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

Document Type: Original Article

Authors

1 Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

2 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

Abstract

Broomrapes (Orobanche spp.) are parasitic weeds and considered as a major limiting factor for the cultivation of various crops in many parts of the world. Due to the special biology of broomrape, including seed production, dispersal and longevity, the control of these species are often extremely difficult. Broomrape poses a serious threat to Iran’s agriculture; therefore exploring potential biological agents for these species are necessary. In this study, samples of infected broomrape plants (brown rot on vertical systems) collected from ten provinces of Iran, over the summer period (2014-2015). Fusarium isolates were identified according to their cultural and morphological characteristics. For phylogenetic analysis, a part of the tef1-α gene was amplified and examined. Based on morphological characters, fourteen species of 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, were identified. F. solani s. l., with 25% frequency, was the most common species among species. Eight species namely F. andiyazi, F. hostae, F. flocciferum, F. foetans, F. lacertarum, F. redolens, F. thapsinum and F. torulosum on broomrape are being reported for the first time on global-scale and F. lacertarumis being reported for the first time in Iran.

Keywords

Main Subjects


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 25ºC 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 26ºC and 30ºC 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. andiyaziF. equiseti, F. flocciferum, F. foetens, F. hostae, F. lacertarum, F. oxysporum, F. proliferatumF. 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.

Species

Isolate

GB Accession no.

