Fusarium species associated with date palm in Saudi ...

Eur J Plant Pathol DOI 10.1007/s10658-016-1095-3

Fusarium species associated with date palm in Saudi Arabia Amgad A. Saleh & Anwar H. Sharafaddin & Mahmoud H. El_Komy & Yasser E. Ibrahim & Younis K. Hamad & Younis Y. Molan

Accepted: 7 November 2016 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Abstract Fusarium is one of the most destructive fungal genera whose members cause many diseases on plants, animals, and humans. Moreover, many Fusarium species secrete mycotoxins (e.g. trichothecenes and fumonisins) that are toxic to humans and animals. Fusarium isolates from date palm trees showing disease symptoms, e.g. chlorosis, necrosis and whitening, were collected from seven regions across Saudi Arabia. After single-sporing, the fungal strains were morphologically characterized. To confirm the identity of morphologically characterized Fusarium strains, three nuclear loci, two partial genes of translation elongation factor 1 α (tef1α) and β-tubulin

Electronic supplementary material The online version of this article (doi:10.1007/s10658-016-1095-3) contains supplementary material, which is available to authorized users. A. A. Saleh : A. H. Sharafaddin : M. H. El_Komy : Y. E. Ibrahim : Y. K. Hamad : Y. Y. Molan Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, P. O. Box 2460, Riyadh 11451, Saudi Arabia A. A. Saleh (*) Agriculture Research Center, Agricultural Genetic Engineering Research Institute, Giza, Egypt e-mail: [email protected] M. H. El_Komy : Y. E. Ibrahim Agriculture Research Center, Plant Pathology Institute, Giza, Egypt Y. K. Hamad Plant Pathology Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt

(tub2), and the rDNA-ITS region, were amplified and sequenced. Of the 70 Fusarium strains, 70 % were identified as F. proliferatum that were recovered from six regions across Saudi Arabia. Fusarium solani (13 %), as well as one strain each of the following species: F. brachygibbosum, F. oxysporum, and F. verticillioides were also recovered. In addition, five Fusarium-like strains were recognized as Sarocladium kiliense by DNA-based data. The preliminary in vitro pathogenicity results showed that F. proliferatum had the highest colonization abilities on date palm leaflets, followed by F. solani. Although F. oxysporum f. sp. albedinis is the most serious date palm pathogen, F. proliferatum and F. solani are becoming serious pathogens and efforts should be made to restrict and control them. In addition, the potential toxin risks of strains belonging to F. proliferatum should be evaluated. Keywords Date palm . Fusarium proliferatum . Fusarium solani . DNA-based data

Introduction Fusarium is one of the most destructive fungal genera causing many diseases on plants, animals, and humans. Some members of Fusarium produce mycotoxigenic secondary metabolites, e.g. deoxynivalenol, nivalenol, T-2 toxin, zearalenone, fumonisins, and fusarins that are highly toxic to plants, animals and humans (e.g. Prouillac et al. 2009; Kvas et al. 2009; Desjardins 2006). Some of these mycotoxins, such as fumonisins, are proven to be

