Inducing Viruses and Phytoplasma on Faba Bean (Vicia Faba L.) in the Gezira State, Sudan

Identification and Molecular Characterization of some Yellow- inducing Viruses and Phytoplasma on Faba bean (Vicia faba L.) in the Gezira State, Sudan

Ibrahim Abd ELhameed Saeed Fadl Elmola

B.Sc.(Honors) in Agriculture and Natural Resources Faculty of Agriculture and Natural Resources University of Gezira (2009)

A Dissertation Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science

in Plant Pathology Plant Pathology Centre Faculty of Agricultural Sciences University of Gezira

June / 2014

Identification and Molecular Characterization of some Yellow- inducing Viruses and Phytoplasma on Faba bean (Vicia faba L.) in the Gezira State, Sudan

Ibrahim Abd EL hameed Saeed Fadl Elmola

Examination Committee: Name Position Signature Dr. Mai Abdalla Ali Abdalla Chair Person ...... Prof. MutwakilAbdelmajeedMahir External Examiner ……………. Dr. Omar Osman Ahmed Elbashir Internal Examiner ……………

Date of Examination :19/6/ 2014

Identification and Molecular Characterization of some Yellow- inducing Viruses and Phytoplasma on Faba bean (Vicia faba L.) in the Gezira State, Sudan

Ibrahim Abd ELhameed Saeed Fadl Elmola

Supervision Committee: Name Position Signature Dr. Mai Abdalla Ali Abdalla Main supervisor ...... Dr. El Shafie El Hassan El Shafie Co-supervisor …………………..

Date of Examination : June / 2014

DEDICATIO

To my mother (Settana and Soul OFmy father) My uncle To My brothers My sisters

My frinds My all those who search of knowledge

Thanks to all my bestowed Allah who endowment methe will and power to accomplish this study. Guidance, encouragement, moral support and constructive criticism of Dr.Mai Abdalla Ali Abdallamy main advisor ,are greatly appreciated. Thanks toEL Shafie El Hassan EL Shafie for his effort in reading and correcting this manuscript .Thanks also extend to Prof. Mutwakil Abdel Mageed Mahir, Thanks also internal examiner, the.Dr. Omar Osman Ahmed Elbashir

In no way could I express my regard and gratitude towho stood behind me and reserved no effort in the execution of the staff of plant pathology center without their assistance this study would be difficult to come into existene.

My thanks are also extended to my friendsClassmates.

Last but not least, special words of thanks to my family and relative.

Identification and Molecular Characterization of some Yellow- inducing Viruses and Phytoplasma on Faba bean (Vicia faba L.) in the Gezira State, Sudan By Ibrahim Abd ELhameed Saeed Fadl Elmola

Abstract In the Sudan, Faba bean is an important food legume crop, for a large proportion of population, regularly consumed by all income groups. Faba bean production is challenged by several biotic constrains. Viral diseases are among the major limiting factors for faba bean production affecting both quantity and quality. This study was aimed at assessing the incidence of the yellow-inducing diseases, identification and characterization of causal agents and screening for resistance among the different faba bean lines/varieties. Field experiments were conducted at the Gezira University Farm for two consecutive winter seasons during 2010/12 .Twelve international and four local varieties of faba bean were screened for genetical resistance. Serology (ELISA) and RT- PCR degenerate primers were used for identification and characterization of the yellow causal agents. Hitherto, this study revealed the presence of four types of symptoms accompanied with or without yellow symptoms. The disease syndrome included dark and light green mottling, stem and leaf blackening, stunting, leaf deformation and phyllody. Serological and molecular characterization showed the occurrence of five causal agents; namely pepper yellow leaf curl luteoviruses, pepper vein yellow luteoviruses, pea seed-borne mosaic potyviruses, tobacco streak ilarviruses and phytoplasma which was found to belong to 16srD subgroup. This is the first report on a phytoplasma16srD subgroup found associated with faba bean plants were deposited in the GenBank database under accession numbers JN233801, JN233803 and JN233804.This study recommended cultivation of Hudaiba cultivar and line C.9/02 which proved to have some degree of tolerance.

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التعريف والتوصيف الجسيئي لبعض الفيروسبث والفبيتوبالزمب المسببت لإلصفرار في الفول المصري ).Vicia faba L( في واليت الجسيرة-السودان.

