Molecular evaluation of resistibility/susceptibility of Saudi date

palm germplasm against Bayoud disease caused by Fusarium

oxysporum f. sp. albedinis

Amgad A. SalehDepartment of Plant Protection, Faculty of Food and Agriculture Sciences, King

Saud University, Riyadh, Kingdom of Saudi Arabia

August, 2013

Introduction

Objectives

Materials and methods

Results & Discussion

On going work

Conclusion

Acknowledgements

Outlines

Date palm is probably the most ancient cultivated tree in the world (Zaid and de Wet, 2002)

Date palm is well adapted for desert climates and can be used to control desertification

Every part of the date palm tree can be utilized (e.g. fruits, trunks, leaves, seeds, etc.)

Dates are the main food in the great desert areas extending from western north Africa to India

Dates can be dried and used in drought situations when other foods may be very limited or unavailable

Dates are rich in carbohydrates, minerals, dietary fiber, vitamins, fatty acids, and proteins

Date palm trees have numerous outstanding characters, e.g. seed ~2,000 years old has recently been germinated (Sallon et al., 2008)

IntroductionDate palm (Phoenix dactylifera L.)

Date palm in Saud Arabia

It is a strategic crop in SA It plays a great role in the cultural, social, and economic

aspects of SA, e.g. dates have been a staple food for the Saudi people for thousands of years

The total cultivated area is around 157,074 hectares (> 18 million trees) with a total production of 986,409 tons of dates (www.moa.gov.sa)

SA produces 16% of the world production of dates making it the fourth largest date producer worldwide (Soliman and Al-Saleh, 2001)

Date palm industry products include food, wood, furniture, cosmetics, and pharmaceuticals

One of the great threats to the yield and health of date palm trees are diseases

There are a variety of organisms that can cause diseases to the date palm trees, e.g. phytoplasmas and fungi

Fusarium oxysporum f. sp. albedinis (FOA) is a fungal pathogen that can cause a very serious and destructive disease to the date palm

The Fusarium wilt disease of date palm is called "Bayoud" disease (Arabic word, "abiadh", meaning white referring to the whitening of the fronds of the diseased trees

FOA is a seed- and soil-borne vascular wilt pathogen The fungus survives in soil as chlamydospores and can remain

dormant in soil for many years

Diseases of date palm

Bayoud disease caused by Fusariumoxysporum f. sp. albedinis

Mohan Jain, 2012

Bayoud disease was first discovered in Morocco in 1870 and then moved to Algeria (Zaid et al., 2002)

In less than one century, FOA destroyed more than 15 million date palm trees (12 m in Morocco and 3 m in Algeria)

Recently, there are reports of a vascular fusariosis disease in Tunisia and Egypt, but these reports have not been confirmed (Djerbi et al., 1982; Freeman and Maymon, 2000)

The best control strategy of controlling FOA is through plant breeding

Quenzer et al. (2001) developed a simple reliable PCR-based diagnostic method, based on mitochondrial plasmids, to discriminate between Bayoud-resistant and susceptible trees

Three mitochondrial plasmid-like DNAs have been isolated from date palm (Benslimane et al., 1994; 1996)

The U plasmid found in all tested date palm cultivars (Benslimane et al., 1994)

The S plasmid (1,454 bp) found only in Bayoud-susceptible cultivars The R plasmid (1,345 bp) found only in Bayoud-resistant cultivars

The nucleotide similarity between the S and R plasmids is > 98%, with the R plasmid lacking a 109-bp DNA segment that may be eliminated by an intra-plasmid recombination event within the S plasmid (Benslimane et al., 1996)

The absence or the presence of these mitochondrial plasmids has been used successfully to evaluate resistance of date palm germplasm against Bayoud disease in Morocco, Tunisia, Syria, and Mauretania (Haider and Nabulsi, 2012; Ould Mohamed Salem et al., 2007; Quenzar et al., 2001)

3' 5'

3'5'

5' 3'

3' 5'

3' 5'

3'5'

3' 5'

3'5'

3' 5'

3'5'

3' 5'

5' 3'

The tree is supposed to be Bayoud-susceptible

The tree is supposed to be Bayoud-resistant

Conventional PCR using oli1 and oli2 primers

indel

265 bp PCR amplicon

373 bp PCR amplicon

Oli1 primer

Oli2 primer

Conventional PCR using oli1 and oli2 primers

FOA has not been currently recorded in Saudi Arabia, but according to the literature, the disease is moving eastward from its origin (Morocco and Algeria) and precautions should be taken to protect the kingdom from this devastating pathogen

The main objective of this study is to develop prophylactic measures to protect Saudi Arabian date palm plantations from this destructive disease:

By evaluating resistibility/susceptibility of date palm trees against Bayoud fungus

By assessing genetic diversity of date palm germplasm using a genome-wide fingerprinting technique (e.g. AFLPs)

Then look for any correlation between AFLP data and mitochondrial DNA markers to improve date palm breeding programs

Materials and methods

Sample collection

Young not fully opened leaflets were collected from different date palm varieties from nine locations across Riyadh region, SA

DNA was extracted from date palm leaves Determination of Bayoud resistibility/susceptibility

using diagnostic PCR primers of mit plasmids Sequencing of PCR amplicons at the Advanced Genetic

Technologies Center, University of Kentucky (AGTC, Lexington, KY, USA)

Data analysis

Map of Riyadh region showing nine locations from which date palm tissues were collected