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potent carcinogenic agents to humans and animals (e.g. Gelderblom et al. 2008; Lemmer et al. 2004; Marasas 2001). In addition, these mycotoxins have been recently detected in soils, surface and drainage waters (Hartmann et al. 2008). In Saudi Arabia, Fusarium species pose serious problems on human, animal and plant health (Gashgari et al. 2010; Al-Abdely 2004; Al-Julaifi and Al-Falih 2001; Musa et al. 2000). Many Fusarium species have been reported as date palm (Phoenix dactylifera L.) pathogens worldwide. The most serious one is F. oxysporum f. sp. albedinis that causes Fusarium wilt of date palm trees (Zaid et al. 2002). Fusarium oxysporum f. sp. albedinis fungus was responsible for loss of more than 15,000,000 trees in Morocco and Algeria between 1870 and 1970 (Djerbi 1982). There are other F. oxysporum formae speciales that attack other palms, namely F. oxysporum f. sp. palmarum, causal agent of lethal disease of queen palm (Syagrus romanzoffiana) and Mexican fan palm (Washingtonia robusta) (Elliott et al. 2010); F. oxysporum f. sp. canariensis, causal agent of Fusarium wilt of Canary Island date palm (P. canariensis) (HernándezHernández et al. 2010); and F. oxysporum f. sp. elaeidis, causal agent of Fusarium wilt of oil palm trees (Elaeis guineensis and E. oleifera) (Flood 2006). The other two most important Fusarium species associate with date palm trees are F. proliferatum and F. solani. Fusarium proliferatum has been reported as a date palm pathogen in many date palm-growing countries, e.g. Saudi Arabia (Abdalla et al. 2000), Spain (Armengol et al. 2005), Canary Islands (Hernández-Hernández et al. 2010), Iran (Mansoori 2012), Iraq (Hameed 2012), Israel (Cohen et al. 2010) and USA (Munoz and Wang 2011). Although F. solani was not as frequently isolated from date palm trees as F. proliferatum, it poses serious threat on date palm plantations. Isolates of F. solani have been recovered from date palm trees in different countries, e.g. Iran (Mansoori and Kord 2006), Iraq (Al-Yasiri et al. 2010), Oman (Al-Sadi et al. 2012), Pakistan (Maitlo et al. 2014), and Egypt (Alkahtani et al. 2011). The other recorded Fusarium species on date palm are either not significantly affecting date palm plantations or wrongly identified. For example, F. moniliforme was recovered from date palm trees and claimed to be associated with Fusarium date palm diseases, e.g. falseBayoud (Khairi et al. 2010; Alkahtani et al. 2011; Mandeel et al. 2005). Recently, it was agreed that the name F. moniliforme, a species name referring to more than 25 different species including F. proliferatum and

F. verticillioides, should not be used as it represents Bunacceptably broad species concept^ (Seifert et al. 2003; Leslie and Summerell 2006). Because Fusarium species are serious threats on date palm planations, the main objective of this study was to isolate and morphologically and molecularly characterize Fusarium pathogens from symptomatic date palm tissues. Preliminary in vitro pathogenicity experiments were conducted to test the potentiality of recovered Fusarium isolates as date palm pathogens.

Materials and methods Isolation, purification and preservation of Fusarium isolates from date palm tissues Leaf and root samples were collected from date palm trees showing foliar disease symptoms, e.g. chlorosis, necrosis and whitening, from seven different regions in Saudi Arabia (Table 1, Fig. 1). The plant samples were thoroughly rinsed with tap water, then washed with sterilized distilled water (sdH 2 O) and cut into 0.5 × 0.5 cm pieces. The leaf and root pieces were surface-sterilized by dipping them in 10 % bleach solution for 3–5 min, rinsing with sdH2O, and placing them onto sterilized filter paper to dry. The surface-sterilized leaf and root pieces were placed onto Difco potato dextrose agar (PDA) (3 pieces per plate) amended with 100 μg ml−1 streptomycin sulfate. The plates were incubated for 7 days at 25 °C. Tentative Fusarium colonies were transferred to Nash-Snyder Fusarium-selective (NS) medium amended with 100 μg ml−1 streptomycin sulfate and 120 μg ml−1 neomycin sulfate (Leslie and Summerell 2006) and the plates were incubated for 7–10 days at 25 °C. Fungal isolates were single-spored and purified cultures were transferred to PDA slants and incubated at 25 °C for three days. Then, 2 ml of 15 % glycerol solution were used to collect both mycelia and spores by scraping slant cultures by using 1-ml micropipette. The glycerol solutions containing mycelia and spores were transferred to 2-ml sterilized cryogenic vials (Thermo Fisher Scientific, Rochester, NY, USA) and then kept at −80 °C for long-term preservation. Morphological characterization of Fusarium isolates To study the morphological characteristics of Fusarium isolates, pure cultures were grown on PDA and

Eur J Plant Pathol Table 1 Fusarium and Fusarium-like isolates recovered from seven date palm-growing regions in Saudi Arabia Region

No. of F. proliferatum F. solani F. oxysporum F. verticillioides F. brachygibbosum Fusarium isolates sp.