إبراهيم عبذ الحميذ سعيذ فضل المولي

ملخص الذراست

انفٕل انًصشي يٍ أْى يحبصيم انبمٕنيبث في انغٕداٌ حيث يشكم انطبك انشئيظ نششيحت كبيشة يٍ يدخًع انغٕداٌ. يٕاخّ إَخبج انفٕل انًصشي في انغٕداٌ انعذيذ يٍ انًشكالث يٍ ضًُٓب األيشاض انفيشٔعيت ٔانخي حؤثش عهي َٕعيّ ٔكًيّ اإلَخبج. نٓزا ْذفج ْزِ انذساعت إني ححذيذ يغخٕيبث اإلصببت نًحفضاث االصفشاس، حعشيف ٔحٕصيف انعٕايم انًغببت نالصفشاس ٔرنك ببعخخذاو انخمُيبث انًصهيت )حمُيّ إنيضا( ٔحفبعم انبٕنيًيشيضانغهغهي (PCR(. حى إخشاء انخدبسة انحمهيت في انًضسعت انخدشيبيت بدبيعّ اندضيشة نًٕعًيٍ يخخبنييٍ 2011/2010)-)201/2011)،ببعخخذاو اثُي عشش صُفب عبنًيب ٔأسبعت أصُبف يحهيت يٍ انفٕل انًصشي نخحذيذ يمبٔيت ْزِ األصُبف نًحفضاث اإلاصفشاس. أٔضحج َخبئح انذساعت ٔخٕد أسبعّ إَٔاع يٍ األعشاض انًشضيت، حضى ْزِ األعشاض، حبشلش بيٍ األخضش انذاكٍ ٔ انفبحح، اعٕداد في األٔساق ٔانغيمبٌ، حشِٕ األٔساق ٔ اإلعخيشاق إضبفت نإلصفشاس. أظٓشث اإلخخببساث انًصهيت ٔاندضيئيت ٔخٕد خًغت إَٔاع يٍ انًغبببث انًشضيت ْٔي فيشٔط اصفشاس ٔحدعذ أٔساق انشطت ٔفيشٔط اصفشاس عشٔق انفهفم ٔانهزاٌ يُخًيبٌ نًدًٕعت )luteovirus(، ٔفيشٔط انخبشلش انًحًٕل بٕاعطت انبزٔس في انبغهت ٔانزي يُخًي ندُظ )potyvirus( ٔفيشٔط حخطيط انخبغ ٔانزي يُخًي ندُظ)ilarviruses( ٔكزنك انفبيخٕبالصيب ٔانخي ٔخذ أَٓب حصُف ححج انًدًٕعّ 16srD.ْزِ أٔل دساعت خيُيت حثبج إصببت انفٕل انًصشي بٓزا انُٕع يٍ انفبيخٕبالصيب ٔلذ حى ٔضع حفبصيهٓب انديُيت ببُك انًعهٕيبث ححج األسلبو JN233804.، JN233803 ،JN233801حٕصي انذساعت بضساعّ انصُف حذيبّ ٔ C.9/02 ٔانخي ٔخذث ببٌ نٓب دسخّ يٍ انخحًم.

List of contents

page

Dedication …..……………………………………..…….….……………………… i Acknowledgement ………………………………………….………..………….… ii English Abstract …………………………………………………..…………..…... iii Arabic Abstract ……………………………………………………….…………... iv List of contents ……………………………………………………….………...…. v List of Table …………………………………………………………….……....… vi List of Figures ……………………………………………………………...………viii CHAPTER ONE …….……………………………………….……..….1 NITRODUCTION………………………………………………………..….…..… 1 1.1. Objectives of the study ……………………………….………..….…. 2 CHAPTER TWO…….…………………………….….. …….….…….3 2.1 Faba bean ………………………………………………………...….………… 3