host

Authors

Fusarium andiyazi

2193

EU620627.1

sorghum grain
Petrovic et al., 2008
Fusarium andiyazi
M051749S2_

KM462947.1

sorghum grain

Funnell-Harris et al., 2014

Fusarium andiyazi
M051946S-3

KM462919.1

sorghum grain

Funnell-Harris, et al., 2015

Fusarium equiseti

XJ-CJ-F11-11

KT224315.1

sugar beet
Wang & Wu et al. , 2015

Fusarium equiseti

UBOCC-A-

KF225018.1

---------
Lecellier, et al., 2013

Fusarium equiseti

MOS879

KP008978.1

Soil

Oskiera, et al., 2014

Fusarium equiseti

ITEM 3190

JF966238.1

Soil

Stepien, et al., 2012

Fusarium flocciferum

VI01420

AJ543572.1

Hordeum 

Kristensen, et al., 2005

Fusarium flocciferum

GS-WW-4-1

KT224194.1

Potato
Wang and Wu, 2015 

Fusarium flocciferum

GS-WW-1-3

KT224192.1

Potato
Wang and Wu, 2015

Fusarium foetens

10-137b

JX298790.1

Begonia elatior

Saurat, et al., 2013

Fusarium foetens

NRRL 52749

JF740825.1

---------

O'Donnell, et al., 2012

Fusarium foetens

NRRL 31852

HM057337.1

Tomato

Huang et al., 2010

Fusarium hostae

NRRL 29889

HM057340.1

---------

Huang, et al., 2010

Fusarium hostae

O-2081

AF331819.1

---------

Geiser, et al., 2001

Fusarium hostae

NRRL29642

AF324322.1

---------

O'Donnell and Geiser, 2000

Fusarium lacertarum

NRRL 52753

JF740828.1

---------

O'Donnell, et al., 2012

Fusarium lacertarum

NRRL 20423

GQ505593.1

cucumber
O'Donnell, et al., 2009
Fusarium oxysporum 
ATCC 16612

KT323866.1

cucumber

Ortiz et al., 2017
Fusarium oxysporum 
CBS 127.73

KF913725.1

Pisum sativum
Bani, et al., 2014
Fusarium proliferatum 
G3-1

KX215078.1

strawberry
Pastrana, et al., 2016

Fusarium proliferatum

M05-1749S-1

KM462938.1

sorghum grain
Funnell-Harris, et al., 2015

Fusarium proliferatum

FV4

KF715258.1

Barley
Molnar, O. 2014

Fusarium redolens

MOS681

KP008977.1

Tomato

Oskiera et al., 2014

Fusarium redolens

NRRL 25123

JF740748.1

Tomato

O'Donnell et al., 2012

Fusarium sambucinum

IM-WL-SD-

KT224139.1

Potato
Wang and Wu, 2015

Fusarium sambucinum

YN-KM-DC

KT224160.1

Potato
Wang and Wu, 2015

Fusarium sambucinum

XJ-YN-1

KT224159.1

Potato
Wang and Wu, 2015

Fusarium solani

NRRL 52778

JF740846.1

---------
O'Donnell, et al., 2012

Fusarium solani

NRRL 25083

JF740714.1

---------
O'Donnell, et al., 2012

Fusarium solani

MOS615

KP008979.1

---------
Oskiera, et al., 2014

Fusarium thapsinum

FT-2

KM589049.1

Tomato
Kandan, et al., 2014

Fusarium thapsinum

M05-1711S-

KM462956.1

sorghum grain
Funnell-Harris et al., 2015

Fusarium thapsinum

M05-1874S-

KM463006.1

sorghum grain
Funnell-Harris, et al., 2015

Fusarium torulosum

NRRL 52772

JF740840.1

---------
O'Donnell, et al., 2012

Fusarium torulosum

F110

JX534443.1

---------
Chen, et al., 2014

Fusarium verticillioides

A71

KY173009.1

---------
Tupaki- et al., 2016

Fusarium verticillioides

638ES

KR905555.1

Maize
Velarde Felix et al., 2015

Fusarium verticillioides

F36

KM598766.1

Maize
Madrigal, et al., 2014

Fusarium scirpi

NRRL 36478

GQ505654.1

---------
O'Donnell et al., 2009

Fusarium scirpi

NRRL 29134

GQ505605.1

---------
O'Donnell et al., 2009

Fusarium scirpi

NRRL 26922

GQ505601.1

---------
O'Donnell et al., 2009

Fusarium sp.

NRRL 52720

JF740802.1

---------
O'Donnell et al., 2012
Fusarium sp.
NRRL 25085

JF740716.1

---------

O'Donnell, et al., 2012

Fusarium sp.

45997

GQ505672.1

---------

O'Donnell, et al., 2009

Fusarium sp.

ITEM 13005

LN901570.1

Wheat
Villani, et al., 2015

Microdochium majus

10149

JX280543.1

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.

Isolate

Species

Sampling region

GB Accession no.

Collection Accession no.

AH1

F. flocciferum

Alborz province

MF588955

ABRII 10265

FSE3-1

F. foetens

Fars province

MF588956

ABRII 10257

HT1-2

F. solani

Alborz province

MF588957

ABRII 10258

KG1

F. torolosum

Kermanshah province

MF611744

ABRII 10259

KG7-1

F. hostae

Kermanshah province

MF611745

ABRII 10260

KK23-2

F. verticillioides

Kurdistan province

MF611746

ABRII 10261

KK27-2

F. andiyazi

Kurdistan province

MF611747

ABRII 10262

KK32-1

F. sambucinum

Kurdistan province

MF611748

ABRII 10263

KK32-2

F. verticillioides

Kurdistan province

MF611749

ABRII 10264

KM4

F. lacertarum

Kermanshah province

MF611751

ABRII 10266

NA2-1

F. equiseti

Hamedan province

MF611752

ABRII 10267

NA9

F. redolans

Hamedan province

MF611753

ABRII 10268

ZCH2-2

F. oxysporum

Zanjan province

MF611755

ABRII 10269

ZCH6

F. proliferatum

Zanjan province

MF611754

ABRII 10270

 

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).

 

Fig. 1. Morphological characters of F. lacertarum. a. A single spore of Macroconidia. b. A clump of clamydospores. c. Front and d. back views of PDA cultured colony. Scale bar: 10 µm.

Fig. 2. Phylogenetic tree showing the relationship of 34 Fusarium species strains based on tef1-α gene sequence using the neighbor-joining (NJ) method. The percentage values of replicate trees in which the linked taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The bootstraps values > 50% were shown next to the branches. Microdochium majus was used as an out-group.

Fig. 3. Phylogenetic tree showing the relationship of 34 Fusarium species strains based on tef1-α gene sequence using the Maximum likelihood (ML) method. The percentage values of replicate trees in which the linked taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The bootstraps values > 50% were shown next to the branches. Microdochium majus was used as an out-group.