Sarocladium kiliense

Besha

1

0

0

0

0

1

0

0

Al-Gouf

1

1

0

0

0

0

0

0

Hael

19

18

1

0

0

0

0

0

AlMadinah Al-Qaseem

12

7

2

0

0

0

0

3

11

6

3

1

0

0

0

1

Riyadh

14

12

2

0

0

0

0

0

AlSharqiya Total

12

5

1

0

1

0

4

1

70

49

9

1

1

1

4

5

Spezieller Nahrstoffarmer agar (SNA) plates (Leslie and Summerell 2006). After seven to ten days of incubation at 25 °C, the fungal isolates were morphologically (e.g. colony color and pigmentations on PDA) and microscopically (e.g. presence/absence of micro-, macro- and chlamydospores and type of conidiophore) examined and then identified to species level according to Leslie and Summerell (2006). Molecular characterization of Fusarium isolates Fungal growth Three to five small pieces of PDA agar containing mycelia and spores were transferred to a 250-ml conical flask containing 50 ml of potato dextrose broth (PDB). The liquid cultures were incubated at 25 °C for 4 days under constant shaking at 100 rpm. The mycelia were collected by filtering the fungal liquid culture through a Whatman No. 2 filter paper. The excess of H2O was removed by pressing the Whatman filter paper containing mycelia between layers of tissue paper. The semidried mycelia were scratched from Whatman filter paper

and put in aluminum foil, wrapped, and kept at −40 °C until they were ground to extract DNA. DNA extraction The mycelia were ground under liquid nitrogen by using a mortar and pestle. The ground mycelia were transferred to 1.5 ml eppendorf tubes. For best results, no more than one-third of the 1.5 ml eppendorf tubes were filled with ground mycelia. Genomic DNA was extracted using CTAB method according to Murray and Thompson (1980) and modified by Saleh et al. (2015). The quality and quantity of DNA was assessed on 1 % agarose gel stained with 1 μg/ml acridine orange (Sambrook et al. 1989). The DNA samples were diluted to a final concentration of 20 ng/μl. PCR and phylogenetic studies Three regions were amplified and sequenced, ITSrDNA and portions of the beta tubulin (tub2) and translation elongation factor 1 alpha (tef1α) genes, with the following primers: ITS4 (5′-TCCT

Fig. 1 Date palm leaves showing yellowing and wilting symptoms from which Fusarium isolates were recovered