2.1.1 Classification……………….……………..………………….…..….………..3

2.1.2 Common Names…………………………………….…………….….….…... 3

2.1.3 Origin………………………………………………………………..……… 4

2.1.4 Morphological ………………….……………………………………………4

2.1.5 Chemical composition…………………………………….………………… 5

2.1.6 Uses……………………………………………………………………..……...6

2.1.7 Ecology ………………………………..…………….…………...……….….. 6

2.1.8 Field Cultivation……………………………………………….….…………..7

2.1.9 Harvesting……………………………………………………………………8

2.1.10 Yields and Economics…………………………………………….……….. 8

2.1.11 Biotic Factors………………………………………………………………. 9

2.2 Faba bean in Sudan……………………………………………...... …..…….10

2.2.1 The growing and sowing dates of faba bean in Sudan…………..…..….10

2.2.2 Method of sowing……………………………………………………...... …10

2.2.3 Seed rate and plant population of faba bean………………….…...... ….11

2.2.4 Pests and diseases of Faba bean……………………………………...……11

2.2.4.2 Diseases ….…………………………………………………….…………11

2.2.4.2.1 Bacterial diseases of faba bean……………………………………… 11

2.2.4.2.2 Fungal diseases of faba bean………………………………….….…..12

2.2.4.2.3 Viral diseases of faba bean……………………………………..…….12

2.2.4.2.3.2Bean yellow mosaic virus (BYMV) ………………………………. 12

2.2.4.2.3.2 Bean yellow mosaic virus (BYMV) …………………………..…….12

2.2.4.3.3.3 Beet western yellows virus (BWYV) …………………….….….….13

2.2.4.3.3.4 Broad bean mottle virus( BBMV) …………………..……..………13

2.2.4.3.3.4.5 Faba bean necrotic yellow virus (FBNY1V) ………………………..……………………………………………………………13

2.2.4.2.3.6 The Genus Ilarvirus ………………………………………….…… 13

2.2.4.2.3.7 Type species Tobacco streak virus (TSV)………………………..…14

2.2.4.2.3.8 The morphology of TSV……………………………………...……15

2.2.4.2.3.9 the symptomatology and host range of TSV……………………….15

2.2.4.2.3.10 The transmission of TSV………………………………………….16

2.2.4.2.3.11 Relations of TSV with Cells and Tissues ………………….…….16

2.2.4.2.3.13 the family Luteoviridae……………………………..……..…...….16

2.2.4.2.3.13 Pepper yellow leaf curl virus (PYLCV) and Pepper vein yellows virus (PeVYV)…………………………………………………………..……….……… 17

2.2.4.2.3.14 The family The Potyviridae ………………………..………...…… 18

2.2.4.2.3.15 Pea seed-borne mosaic virus (PSBMV)……………………….…. 19

2.2.5 faba bean (phyllody)…..……………………………….…..….…,…….… 20 CHAPTER THREE …………………………..…………..………… 21 3. MATERIALS AND METHODS ………………………………..…...... 21 3.1 Field experiment………………………………………………………….……21 3.2. Seed testing (for which room) ………………………………….………..…...22 3.3 Laboratory tests …………………………………………………………… 22 3.3.1 Serological test …………………………………………………………… 22 3.3.2 Different buffers and steps for applying Double antibody sandwich ELISA (DAS-ELISA) test ………………………………………………………….….….. 23

3.3.3. Molecular tests …………………………………….………………….…….24 3.3.4 Molecular analysis ……………………………………………..…..24 CHAPTER FOUR….…………………………….……………,,…………,,..… 26 4. 1 RESULTS AND DISSCUSION …………….……………..…..…….………… 26 4.1 Field experiment ……………………………………..…………………………26 4.2 Serological test………………………………………………………..…………26 4.3 Molecular analyses ………………………………………….………………… 26 4.3.1 First season …………………………………………………………..……… 26 4.3.2 Second season ………………………………………………..……………… 26 4.2. DISCUSSION………………………………………………………………… 35 CHAPTER FIVE ……………………………………………………...36 CONCLUTIONS AND RECOMMENDATIONS………….…………...... …….. 39 5.1 Conclusions ……………………………………………………………………. 39 5.2 Recommendation ……………………………………………………………… 39 REFERNCES …………………………………….………………..……..………... 40

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List of Table Table page Table one …………………………………………………………….…..……….….22 Table tow …………………………………………………………………………… 29 List of Figures Figure page Figure one…………………………….………………………………………….… 30 Figure tow ………………………………………..………………………………... 32

