were performed only for the isolates from hosts which had symptoms of Fusarium infections in field samplings.Local cultivars of Achillea wilhelmsii, Mentha piperita and Ziziphora cliniopodiodes were included in the pathogenicity tests. The following experiments were designed. The isolates of F. nygamai, F. oxysporum and F. solani were selected to test for pathogenicity on Achillea wilhelmsii. The isolates of F. brachygibbosum, F. equiseti, F. proliferatum and F. redolens were selected for pathogenicity test on Mentha piperita. The Fusarium solani and F. proliferatum isolates were also selected to test on Ziziphora cliniopodiodes plants. 

Five plants for each isolate/plant species combination were inoculated. The pathogenicity of the isolates was tested using root-dip inoculation method (Biles & Martyn 1989). Two isolates from each fungal species were used for inoculum preparation. The inoculum was prepared by growing cultures in potato dextrose broth (PDB) shaking at 180 rpm for 3-5 days. The cultures were filtered through two layers of sterile cheesecloth and then spore concentrations were adjusted to 5 × 10⁶ spores ml^-1 using a hemocytometer. For inoculations, few months old plants grown in autoclaved potting mix were removed and washed in running tap water. Afterward, the wounded root tips were dipped into the spore suspension for 30 min, then planted into pots containing sterile potting mix. Sterile PDB was used for controls. Plants were incubated at 20^°C for 24 h, then transferred to greenhouse. Plants were inspected daily for symptoms.

RESULTS

Morphological and Phylogenetic identification of Fusarium isolates

Amplification of the TEF1-α gene fragment of approximately 700 bp from all isolates was conducted successfully using primers ef1 and ef2. To elucidate phylogenetic relationships among the isolated Fusarium spp. and to aid their accurate identification, a Bayesian tree was constructed using TEF1-α sequences (Fig 1).

Table 1. Fusarium spp. isolates used in phylogenetic analysis, isolated from medicinal plants of Lamiaceae and Asteraceae in Kerman, Iran.

The phylogenetic reconstruction revealed eight species complexes namely Fusarium solani species complex (FSSC), Gibberella fujikuroi species complex(GFSC), Fusarium redolens species complex (FRSC), Fusarium incarnatum-equiseti species complex (FEISC), Fusarium sambucinum species complex (FSSC), Fusarium tricinctum species complex (FTSC), Fusarium oxysporum species complex (FOSC) and F. merismoides species complex (FMSC). Maximum parsimony analysis yielded a tree with a topology similar to Bayesian tree. The analysis of tree constructed based on the Bayesian inference (BI), is presented here because of the slightly better clade support.

In the present study, the number of 121 Fusarium spp. isolates was characterized from selected medicinal plants of Lamiaceae and Asteraceae families (Teucrium polium, Ziziphora tenuior, Thymus carmanicus, Mentha piperita, Ziziphora cliniopodiodes, Achillea wilhelmsii and Artemisia dracunculus) collected in Kerman, Iran, based on morphological and molecular criteria. The following species were isolated: Fusarium oxysporum, F. solani, F. nygamai and F. redolens, F. proliferatum, F. brachygibbosum, F. equiseti, F. merismoides and species of F. tricinctum species complex (Table 2).

The strain F. solani was the most dominant isolated species (24.8%) across all the sampled plant species, followed by F. proliferatum (19%). Other most frequently isolated species recovered from collected samples that were outside the scope of this study was Rhizoctonia solani. Species belonging to F. tricinctum and F. solani species complexes were recovered almost from rhizosphere soil of most plant species. Twenty-six isolates were obtained from Achillea wilhelmsii plants showing stem base yellowing and leaf necrosis. The number of eleven isolates belonged to F. nygamai and the remaining isolates were members of F. solani as well as isolates of F. tricinctum species complex and isolates of F. oxysporum. Thirty eight isolates which were associated with Mentha piperita were identified as F. brachygibbosum, F. redolens, F. equiseti, F. proliferatum and species of F.tricinctum species. Twelve isolates of F. solani and F. proliferatum were associated with Ziziphora cliniopodiodes. Besides, Ziziphora tenuior was associated with 17 Fusarium isolates belonging to F. solani, F. proliferatum and few isolates of F. tricinctum species complex (Table 2). Thymus carmanicus and Artemisia dracunculus did not associate with any Fusarium species except for the few isolates belonging to F. tricinctum species complex being isolated from rhizosphere soil dilutions. Most Fusarium isolates being nonpathogenic to their hosts were grouped in the same clade with the pathogenic isolates from other hosts (Fig. 1).

Pathogenicity tests

Among the examined fungal species, Fusarium nygamai isolates (MB4 and MB8, Fig 2) were pathogenic to Achillea wilhelmsii and foliar symptoms were observed 15 days after inoculation while non-inoculated plants remained asymptomatic. To complete Koch’s postulates, the tested isolates were consistently re-isolated from symptomatic tissues. The inoculated plants showed wilt symptoms similar to those observed in the field. Foliar yellowing symptoms began at the stem base and progressed throughout the whole plants, occasionally resulted in necrotic leaves. Examination of the roots showed necrotic lesions in tap roots. No symptoms were observed in Achillea wilhelmsii plants inoculated with F. solani, F. oxysporum and F. tricinctum species complex isolates. However, in the plants inoculated with F. solani, mild yellowing at the stem base was observed after 18 days of inoculation and the tested isolates (MB9 and MB12) were recovered from Achillea wilhelmsii tissues on solid media. No vascular discoloration in the tissues were observed.

The Fusarium redolens and F. brachygibbosum isolates were pathogenic to Mentha piperita. Fusarium brachygibbosum isolates (O16 and O19, Fig 2) caused whole plant decline 12 days after inoculation, whereas no symptoms were observed in the control plants. In one case, the plant showed stem lesions up to few cm long as well as stem dieback. The fungus was re-isolated from the symptomatic tissues but not from the control samples. In pathogenicity tests of F. redolens, yellowing and few necrotic root lesions were observed on Mentha piperita 15 days after inoculation with F. redolens isolates (O31 and O46, Fig 2), whereas no symptoms were observed in the control plants. In addition, no symptoms were observed in the control plants and in the plants inoculated with isolates of F. proliferatum and F. equiseti having been proved to be nonpathogenic on M. piperita. Isolates of F. solani (MKK1) and F. proliferatum (O30) being obtained from Ziziphora cliniopodiodes plants with foliar yellowing and Fusarium-like symptoms did not show any symptoms in inoculated plants. No Fusarium isolates were recovered from inoculated Z. cliniopodiodes tissues as well.

Table 2. Fusarium spp. recovered from plants of Lamiaceae and Asteraceae in Kerman, Iran.

^a. Numbers in parentheses are the numbers of isolates recovered from each plant species.

Fig. 1. Bayesian tree generated from the nuclear TEF1-α sequences. Numbers on the branches are posterior probabilities. The tree was rooted to Fusarium merismoides. Isolates being sequenced in this study are shown in bold.

Fig. 2. Fusarium nygamai (a-e): a. Colony on PDA incubated for five days at 24 °C; b. Macroconidia; c-d. False heads and microconidia in chain; e. Chlamydospores; Fusarium brachygibosum (f-h): f. Colony incubated for five days at 24^°C grown on PDA; g. Chlamydospore. h. Macroconidia; Fusarium redolens (i-l): i. Colony incubated for seven days at 24^°C grown on PDA; j. Macroconidia; k. Micoconidia. l. False heads; Scale bars = 10 μm