F. solani Madinah region

F. proliferatum Qaseem region

F. proliferatum Hael region

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CCGCTTATTGATATGC-3′) and ITS5 (5′-GGAA GTAAAAGTCGTAACAAGG-3′), or T1 (5′-AACA TGCGTGAGATTGTAAGT-3′) and T2 (5′-TAGT GACCCTTGGCCCAGTTG-3′), or EF1 (5′-ATGG GTAA GG AR GA CAA GA C-3 ′) and EF2 (5′GGARGTACCAGTSATCATGTT-3′), respectively (White et al. 1990; O’Donnell and Cigelnik 1997; O’Donnell et al. 1998b). The PCR reaction contained 1 ng/μl genomic DNA, 1 × PCR buffer (Bioline US Inc., Taunton, Massachusetts, USA), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM of each primer, and 1.0 unit Taq DNA polymerase (Bioline) in a 30 μl reaction volume. The PCR program was: one cycle at 94 °C for 3 min, 30 cycles of 94 °C for 1 min, 55 °C for 1 min (ITS-rDNA) or 53 °C for 1 min (tub2) or 56 °C for 1 min (tef1α), and 72 °C for 1 min, and a terminal incubation at 72 °C for 5 min. All the PCR amplifications were carried out in a TC-412 thermocycler (Techne, Cambridge, United Kingdom). The amplification products were electrophoretically separated on 1.5 % agarose gel in 0.5× TBE buffer stained with 1 μg/ml acridine orange. HyperLadder IV molecular weight marker (BioLine) was used alongside the PCR products. The gels were visualized under UV and photographed. PCR amplicons were cleaned and sequenced at the University of Kentucky Advanced Genetic Technologies Center. The DNA sequences were edited and aligned using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Single nucleotide polymorphisms (SNPs) within and among fungal DNA sequences were also detected using BioEdit. The phylogenetic analyses were conducted using PAUP software (Swofford 2002). Neighbor-joining (NJ) and parsimonious (using heuristic search) trees were constructed for aligned DNA sequences for both individual and combined sequences using PAUP. Bootstrapping with 1000 replications was used to support internal branches of phylogenetic trees (Felsenstein 1985). Consistency and retention indices (CI and RI, respectively) were calculated for parsimonious trees using PAUP. Colonization ability of Fusarium isolates on detached date palm leaflets A closed young leaf was detached from the middle portion of a date palm tree grown at the King Saud University campus. Leaflets were removed, wiped with 70 % ethanol-moistened cotton, and cut into pieces of ca

4 cm in length. Four leaflet cuttings were placed in 9-cm Petri plates containing two sterilized filter papers soaked in 2 ml sdH2O. Five mm diameter fungal disks were cut from edges of 5-day-old Fusarium colonies and placed at the middle of each leaflet cutting, one disk/leaflet cutting. Then, the plates containing inoculated leaflet cuttings were incubated at 25 ± 2 °C, under the lab conditions, for four weeks in closed plastic case to maintain high humidity level. For control treatment, the leaflet cuttings were placed in Petri plates containing two sterilized filter paper. The fungal colonization ability was determined after four weeks of incubation at 25 °C as follows, A = 0 % no colonization, B = 25 % colonization of a leaflet cutting, C = 50 % colonization of a leaflet cutting, D = 75 % colonization of a leaflet cutting, E = 100 % colonization of a leaflet cutting. The average of colonization ability was determined by summing the colonization percentages of four leaflet cuttings and divided by 4.

Results Isolation and morphological characterization of Fusarium isolates A total number of 70 fungal isolates were recovered from date palm trees showing disease symptoms, e.g. leaflets yellowing and vascular bundle browning of the rachis (Fig. 1). Most of the isolates grew very well on NS and PDA media. However, few isolates, e.g. E166, Q53, M61, and M114, showed very restricted growth on PDA and produced only yeast-like microconidia on long monophailide conidiophores (Table 2s). Based on the culture morphology and microscopic examination, the fungal isolates were generally placed into two groups, the first group containing Fusarium isolates and the second group containing Fusarium-like isolates (E166, Q53, M61, and M114). Further, most of the Fusarium isolates were identified as the following species: F. proliferatum, F. solani, F. brachygibbosum, F. oxysporum and F. verticillioides (Table 1). Four isolates recovered from Al-Sharqiya could not be assigned to the species level and designated as Fusarium sp. Isolates of F. proliferatum were recovered from most regions, followed by F. solani recovered from five regions. Fusarium brachygibbosum (Besha) F. oxysporum (Al-Qaseem) and F. verticillioides (Al-Sharqiya) were represented by only one isolate (Table 1).

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Molecular characterization of Fusarium isolates

tef1α

Sequences from three regions belonging to rDNA, and the nuclear genes tef1α and tub-2 were used to confirm morphological identifications of Fusarium isolates.