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CHAPTER ONE

1- INTRODUCTION

Leguminosae, is one of the largest families of the flowering plants with approxitly. 18,000 species, and 650 genera Polhill and Raven, (1981). Legumes are second only to the grasses (cereals) in providing food globally. The seeds of legumes are rich protein; this considered as valuable food resource. Legumes constitute the major dietary protein for millions of people in Sub-Saharan Africa, besides being an important component of animal feeds and their direct contribution to the soil fertility via nitrogen fixation. In Sudan Faba bean (Vicia faba). is considered as the most important legume, which constitutes the main dish for a large sector of population. The average per capita consumption was found to be 2.25 kg/month in the urban area Yousif, (1988). It hasshare the biggest area compared to other legume crops, that range between 20,000 - 35,000ha in some season, with anaverage yield of approx. 1.86/ha Salih et al., (1995).The traditional area of Faba bean production is northern region in Small pump irrigated schemes. Faba bean production in the River Nile State is faced by many constraints that contribute to the low productivity . There include poor cultural practices, lack of high yielding cultivars, stresses inflicted by harsh a biotic and biotic factors, e.g. high temperature, diseases, insect, weeds and intrinsic factors pertaining to the sensitivity of the species to sudden changes in climatic conditions Salih, (1995).Recently a limited effort was made to extend faba bean production to other non-traditional areas, in the several days in Gezira and plain new Halfa.Viral diseases remain among the major constraints to faba bean production in the Sudan. A number of viral diseases were reported in different parts of the country,thes includeBean yellow mosaic virus (BYMV) Ali et al., (2000), Faba bean necrotic yellow virus (FBNYV) Makkouk, et al.,(2003a), Tobacco streak virus (TSV) Ali et al.,(2008), besides other unknown yellow- inducing ones. Most of these viruses occur as a sudden outbreak in most of the commercial farms resulting inarticle or total crop failure.

1.1 the current study was under bale to full the:

• Assessment of incidence of the most commoYellow-inducing viruses affecting faba bean; • Identification and molecular characterisation of the main virus(es) and phytoplasma that infects faba bean and represent serious constraints to its cultivation and production in Sudan; • Screening for resistance to viruses among faba bean germplasm

CHAPTER TWO

LITERATURE REVIEW

2.1 Faba bean

2.1.1 Classification:

Faba bean belong to the Species: Vicia faba L., of the Family Leguminosae

Kingdom: plantae

Division: Magonoliophyta

Class: Magnoliopsida

Order: Fabales

Family: Fabaceae

Subfamlily: Faboideae

Tribe: Vicieae

Genus: Vicia

Species: Vicia Faba

Binomial name: Vicia Faba L.

Synonyms: Faba sativa moench

2.1.2 Common Names:

Faba bean is known by different local names depending on location.Broad bean, Faba bean, Horse bean, Windsor bean, Tick beans (small types), Bakela (Etheopia), Boby kurmouvje (former USSR), Faveira(Portugal) Ful masri (Sudan), Feve (French) and Yeshil Bakla (Turkey) Bond et al.,(1985).

2.1.3 Origin:

Faba bean is assigned to the Central Asian, Mediterranean and South American centers of Diversity. Cubero (I974) postulated a Near Eastern center of origin, with four radii (1) to Europe (2) along the North African coast to Spain, (3) along the Nile to Etheopia, and (4) from Mesopotamia to India. Secondary centers of diversity are postulated in Afghanistan and Etheopia. However, Ladizinsky (1975b) reported the origin to be Central Asia. The wild progenitor and the exact origin of faba bean remain unknown. Several wild species (V. narbonensis L. and V. galilaea, Plitmann and Zohary) are taxonomically closely related to the cultivated crop, but they contain 2n = 14 chromosomes, whereas cultivated faba bean has 2n = 12 chromosomes. Numerous attempts to cross the wild species to cultivated faba bean have failed Bond et al.,(1985).

