Morphological and phylogenic analysis of Fusarium species associated with vertical system of Orobanche spp.

INTRODUCTION

Broomrapes (Orobanche spp.) are one of the most important weeds around the world (Ghotbi et al., 2011). Broomrapes are distributed in more than 80 countries and invaded almost 16 million hectares of agricultural lands around the world. Depending on rate and the amount of infection, broomrape can reduce yield quality and quantity between 30 to 100 percent. Broomrapes only germinate in response to specific chemicals released by the host plants (Perez-de-Luque et al. 2010). After germination, the seedlings attach to the host roots by the production of specialized feeding structures, described as haustoria to develop and accumulate nutrient resources from host plant (Joel et al. 2007). Therefore, broomrape is a damaging and destructive weed to crops and is difficult to control.

Although broomrapes are efficient in mechanisms such as seed production, dispersal and longevity and host roots attachment ability (reaching and entering the vascular tissue and underground development), the control of these species are extremely difficult (Montazeri, 2011; Amsellem et al. 2001b; Mazaheri & Ershad, 1995).  Despite the various control practices against broomrapes, such as cultural and mechanical methods, soil fumigation, soil solarization, trap crops and resistant cultivars (Jacobsohn et al. 2001), these available control techniques have not yet proven to be as effective, economical and applicable as expected (Alejandro et al. 2010; Goldwasser & Kleifeld 2004). Phytopathogenic fungi such as Fusarium species (F. oxysporum Schelcht, F. oxysporum f. sp. orthoceras, F. solani Mart., F. arthrosporioides Sherb., F. nygamai Burgess & Trimboli and F. semitectum Berk. & Ravenel, especially F. semitectum var. majus) were reported to be associated with Orobanche spp. These Fusarium species have shown significant pathogenicity against Orobanche spp. when tested under controlled or field conditions (Amsellem et al. 2001b; Bedi & Donchev, 1991; Cohen et al. 2002; Muller-Stover et al. 2002). These fungal pathogens demonstrate their potential ability to be used as bio-herbicides. 

TEF is a protein that is translated into an essential part of the encoding high phylogenetic region (Geiser et al. 2004). This gene first was used as a marker for identification and phylogenetic relationship of the species belongs to noctuid moths subfamily Heliothinae in Lepidoptera Order (Cho et al. 1995). Since, the issue of broomrape has caused serious problems to Iran’s agriculture; this study introduce a proper method to overcome the problems associated with Orobanche spp. The aim of this study was to identify Fusarium species associated with Orobanche species that potentially used as biological control agents of broomrape using indigenous antagonistic Fusarium species.

MATERIALS AND METHODS

Sampling

Infected broomrape plants (Orobanche spp.) with vertically brown rot symptoms were randomly collected from tomato farms in ten provinces of Iran, including Alborz, Tehran, Kermanshah, Kurdistan, Hamadan, Zanjan, East Azerbaijan, Razavi Khorasan, Fars and Markazi provinces, during summertime in 2014-2015. Samples were picked up from their roots using trowel and transferred to the laboratory in paper packets.

Fungal isolation and preservation

Isolation of fungi was carried out according to Nash and Snyder (1962) medium. The roots were separated and rinsed in tap water for 20 minutes to wash away soil particles. The Root part of each samples were then cut into 2 cm pieces and the sterilization steps took place. The pieces were soaked at 1% sodium hypochlorite for 2 minutes and rinsed in sterilized distilled water and air dried on sterile filter paper. The disinfected pieces were cut into 2 cm pieces, placed on Peptone PCNB Agar (PPA) medium and incubated at 25^ºC in the dark for 3 days. The isolates were sub-cultured into Water Agar (WA) medium and a tip of the hyphae was picked up and transferred to PDA (potato dextrose agar) medium. The purified isolates were then stored on sterile filter papers at -20^°C. Fungal isolates were deposited in fungal culture collections of University of Tehran (UTFC).

Morphological characterization

Isolates of Fusarium were identified according to their cultural and morphological characteristics as described by (Gerlach & Nirenberg, 1982; O'Donnell et al., 2004; Leslie & Summerell, 2006; Saremi, 2005). The isolates were grown on PDA medium to determine their growth rate and colony pigmentation, so the cultures were incubated at 26^ºC and 30^ºC for 7–10 days in the dark. Colony diameter was measured and Colony color recorded with naked eyes. Isolates were also placed on CLA and SNA, then incubated for 14 days under fluorescent and near-ultraviolet lights conditions to investigate the presence and shape of the macroconidia, microconidia and chlamydospores.