Generally, the tef1α PCR amplicons ranged in size between 667 and 703 bp for Fusarium isolates, whereas Fusarium-like isolates generated one tef1α haplotype of 716 bp in size. Although exon 2 and 3 for F. proliferatum isolates had no SNPs, the nucleotide variation in intron regions resulted in 12 different tef1α haplotypes; most of them (8 out of 12) were singletons, i.e. each haplotype was represented by one isolate. The isolates belonging to F. solani generated tef1α amplicons ranging in size between 691 and 703 bp with four different haplotypes due to the nucleotide variation in intron regions. In addition to nucleotide variation in intron regions among different Fusarium species, synonymous SNPs were also detected in exon 2 and 3 regions (Fig. 4). Most of the SNPs detected in Fusarium-like isolates were silent mutations except three successive ones (CGC → AAG) at the end of exon 3 resulted in changing Arg to Lys (Fig. 4).

rDNA-ITS PCR amplicons of rDNA-ITS region from Fusarium isolates ranged in size from 526 to 551 bp, whereas one haplotype of 560 bp in size was generated from five Fusarium-like isolates. When the rDNA-ITS sequence of Fusarium-like isolates was searched in NCBIGenBank database, the 100 % homologous sequence was related to Sarocladium kiliense (accession number KM231849). In addition to the DNA variations in ITS1 and ITS2 regions within and among different fungal species, Fusarium-like isolates (S. kiliense) had 5 unique SNPs at positions 123, 125, 127, 130, 146 of 5.8S gene (Fig. 2). Most of the Fusarium isolates had identical 5.8S sequences except F. solani isolates, which had three unique SNPs at positions 26, 51 and 158, two of them overlapped with S. kiliense isolates (26 and 51) (Fig. 2). Tub-2 The tub-2 PCR amplicons of fungal isolates had different fragments sizes. Almost all F. proliferatum isolates had 558 bp PCR products. However, R99B isolate gave 556 bp fragment with four synonymous SNPs in exons 3 and 4 (Fig. 3). Fusarium solani isolates generated 510 bp tub-2 fragments with four different haplotypes. The Fusarium-like isolates generated an identical 642 bp tub-2 fragment. All the SNPs detected in exons 2, 3, and 4 among different fungal species were in the third position of codons with no change in amino acid sequence (Fig. 3).

Phylogenetic studies using DNA sequence Individual or concatenated DNA sequences obtained from the three nuclear regions were used to construct NJ and parsimonious phylogenetic trees. Generally, there was no significant difference between NJ and most parsimonious phylogenetic trees using individual genes where the clade of Fusarium isolates received 100 % bootstrap values. Fusarium-like isolates were treated as the outgroup taxon. The Fusarium isolates were divided into two clusters supported with 100 % bootstrap value each (Fig. 5). The first cluster contained F. proliferatum, F. verticillioides and F. oxysporum, whereas the second cluster contained F. solani, F. brachygibbosum and Fusarium sp. isolates (Fig. 5).

Fig. 2 Alignment of 5.8S rDNA from different Fusarium and Fusarium-like (S. kiliense) isolates recovered from date palm in Saudi Arabia

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Fig. 3 Alignment of exons 2, 3 and 4 of tub-2 gene from different Fusarium and Fusarium-like (S. kiliense) isolates recovered from date palm in Saudi Arabia. Dashes refer to the positions of introns 2 and 3, whereas dots indicate that nucleotide residues are identical

at these positions. Single nucleotide polymorphisms (SNPs) are in the third positions and are synonymous substitutions with no change in amino acid sequence

Colonization ability of Fusarium isolates on detached date palm leaflets

and F. oxysporum, were also able to colonize leaflet cuttings (Table 2s). Two Fusarium-like isolates had low virulence.

Generally, F. proliferatum isolates showed the highest virulence on date palm leaflets compared with the other Fusarium isolates. The mean virulence of pathogenic F. proliferatum isolates ranged between 6 % (e.g. H45) and 81 (e.g. H90) (Fig. 6, Table 2s). However, few isolates of F. proliferatum were not able to colonize date palm leaflet cuttings, e.g. M119 (Fig. 6). Eight out of the 9 F. solani isolates were able to colonize leaflet cuttings with different degrees of virulence ranging between 6 and 56 % (Table 2s). The other isolates of Fusarium, including F. verticillioides

Discussion Fungi are associated with most reported date palm diseases worldwide. Fusarium is the most important genus amongst these fungi. Three main Fusarium species were recorded as date palm pathogens: F. oxysporum, F. proliferatum and F. solani (e.g. Zaid et al. 2002; Abdalla et al. 2000; Mansoori and Kord 2006). The most serious date palm Fusarium pathogen is F. oxysporum f.