2.1.4 Morphology:

Vicia faba is an annual herb with coarse and upright stems, unbranched 0.3-2 m tall, with 1 or more hollow stems from the base Bond et al.,(1985); Duke, 1981and Heath, et al.,(1994). The leaves are alternate, pinnate and consist of 2-6 leaflets, each up to 8 cm long.Unlike most other members of the Genus. it is possess on tendrils or with rudimentary marking or magrin tendrils Kay,(1971) and Bond et al.,(1985). Flowers are large, white with dark purple ranges, borne on short pedicels in clusters of 1-5 on each axillary raceme, usually between 5 and 10th node; 1-4 pods develop from each flower cluster and growth is indeterminate, though determinate mutants are available. About 30% of the plants in a population are cross-fertilized and the main insect pollinators arebumblebees. There is a robust tap root with profusely branched secondary roots Bond et al.,(1985). Based on seed size, two subspecies were recognized, paucijuga and faba. The later was subdivided into var. minor with small rounded seeds (1 cm long), compestris patho var . equina with medium sized seeds (1.5 cm) and compestris patho var. major with large broad flat seeds 2.5 cmKay, (1971) and Bond et al., (1985). Cubero (1974) suggested four subspecies, namely: minor, equina, major, and paucijuga. Taxonomically the crop belongs to section faba of the Genus Vicia Bond et al., (1985) and Smart, (1990).

2.1.5 Chemical composition:

Wide variation of protein content (20-41%) has been reported Chaven et al.,(1989). When spring and winter Faba beans are compared, winter beans have slightly higher concentrations of protein than the later Bond et al.,(1985). Protein concentration is influenced by both genetic and environmental factors and it has been reported that inheritance of this trait is additive with some partial dominance Bond et al.,(1985). Amino acid content as mg g-1 of nitrogen varies from 36-69 mg for methionine, 44- 94 mg for cystine and 333-400 mg for lysine Chevan et al.,.(1989). Legumin is the predominant globulin and has a larger proportion of arginine, threonine and tryptophan Hulse, (1994). Utilizable protein, protein digestibility and biological value are reported to vary from 14.8-15.5%, 82-92% and 45-55%, respectively Hulse, (1994). Faba bean contains small amounts of several possible ant nutritional factors; however, their effects are less acute, and protease inhibitors are at much lower (2%) concentrations compared to soybeans Lawes, (1980) and Bondetal.,(1985). Inhalation of the pollen or ingestion of the seeds may incite the condition known as favism, a severe hemolytic anemia, perhaps causing collapse Smart, (1990). It is an inherited enzymatic deficiency occasional among Mediterranean People (Greek, Italian, and Semitics); the genetic disorder occurs in about 1% of whites, 15.0% of blacks Duke, (1981). The main factors responsible for fauvism, which can occur in susceptible people, are believed to be glycoside vicinal and convicine and their hydrolytic derivatives divicine and isouramil, respectively. These anti-nutritional factors render the red blood cells of glucose -6- phosphate dehydrogenase deficient patients vulnerable to oxidation and destruction Bond et al.,(1985); Hussein and Saleh, ( 1985); Smart, 1990) which are uncommon in cooked beans Lawes, (1980). The whole dried seeds contain (per 100 g) 344 calories, 10.1% moisture, 1.3 g fat, 59.4 g total carbohydrate, 6.8 g fiber, 3.0 g ash, 104 mg Ca, 301 mg P, 6.7 mg Fe, 8 mg Na, 1123 mg K, 130 mg b-carotene equivalent, 0.38 mg thiamine, 0.24 mg riboflavin, 2.1 mg niacin, and 162 mg tryptophane. Flour contains: 340 calories, 12.4, % moisture, 25.5 g protein, 1.5 g fat, 58.8 g total carbohydrate, 1.5 g fiber, 1.8 g ash, 66 mg Ca, 354 mg P, 6.3 mg Fe, 0.42 mg thiamine, 0.28 mg riboflavin, and 2.7 mg niacin. The fatty acid composition of broad bean oil has been reported as 88.6% unsaturated Duke, (1981). The amino acid content except for methionine is reasonably well balanced Bond et al., (1985). Haema gglutinins (lectins), although found in many legumes,

5 concentration is higher in faba bean and can be troublesom. These substances are destroyed during the normal food preparation process (heat) Hussein and Saleh, (1985). Similarly, oligosaccharides mainly stachyose, raffinose and verbascose are also more found occur in faba bean; these molecules contain glucose and galactose residues which can persist in sugar metabolism pathway in digestive tracts. They ferment and produce methane and other gases causing discomfort and abdominal pains. Faba bean contains other objectionable factors including, cyanogens, favogens, phytic acid, tannins, and tripsin inhibitors Williams et al.,(1988).