Phylogenetic analysis

DNA extraction: Liquid cultures were initiated by adding 2 pieces of 5 days old fungal cultures to 250-mL Erlenmeyer flasks containing 100 mL PDB medium (potato dextrose broth plus 2 g yeast extract per liter). Flasks were incubated at room temperature approximately 25°C on a rotary shaker for 6–8 days. Mycelium was collected by filtration through the sterile filter paper with a vacuum funnel. Mycelia were harvested, frozen and stored at −20^°C. DNA was extracted using a modified hexadecyl trimethyl-ammonium bromide (CTAB) procedure (Doyle and Doyle 1987). The DNA was visualized on a 1% agarose gel (wt/v) (Boehringer Mannheim) stained with ethidium bromide and viewed under ultra-violet light. DNA concentrations were estimated by comparing the intensity of ethidium bromide fluorescence of the DNA sample to a known concentration of lambda DNA marker (marker III, Roche Diagnostics). Extracted DNA (50–90 ng) was used as the template for the PCR reaction.

Molecular characterization

A part of the tef1-α gene was amplified by PCR using the primers Ef1 F (5^'-ATGGGTAAGGAGGA CAAGAC-3^') and Ef2 R(5^'GGAAGTACCAGTGAT CATGTT-3^') (O’Donnell et al. 1998) in a final volume of 25 μL containing 50-60 ng of DNA, 0.1 μM of each primer, 150 μM dNTP, 3 U Taq DNA polymerase and PCR reaction buffer. Amplifications were conducted in a Master-cycler (Eppendorf) with an initial denaturation of 5 min at 95^°C followed by 35 cycles of 60 s denaturation at 95^°C, 75 s annealing at 56^°C, 60 s extension at 72^°C and a final extension of 7 min at 72^°C. The presence of PCR products was confirmed by gel electrophoresis. The tef1-α amplicons were sequenced by Macrogene Co. (South Korea) using the two PCR primers as sequencing primers. Sequence identities were determined using Blast analysis from NCBI available online and most identic sequences from each species were recorded together with their information to use in phylogenetic analysis (table 1).

Sequence analysis

Sequences were aligned and compared by Kimura’s two parameters distance model and the neighbor-joining (NJ) and Maximum Likelihood (ML) methods with Tamura-Nei distance model using the program MEGA ver. 6.0 software (Gouy et al. 2010). The topology of the resulting tree was tested by bootstrapping with 1000 re-samplings of the data.

RESULTS AND DISCUSSION

 A total of 203 isolates from 385 collected samples were identified as the genus of Fusarium. Based on morphological characters, only fourteen Fusarium species, including Fusarium, including F. andiyazi, F. equiseti, F. flocciferum, F. foetens, F. hostae, F. lacertarum, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. solani s. l., F. thapsinum, F. torulosum and F. verticillioides, have been identified. Fusarium solani, F. oxysporum and F. redolense with 25%, 20% and 15% frequency are common among all the species, respectively (table 2).

Table 1. Information of refer sequences from NCBI gene bank used in phylogenic analysis.