Generally, five female trees were sampled per cultivar per farm

Total # Varieties Unknown ♀ ♂ trees ♀ trees Location

61 13 3 14 44 Al-Dawadmi

133 26 5 9 119 Alghat

71 15 1 0 70 Al-Guwaiya

98 23 4 10 84 Alkharj

73 13 0 2 71 Almajmaa

80 9 1 6 73 Dariya

104 30 0 0 104 Muzahmiya

114 42 6 2 106 Shaqra

108 13 0 7 101 Wadi Al-Dawasir

842 76 20 50 772 Total

Results and Discussion

Results & Discussion

Total number of sampled trees was 842These tress had female (792/842) and male (50/842) individuals772 known trees representing 76 different varieties20 unknown female trees

DNA extracted from all samples

430 trees were tested by PCR to determine their resistibility/susceptibility against Bayoud fungus

R353

S43

NTR34

A representative 1.8% agarose gel stained with 1 µg/ml acridine orange showing Bayoud resistibility/susceptibility PCR amplicons generated from different date palm varietiesLanes M: 1kb DNA Ladder RTULanes 1-11: date palm trees representing different varietiesLane12: negative control (NC)Lane 13: positive controlS: susceptible and R: resistant

Determination of resistibility/susceptibility of Saudi date

palm germplasm

Fusariosis resistibility No. of R trees Date palm varietyR 1 AsselaR 13 BarhiR 1 DahmaliaR 18 DekhanyR 3 DhabahR 2 EnabR 1 Fankha kabeerR 1 HakiR 3 HamraaR 4 JafirR 15 khalas Al-Ahsa'aR 13 khalas AlwadiR 28 KhodryR 6 KhoshkhashR 5 MaqfaziR 8 MaktoumiR 5 MestaniR 2 Nabtat AliR 2 Nabtat SulimanR 7 Nabtat SultanR 2 RushodiR 16 SariR 3 SefriR 2 ShahaliR 1 ShaisheeR 1 ShebebaR 2 Sukkari AhmerR 25 SukkariR 1 Sulaj AishR 7 SullajR 1 ThaweeR 5 Um alkhashabR 1 WannanaR 2 ZamliR 6 Zawi

Date palm varieties (female known trees) that are expected to

be fusariosis resistant to Bayoud fungus

213 trees35 varieties

Fusariosis susceptibility

No. of tested trees

Date palm variety

S 3 ShaisheeS 2 khesabS 1 LahmiaS 1 MutwahS 1 QatarahS 7 Ruzeizi

Date palm varieties that are expected to be fusariosis susceptible to Bayoud fungus

15 trees6 varieties

R/S Fusariosis susceptibility

Fusariosis resistibility Date palm variety

R? 9:2 2 9 Hulwa

R? 33:1 1 33 Khalas

R? 10:1 1 10 Menefee

R? 20:1 1 20 Naboot saif

R? 9:1 1 9 Rotan

R? 19:2 2 19 Segae

S? 1:2 2 1 Dagel

S? 1:7 7 1 Hilali

R/S 1 1 Shaqra

R/S 3 25 Male trees

R/S 7 12 Unknown

To be fusariosis resistant or not to be fusariosis resistant

Sequencing of diagnostic PCR amplicons

67 diagnostic PCR amplicons were sequenced

The sizes of PCR amlicons amplified from S- and R-plasmids were 357 (373) and 265 bp, respectively

There was no nucleotide polymorphisms detected within either S or R sequences obtained from Saudi date palm germplasm

One representative sequence from S- and R-plasmids were deposited in GenBank under the accession numbers KC771885 (S-fragment) and KC771886 (R-fragment)

DNA sequence of the PCR amplicon of R-plasmids was 100% identical to DNA sequences deposited in GenBank database

The DNA sequence of PCR amplicons generated from S-plasmid was 97.6% identical to GenBank DNA sequences obtained from Bayoud-susceptible date palm varieties

DP2-Shaishe V1DP-S DP90_Sullaj V3DP-R

94.9 96.4 100 V3DP-R

94.9 96.4 0 DP90-Sullaj

97.6 3.6 3.6 V1DP-S

2.4 5.1 5.1 DP2-Shaishe

DNA sequence homology and DNA distance matrix among mitochondrial R- and S-plasmids of resistant and susceptible date palm cultivars

DNA alignment of 4 sequences generated from date palm mitochondrial plasmidsDP90 and DP2 from Saudi date palm germplasm (this study) whereas V3DP-R and V1DP-S retrieved from GenBank database

I and II are unique indel mutations in S-plasmid from Saudi

date palm

* are unique SNPs in S-plasmid from Saudi

date palm

Part of ORF2 region(1-138)

Two mutations, one SNP and one indel

Arrows pointed to the 109 indel region

SNP (CAA→CTA) L→Q

Indel →41bp gap

I

II

Continue evaluating resistibility/susceptibility of date palm trees against Bayoud fungus using mit primers

We have started genotyping date palm germplasm using AFLPs technology

Look for any correlation between AFLP data and mitochondrial DNA markers to improve date palm breeding programs

Poster Session: Population Biology Genetics "529-P"

Morphological and molecular characterization of Fusarium isolates collected from date palm in Saudi Arabia

On going work

The genetic assessment of the resistibility/susceptibility of date palm germplasm in Saudi Arabia will enable the authorities to take a rapid response in case of Bayoud disease outbreaks

This work is also very important to Saudi national security and economy because the date palm industry is very important socially, economically and scientifically to the Kingdom

Conclusion

Dr. Younes Molan Dr. John LeslieDr. Mahmoud El_KomyMr. Anas EranthodiMr. Anwar Hamoud

This research is financially supported by the National Plan for Science, Technology & Innovation (NPSTI) program, King Saud University, Riyadh, Saudi Arabia (project # 11-AGR1475-02)

Acknowledgements

Thank you