2.1.6 Uses: faba bean is used as human food in developing countries and as animal feed, mainly for, horses, poultry and pigeons in industrialized countries. It can be used as a vegetable, either green or dried, fresh or canned. It is a common breakfast food in the Middle East, Mediterranean region, China Ethiopia and SudanBond et al., (1985). The most popular dishes of faba bean are Medamis (stewed beans), Falafel (deep fried cotyledon paste with some vegetables and spices), Bissara (cotyledon paste poured onto plates) and Nabet soup (boiled germinated beans) Jambunathan et al., (1994). Feeding value of Faba bean is high, and is considered in some areas to be superior to field peas or other legumes. It is one of the most important winter crops for human consumption in the Middle East. Faba bean has been considered as a meat extender or substitute and as a skim-milk substitute. Sometimes grown for green manure, but more generally for stock feed. Large-seeded cultivars are used as vegetable. Roasted seeds are eaten like peanuts in India Duke, (1981). Straw from faba bean harvest fetches a premium in Egypt and Sudan and is considered as a cash crop Bond etal., (1985). The straw can also be used for brick making and as a fuel in parts of Sudan and Ethiopia. In the past, faba beans for human consumption, feed for horses (commonly known as horse beans); green manuring and feed for other stock were important uses in southern United States and along the U.S. Pacific Coast.

2.1.7 Ecology:

Faba bean requires a cool season for best development. It is grown as a winter annual in warm temperate and subtropical areas; hardier cultivars in the Mediterranean region tolerate winter temperatures of -10°C without serious injury whereas the most

6 hardy European cultivars can tolerate up to -15°CRobertson, (1996). It can be grown anywhere . Well-adapted to wetter portions of cereal-growing areas of western Canada and elsewhere. Tolerates nearly all soil type; grows best on rich loams. Moderate moisture supply is necessary Duke, (1981). They are considered to be the least drought resistant of legume crops; however, cultivars with high water use efficiency, have been developed at ICARDA Robertson, (1996). Moisture requirement is highest about 9-12 weeks after establishment. Faba bean is more tolerant to acid soil conditions than most other legumes. Can be grown in nearly all parts of the United States without limiting. Growing seasons should have little or no excessive heat, optimum temperatures for production range from 18 to 27°C (65- 85°F) Duke, (1981). Rainfall of 650-1000 mm per annum evenly distributed is ideal Kay, (1971). The maturity period ranges from 90-220 days depending upon the cultivars and climatic conditions Bond et al.,(1985).

2.1.8 Field Cultivation:

All though, faba bean has been cultivated in many countries, 60% of total world production comes from China (FAO, 1994). The date of introduction of Vicia faba var. minor to China is believed to be around 100 BC (Bond et al.,1985). In localities having no hard frosts, most cultivars can be grown in fall and survive the winter. In northern localities or at high elevations further south, Faba bean is planted in early spring, when ground can be worked, at the same time as the earliest ordinary spring crops. Large-seeded cultivars are sown with planters used for lima beans, while small- seeded cultivars can be sown with a common corn planter. In some areas, such as Ethiopia and Sudan, broad bean seeds are broadcast. At seeding time, fields are plowed shallow and seeds are dropped into every second or third furrow. Seeds are usually sown 5-10 cm deep in rows 75 cm apart, with seeds 15 cm apart in the rows. Rows60 cm apart, or even closer, give good results under favorable conditions. Small- seeded cultivars are planted at 90-122 kg/ha, and large-seeded cultivars at 78-90 kg/ha. In United Kingdom, 450 kg seed/ha produces maximum yields. Yields are economically optimal at a seeding rate of 225-340 kg/ha for large seeded cultivars, and satisfactory at 190 kg/ha for small-seeded cultivars Duke, (1981). For green manure or forage, small-seeded cultivars are usually broadcast. Murinda and Saxena (1985) reported that faba bean fixes more nitrogen (135 kg N ha-1) than lentil and

7 chickpea. Inoculation is not always practiced if effective strains of Rhizobia sp are present e.g. Europe in general and Middle East Murinda and Saxena, (1985); Bond et al., (1985). Applications of P and K in southern United States Duke, (1981), and Nitrogen in Africa and Middle East Murinda and Saxena, (1985) as starter fertilizers before or at seeding time have been reported to be beneficial for vigorous early growth. Early deep sowing into a well-drained firm seedbed gives best results. Faba beans should be cultivated during their growing period. When planted in 60 cm rows or closer, special machinery is necessary for cultivation. When planted in 90 cm rows, ordinary cultivators can be used. In United Kingdom, Thiram and Captan are recommended as fungicides; Chlorpropham plus Diuron or Fenuron, or Simazine, as pre-emergence herbicides, and Dinoseb-acetate as a post-emergence herbicide Kay, 1979 and Duke, (1981).