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Fig. 4 Alignment of exons 2 and 3 of tef1α gene from different Fusarium and Fusarium-like (S. kiliense) isolates recovered from date palm in Saudi Arabia. Dashes refer to the positions of introns 2, whereas dots indicate that nucleotide residues are identical at

these positions. Single nucleotide polymorphisms (SNPs) are synonymous substitutions with no change in amino acid sequence, except three successive SNPs (CGC → AAG) that result in an Arg to Lys change at the end of exon 3

sp. albedinis but until now it has been restricted to North Africa (Zaid et al. 2002; Djerbi 1982). In the present study, fungal strains belonging to five Fusarium species, F. proliferatum, F. solani, F. brachygibbosum, F. oxysporum and F. verticillioides, were recovered from date palm tissues showing fungal disease symptoms, from different regions in Saudi Arabia. The most frequently isolated species was F. proliferatum, recovered from almost all the surveyed regions. Many recent studies have shown that pathogenic isolates of F. proliferatum affecting date palm plantations were recovered from many countries including Spain, Canary Islands, Iran, Iraq and United States (Armengol et al. 2005; Hernández-Hernández et al. 2010; Mansoori 2012; Hameed 2012; Munoz and Wang 2011). The disease symptoms produced by F. proliferatum on date palm trees included wilt symptoms and dieback (Armengol et al. 2005; Hernández-Hernández et al. 2010; Abdalla et al. 2000). In addition, F. proliferatum can cause a serious fungal disease on Iranian and Israeli date palm trees called date bunch fading disease (Mansoori 2012; Cohen et al. 2010). Other palm trees, such as queen and Mexican fan palms, can be also

attacked by F. proliferatum strains (Elliott et al. 2010; Hernández-Hernández et al. 2010; Polizzi and Vitale 2003). The second predominant Fusarium species recovered from Saudi date palm trees was F. solani. It was isolated from five date-palm growing regions in Saudi Arabia. Fusarium solani is considered the third most important Fusarium on date palm worldwide. Strains of F. solani were the main causal agents of the following diseases: yellow death in Iran (Mansoori and Kord 2006), yellowing and deterioration of palm fronds that resulted in death of palms in Iraq (Al-Yasiri et al. 2010), and sudden decline disease of date palm in Pakistan (Maitlo et al. 2014). In addition, F. solani strains were isolated from date palm fruits, roots and rhizosphere soils in Egypt, Oman, Saudi Arabia and Bahrain (Alkahtani et al. 2011; Al-Sadi et al. 2012; Bokhary 2006; Mandeel et al. 2005). The other Fusarium species ( F. b r a c h y g i b b o s u m , F. o x y s p o r u m a n d F. verticillioides) recovered in this study were represented by single strains. Strains of F. brachygibbosum and F. oxysporum had been isolated previously from date palm roots showing root rot/necrosis symptoms (AlSadi et al. 2012; Mandeel et al. 2005). However,

Eur J Plant Pathol Fig. 5 One of the most parsimonious trees based on combined sequence data of rDNA-ITS, tub-2 and tef1α fragments obtained from Fusarium and Fusarium-like isolates recovered from date palm trees. The five Fusarium-like isolates were used as the outgroup taxon. rDNA-ITS, tub-2 and tef1α sequences of F. proliferatum NRRL 22944 isolate were obtained from NCBI-GenBank. Numbers of tree branches represent bootstrap values. Bar scale at the bottom of the tree represents 10 nucleotides change

Tree length = 1230 CI = 0.787 RI = 0.926

F. proliferatum

100 100

Fusarium isolates

98

F. verticillioides F. oxysporum

100

99

F. solani

100 100 98

100

Fusarium sp. F. brachygibbosum Fusarium sp.