 

Phylogenetic analysis

A phylogenetic tree with 3 clades including clades A, B and C was drown during phylogenic analysis of tested taxa based on NJ method (Fig. 2). Clade A was containing Gibberella fujikuroi species complex and divided into four subclades. Sublade A1 showed strain of F. andiyazi that were supported by bootstrap value of 99%, subclade A2 consisted of isolates F. verticilliodesand subclad A3 including strains of F. foetans, F. oxysporum and F. thapsinum. An isolate which was morphologically identified as F. verticilliodes (kk 23-2), according to no observation of globose microconidia, was placed within the F. thapsinum isolates from NCBI and subclade A4 contained strains of F. proliferatum species.

Clad C is a group with members of Gibbosum complex of Fusarium species and divided into four subclades names C1, C2, C3 and C4. Subclade C1 was supported by the strong bootstrap value of 94% and included F. laceratum strains, Subclade C2 included strains of F. scirpi and was located as sister group of subclade C3 including, strains of F. equiseti and some Fusarium sp. isolates.

Clade B includes other species and divided into four subclades (B1, B2, B3 and B4). Subclade B1 included the strain HT 1-2 and demonstrated high similarities of tef1-α gene sequence to the referred isolates of F. solani, subclade B2 contained two species of F. hostae and F. redolens by strong bootstrap value of 98%, subclade B3 consists of F. torulosum and F. flociferatum strains, subclade B4 consisted of strain KK32-1 which gave high similarity of tef1-α gene sequence to the referred F. sambucinum from NCBI and was supported by the strongest bootstrap value of 100%.

Maximum likelihood (ML) analysis of the tef1-α gene sequence alignment recovered a tree with significant similarities to the phylogenetic tree in NJ method (Fig. 3). However, there are some differences between the two trees. For example, subclade B1 in NJ analysis which includes F. solani strains is located as a separate clade in maximum likelihood method that was named as clade D. Subclade B4 consisted of strain KK32-1 and strains of F. sambucinum was placed in clade C in maximum likelihood analysis by the strongest bootstrap value of 100%. The location of isolate KM4 (F. lacertarum) in ML tree is the same as NJ tree.

In this study, in addition to the morphological identification, the sequence data analysis of the tef1-α region was employed. The molecular data and phylogenetic relationships allow reliable differentiation between the major Fusarium species. For this purpose, sequence of tef1-α gene was used to assess 15 identified species of genus Fusarium isolated from broomrape samples. Use of translation elongation factor 1α (tef1-α) and β-tubulin genes can lead to more clear fungal identification (Vitale et al. 2011; Wang et al. 2011). Overall, Molecular analysis had harmony with morphological grouping, except for isolate kk23-2 in clade A and subclade A3, where F. verticilliodes isolate (kk23-2) was located between the F. thapsinum isolates. Some strains of F. thapsinum that do not produce diagnostic yellow pigment, is morphologically identical to F. verticillioides therefore in similar situations molecular characterization can be helpful in identifying these isolates (Leslie & Summerell, 2006). This sort of discrepancy between morphological and molecular data in fungal studies has been seen frequently (Darvishnia 2013; Watanabe 2013). Although the morphological species concept does not completely reflect the phylogenetic tree of the genus Fusarium (O'Donnell et al., 2000), this does not imply that morphological characteristics are not useful for identification and taxonomy. To identify unknown species, morphological characteristics can be widely applied to any species, not only the genus Fusarium but also to other fungi (Taylor et al. 2000). Fusarium isolates can be initially classified on the basis of morphological similarity, with the awareness that sections are in fact a means of artificial grouping, but the morphological approach fails to detect many biological factors but phylogenetic approach can be useful in detection of this factors (Liddell, 2003).

Due to the deficiencies and problems within morphological identification knowledge and also due to the large number of fungal species and inadequate available morphological information, the use of molecular information can be helpful (Davari et al. 2013). So, we need to construct more reliable taxonomic system in combination with the morphological, phylogenetic, toxicological, biological, and other recognition methods. The use of another gene in molecular identification can be useful in better identifying and better phylogenetic analyses.

 

ACKNOWLEDGEMENTS

This research was supported by University of Zanjan and the authors wish to acknowledge Research Institute of Modern Biological Techniques at University of Zanjan for the use of their equipments.

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