2.1.9 Harvesting:

When the crop is meant for dry seed, it is harvested fully mature, and heretical grown for consumption as a vegetable, it is harvested green. The most common harvesting system is to pull and thresh the crop by hand; however, hand harvesting is cheched costly compared to mechanized harvesting Diekmann and Papazian, (1985). Time of harvest depends onor mechanical methods are used. Beans mature 90-220 days after planting based on localities Bond et al., (1985). Harvest can be delayed a little longer for hand than for mechanical harvest. In either case, the crop should not be cut until the lower pods are matured and the upper ones fully developed. If harvest is delayed until the upper pods are ripe, there can be great losses from shattering. The crop should be cut on cloudy days or cut at night. Large seeded cultivars are threshed with a common bean thresher with special adjustments to the cylinder Duke, (1981). Small-seeded types can be threshed without difficulty using a cutter bar and stationary thresher Diekmann and Papazian, (1985). After threshing, the seed is cleaned with ordinary fanning mills. For canning, beans are soaked and allowed to swell and then are picked by hand to avoid hard seeds Duke, (1981).

2.1.10 Yields and Economics:

The lack of adequate pollination culture and reduced seed setting were found to contribute substantially to faba beanyield. Flower drop seed abortion, disease such as

Botrytis fabae, Ascochyta fabae, Uromyces fabae, Orobanchecrenata, and Aphis fabae are also major constraints to yield. Abiotic factors such as drought, high temperature, inadequate supply of nutrients, salinity and excessive moisture also play an important role. Yields are closely correlated with the number of pods per plant. China was the largest producer with estimated annual production ranging from 2.4-2.6 million MT (1161-1447 kg/ha) from 1979 to 1994 (FAO, 1994). Argentina reported the highest productively record˃9000 kg/ha from 1992 to 1994, followed by Switzerland (3350-4375 kg/ha), France (3000-3900 kg/ha) and Belgium (3350-3750 kg/ha) during the same years (FAO, 1994). Faba beans are grown in home gardens and mostly used as a green vegetable when the seeds are still succulent. Faba bean production in the world is concentrated in nine major agro-ecological regions, namely; Northern Europe, Mediterranean, the Nile valley, Ethiopia, Central Asia, East Asia, Oceana, Latin America and North America Bond et al., (1985). There has been a 50,000 tons increase in production in Australia, a 50,000 tons increase in EEC, and a 210,000 tons increase in West Asia and North Africa (WANA) from 1982 to 1992 Oram and Agcaoili, (1994). However, the same authors reported that there was a 25% decrease in area sown to faba bean in China alone, the largest producer, and a decrease in production of 201,000 tons in Africa during the same period Oram and Agcaoili, (1994). Large seeded green types are canned. It is the second ranking food legume in Europe Picard et al., (1988).

2.1.11 Biotic Factors:

Faba bean subjected to infect by different fungal viral pathogen. The most important fungal diseases chocolate leaf spot (Botrytis faba and B. cinerea), rust (Uromyces viciae fabae), black root rot (Thielaviopsis basicola), stem rots (Sclerotina trifoliorum, S. sclerotiorum), root rots and damping-off (Rhizoctonia spp.), downy mildew (Pernospora viciae), pre-emergence damping-off (Pythium spp.), leaf and pod spots or blight (Ascochyta fabae), foot rots (Fusarium spp.) Van Emden et al., (1988).Bacterial diseases in faba bean comprisedofBacteriumphaseoli, B. viciae, Erwinia phytophthora, and Psuedomonas viciae Van Emden et al., (1988). Viral diseases include Bean leaf roll virus (BLRV) Bean yellow mosaic virus (BYMV), Beantrue mosaic virus (BTMV), Faba bean necrotic yellow virus (FBNYV)tobacco streak virus (TSV)Makkouk et al., (2003).Among the insect pests, groundnut aphid