Fusarium andiyazi

sorghum grain

Petrovic et al., 2008

Fusarium andiyazi

M051749S2_

sorghum grain

Fusarium andiyazi

M051946S-3

sorghum grain

XJ-CJ-F11-11

sugar beet

Wang & Wu et al. , 2015

UBOCC-A-

---------

Lecellier, et al., 2013

MOS879

Oskiera, et al., 2014

ITEM 3190

VI01420

Hordeum 

GS-WW-4-1

Potato

Wang and Wu, 2015 

GS-WW-1-3

Potato

Wang and Wu, 2015

10-137b

Begonia elatior

NRRL 52749

O'Donnell, et al., 2012

NRRL 31852

Tomato

NRRL 29889

O-2081

NRRL29642

O'Donnell and Geiser, 2000

NRRL 52753

O'Donnell, et al., 2012

NRRL 20423

cucumber

O'Donnell, et al., 2009

Fusarium oxysporum 

ATCC 16612

Ortiz et al., 2017

Fusarium oxysporum 

CBS 127.73

Pisum sativum

Bani, et al., 2014

Fusarium proliferatum 

G3-1

strawberry

Pastrana, et al., 2016

M05-1749S-1

sorghum grain

Funnell-Harris, et al., 2015

FV4

Barley

Molnar, O. 2014

MOS681

Oskiera et al., 2014

NRRL 25123

O'Donnell et al., 2012

IM-WL-SD-

Potato

Wang and Wu, 2015

YN-KM-DC

Potato

Wang and Wu, 2015

XJ-YN-1

Potato

Wang and Wu, 2015

NRRL 52778

---------

O'Donnell, et al., 2012

NRRL 25083

---------

O'Donnell, et al., 2012

MOS615

---------

Oskiera, et al., 2014

FT-2

Tomato

Kandan, et al., 2014

M05-1711S-

sorghum grain

Funnell-Harris et al., 2015

M05-1874S-

sorghum grain

Funnell-Harris, et al., 2015

NRRL 52772

---------

O'Donnell, et al., 2012

F110

---------

Chen, et al., 2014

A71

---------

Tupaki- et al., 2016

638ES

Maize

Velarde Felix et al., 2015

F36

Maize

Madrigal, et al., 2014

NRRL 36478

---------

O'Donnell et al., 2009

NRRL 29134

---------

O'Donnell et al., 2009

NRRL 26922

---------

O'Donnell et al., 2009

NRRL 52720

---------

O'Donnell et al., 2012

Fusarium sp.

NRRL 25085

O'Donnell, et al., 2012

45997

ITEM 13005

Wheat

Villani, et al., 2015

10149

Triticum sp.

Jewell, & Hsiang, 2012

Fungi associated with Orobanch spp. including Alternaria spp., Bipolaris austransis, F. equiseti, F. oxysporum, F. semitectum, F. solani, Rhizoctonia solani, Ulocladium atrum and Verticillium allbo-atrum, were isolated from O. aegyptiaca (Mohammadi et al. 2014). Fusarium species are well distributed across many geographical regions and substrates, and also widely distributed in different soils, plants and air (Booth 1971; Burgess et al. 1994; Nelson et al. 1994; Summerell et al. 2003). So, in this research different species of genus Fusarium associated with Orobanch spp. from different geographical regions of Iran were identified.

The morphological identification of the Fusarium species was confirmed by the sequencing of tef1-α gene. So, the Standard Nucleotide BLAST search for similarities showed the similarity percentage of the strains ranged from 98 to 99 percent. The tef1-α sequence of Fusarium strains were searched for homology in GeneBank database. Then, the result of tef1-α gene sequencing demonstrates that all tested isolates belong to the genus Fusarium. Also, the similarities of tef1-α sequence between our isolates and the reference sequences from the GeneBank, were supported by bootstrap values of more than 50 percent. All the analyzed sequences data were deposited in the GeneBank database. To our knowledge, this is the first report of eight species including F. redolens, F. torulosum, F. hostae, F. foetans, F. andiyazi, F. flocciferum, F. lacertarum and F. thapsinum on Orobance spp. in global scale. Idetification of F. lacertarumis from Orobanch spp. is recorded for the first time for the mycobiota of Iran.

Table 2. Information of fifteen species of Fusarium isolated from infected broomrapes.

Fusarium lacertarum Subrahm., Mykosen 26 (9): 478. 1983.

Colonies on PDA were reached to 38-44 mm average growth after three days at 26°C. Mycelia were white and the bottom of the colony showed brown-orange pigmentation after seven days. On CLA, the aerial mycelium produced solitary chlamydospores with 7.7-12.3µm diameters, and no microconidia was formed. Macroconidia originated from abundant sporodochia with strong orange color, in short monophialids, usually with not more elongated apical cells, rarely curved and the basal cell of macroconidia presented a foot form. Apicali cell form in hook form, smoothly curved, 5 septa, 42-58×3.5–4.5 µm (Fig. 1). Morphological characters of F. lacertarum approved by sequencing results of tef1- α part gene. 

Specimens examined. IRAN, Kermanshah Province, Mahidasht, isolated from crown and root of Orobanche aegyptiaca, 28 July 2014, A. Rostami, (IRAN UTFC-FO11, isolate KM4). 

In this study, based on molecular data and morphological characterization, F. lacertarum is reported for the first time in Iran and also on broomrape across the world. Morphology of examined specimens agrees with the description provided by Poletto et al. (2015).