1. Molecular evaluation of resistibility/susceptibility of Saudi date palm germplasm against Bayoud disease caused by Fusarium oxysporum f. sp. albedinis
Amgad A. Saleh
Department of Plant Protection, Faculty of Food and Agriculture Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia
August, 2013

2. Outlines Introduction Objectives Materials and methods
Results & Discussion
On going work
Conclusion
Acknowledgements

3. Introduction Date palm (Phoenix dactylifera L.)
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)

4. 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 (
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

5. Diseases of date palm
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

6. Bayoud disease caused by Fusarium
oxysporum f. sp. albedinis
Mohan Jain, 2012

7. 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

8. 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)

9. The tree is supposed to be Bayoud-resistant
373 bp PCR amplicon
The tree is supposed to be Bayoud-susceptible 3' 5' 5' 3'
Conventional PCR using oli1 and oli2 primers
Oli1 primer 5' 3' 3' 5' 5' 3'
indel 3' 5' 5' 3'
Oli2 primer 3' 5' 5' 3' 3' 5'
Conventional PCR using oli1 and oli2 primers
The tree is supposed to be Bayoud-resistant 3' 5' 5' 3'
265 bp PCR amplicon

10. 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

11. 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

12. Map of Riyadh region showing nine locations from which date palm tissues were collected
Generally, five female trees were sampled per cultivar per farm

13. Results and Discussion
Total
# Varieties
Unknown ♀
♂ trees
♀ trees
Location 61 13 3 14 44
Al-Dawadmi
133 26 5 9
119
Alghat 71 15 1 70
Al-Guwaiya 98 23 4 10 84
Alkharj 73 2
Almajmaa 80 6
Dariya
104 30
Muzahmiya
114 42
106
Shaqra
108 7
101
Wadi Al-Dawasir
842 76 20 50
772

14. Results & Discussion Total number of sampled trees was 842
These tress had female (792/842) and male (50/842) individuals
772 known trees representing 76 different varieties
20 unknown female trees
DNA extracted from all samples
430 trees were tested by PCR to determine their resistibility/susceptibility against Bayoud fungus R
353 S 43
NTR 34

15. Determination of resistibility/susceptibility of Saudi date palm germplasm
A representative 1.8% agarose gel stained with 1 µg/ml acridine orange showing Bayoud resistibility/susceptibility PCR amplicons generated from different date palm varieties
Lanes M: 1kb DNA Ladder RTU
Lanes 1-11: date palm trees representing different varieties
Lane12: negative control (NC)
Lane 13: positive control
S: susceptible and R: resistant

16. Fusariosis resistibility
No. of R trees
Date palm variety R 1
Assela 13
Barhi
Dahmalia 18
Dekhany 3
Dhabah 2
Enab
Fankha kabeer
Haki
Hamraa 4
Jafir 15
khalas Al-Ahsa'a
khalas Alwadi 28
Khodry 6
Khoshkhash 5
Maqfazi 8
Maktoumi
Mestani
Nabtat Ali
Nabtat Suliman 7
Nabtat Sultan
Rushodi 16
Sari
Sefri
Shahali
Shaishee
Shebeba
Sukkari Ahmer 25
Sukkari
Sulaj Aish
Sullaj
Thawee
Um alkhashab
Wannana
Zamli
Zawi
Date palm varieties (female known trees) that are expected to be fusariosis resistant to Bayoud fungus
213 trees
35 varieties

17. Fusariosis susceptibility
Date palm varieties that are expected to be fusariosis susceptible to Bayoud fungus
Fusariosis susceptibility
No. of tested trees
Date palm variety S 3
Shaishee 2
khesab 1
Lahmia
Mutwah
Qatarah 7
Ruzeizi
15 trees
6 varieties

18. To be fusariosis resistant or not to be fusariosis resistant
R/S
Fusariosis susceptibility
Fusariosis resistibility
Date palm variety
R? 9:2 2 9
Hulwa
R? 33:1 1 33
Khalas
R? 10:1 10
Menefee
R? 20:1 20
Naboot saif
R? 9:1
Rotan
R? 19:2 19
Segae
S? 1:2
Dagel
S? 1:7 7
Hilali
Shaqra 3 25
Male trees 12
Unknown

19. Sequencing of diagnostic PCR amplicons
67 diagnostic PCR amplicons were sequenced
The sizes of PCR amlicons amplified from S- and R-plasmids were (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 KC (S- fragment) and KC (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

20. DNA sequence homology and DNA distance matrix among mitochondrial R- and S-plasmids of resistant and susceptible date palm cultivars
DP2-Shaishe
V1DP-S
DP90_Sullaj
V3DP-R
94.9
96.4
100
DP90-Sullaj
97.6
3.6
2.4
5.1

21. I II
I and II are unique indel mutations in S-plasmid from Saudi date palm
* are unique SNPs in S-plasmid from Saudi date palm
Arrows pointed to the 109 indel region
Part of ORF2 region
(1-138)
Two mutations, one SNP and one indel
SNP (CAA→CTA)
L→Q
Indel →41bp gap
DNA alignment of 4 sequences generated from date palm mitochondrial plasmids
DP90 and DP2 from Saudi date palm germplasm (this study) whereas V3DP-R and V1DP-S retrieved from GenBank database

22. On going work
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

23. Conclusion
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

24. Acknowledgements Dr. Younes Molan Dr. John Leslie Dr. Mahmoud El_Komy
Mr. Anas Eranthodi
Mr. 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-AGR )

25. Thank you