Fusarium-like isolates

F. verticillioides has not been isolated from date palm and this is the first report of isolating it from date palm. The identity of the Fusarium strains recovered in this study was confirmed by DNA sequences obtained from tef1α, tub-2 and rDNA-ITS regions. DNA sequences of different fungal nuclear regions including, rDNA, polyketide synthase and tef-1α, have been used to confirm species level of Fusarium strains isolated from date palms (Abdalla et al. 2000; Stępień et al. 2011; AlSadi et al. 2015). It had been claimed that F. moniliforme is associated with false-Bayoud and other date palm diseases (Khairi et al. 2010; Alkahtani et al. 2011; Mandeel et al. 2005). However, this taxonomic identity is no longer in use (Seifert et al. 2003) and by using molecular data along with morphological traits, the identification of fungal taxa is possibly available up to the species level. In the current study, the phylogenetic analysis also confirmed the close relationship between strains of F. proliferatum and F. verticillioides, two members of Gibberella-fujikuroi species complex (section Liseola), and F oxysporum (section Elegans) where their clade received 100 % bootstrap value.

Generally, phylogenetic analysis based on DNA sequences of rDNA, tef1α, tub-2 supports the lineage that comprise fungal strains belonging to G. fujikuroi and F oxysporum species complexes (O’Donnell et al. 1998a; O’Donnell et al. 2000; Leslie and Summerell 2006). Although the five Fusarium-like strains were morphologically very similar to Fusarium ones, their DNA sequences showed that they are S. kiliense (syn. Acremonium kiliense). Most of the SNPs detected in protein-coding regions were synonymous. The only three successive SNPs in tef1α exon 3 (CGC → AAG) resulted in changing Arg to Lys, a conservative mutation that most probably would not affect the function of the protein (Klumpp et al. 2003; Betts and Russell 2003). In addition to the soil saprophytic nature of Sarocladium species, plant endophytic and pathogenic strains have been recorded (e.g. Yeh and Kirschner 2014; Summerbell et al. 2011). In order to effectively control date palm diseases caused by Fusarium species, especially those belonging to F. proliferatum and F. solani, it is essential to investigate how these fungal isolates reached upper parts of

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Conclusion

Control

H90 (FP)

Both F. proliferatum and F. solani are becoming important pathogens of date palm and efforts should be made to restrict and control them. The preliminary in vitro pathogenicity results showed that F. proliferatum had the highest virulence on date palm leaflets, followed by F solani. Studies are being executed to assess the correlation of the aggressiveness of F. proliferatum strains with their abilities to produce cell-wall degrading enzymes as well as their toxin production abilities (unpublished data). Acknowledgment This Project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (11-AGR1475-02).

M119 (FP)

H13 (FS)

E44 (FP)

E140B (Fsp)

Fig. 6 Date palm detached leaflet cuttings showing Fusarium colonization of different fungal isolates collected from trees showing disease symptoms in different regions of Saudi Arabia. (FP): F. proliferatum, (FS) F. solani and (Fsp) Fusarium sp

the palms. Insects are amongst the key players in dispersing plant pathogens. There are many insect pests attacking date palm, e.g. red palm weevil (Rhynchophorus ferrugineus) and white scale (Parlatoria blanchardii), which may play a role in disseminating these Fusaria. Khudhair et al. (2014) showed that F. proliferatum can be transmitted by the date palm borer (Oryctes elegans) where the infested trees showed wilt symptoms and high infection rates by the pathogen. Many studies have reported that insects can vector plant pathogenic fungi (Doval et al. 1976; Gillespie and Menzies 1993; Jarvis et al. 1993; Meldrum et al. 2013; Salgado-Neto et al. 2016; Stanghellini et al. 1999). In addition, Fusarium isolates can be transmitted during either the pollination of female trees or trimming the trees using infected tools.

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