(Aphis cracivora), Black bean aphid (Aphisfabae), pea aphid (Acyrthosiphon pisum), pea thrip (Thrip kakosrobustus), cowpea bean beetle(Callosobruchusmacculatus), seed weevils (Apion spp.), bean weevil (Sitonialineatus), and Egyptian leaf worm (Spodoptera littoralis) are importantWeigand and Bishara, (1991). The Important nematodes include Meloidogyne incognita, M. javanica,Pratylenchus spp, Trichodorus spp, and Xiphenema spp. The parasitic plant Broomrape (Orobanche crenata) considered as a serious problem in the Mediterrannean region.A disease caused by phytoplasma was also recorded on Faba bean.

2.2 Faba bean in Sudan:

2.2.1 The growing and sowing dates of faba bean in Sudan:

Last and Nour (1961) and many researchers, reported that faba bean is grown mainly in the northern part of the Sudan, where winter season is relatively longer which factor of vegetable and productive growth. Biotic factor (diseases and insect pests) which are themselves influence by the weather conditions, affect the optimum sowing date of the crop. Ibrahim and Ali (1993) reported the optimum sowing date of the crop in Hudeiba, (Northern Sudan) range between 13th October to 10th November and that the variation in grain yield, was highly associated with the variation in biomass yield at maturity, harvest index, plant height at maturity, single kernel weight and the number of seed per pod. A study conducted at river Nile in Ed Damer, by Ageeb, (1977) revealed highly significant effects of sowing date, irrigation interval and their interaction on seed yield. They also reported that, the optimum sowing date, varied with irrigation interval, being in the second half of October, when the crop is irrigated every week and in the first week of November, when the crop is irrigated every two weeks.

2.2.2 Method of sowing:

Salih et al., (1995) reported that the method of sowing of faba bean in the research station (Hudieba,Shambat and Shendi ) is better, when the crop is planting on ridges 60 cm apart .This method was compared with the traditional farmers practice (sowing on flat ), at various locationssuch asAliab,Saiyl,shendi,Hager Elasal and Basbeer using on-farm trials in farmers field. The method of sowing on ridges were found not

11 to be superior to the farmers practice in the traditional faba bean growing areas, where adequate land preparation is made.

2.2.3 Seed rate and plant population of faba bean:

Ishag, (1970), reported that faba beans are known to be highly variablein their reports to plant population. Last and Nour(1961), found that increasing seed rate from 81.6 to 163.2 kg/ha increased the seed yield of a local variety (B.F.M.) by 48% and that of an introduced variety (Rebaya 34) by 18%. Hawtin and Webb (1982) stated that no significant differences in the seed yield of the two varieties B. F. M and Rebaya 40, when were sown at seed rates of 73.3 or 110.4 kg/ha. Ageeb (1976;1977) at Hudeiba research station, found that the variation in row spacing using 60,40 and 20 cm and plants spacing 20,10 and 5 cm and the numbers of plant per hole, using 2 plant, no significant effect was obtained on the seed yield of variety Hudaiba 72. This probably due to the variation in the number of plant per hole.

2.2.4 Pests and diseases of Faba bean:

2.2.4.1 Insect Pests:

Salih et al., (1995) stated that the insect pests such as aphid, leaf miner, cutworms, pod borer, represent one of the major biotic constrains, that limit the production of cool season food legumes in the Sudan. The rate of infestation of the wide range of herbivores, which attack every part of the plants, and the magnitude of damage the crops, which can be tremendous, vary depending on area, season and cultural practices.

2.2.4.2 Diseases:

2.2.4.2.1 Bacterial diseases :

Several bacterial diseases were recorded to affect the Economic important origin variety production constrains of the Faba beans around the world. These diseases include the halo blight caused by Pseudomonas syringae pv. phaseolicola, bacterial brown spot (Pseudomonas syringae pv. syringae), common bacterial blight (Xanthomonas campestris pv. phaseoli) and the bacterial wilt (Curtobacterium flaccumfaciens subsp. flaccumfaciens, most of these bacterial diseases are seed-borne,

11 thus their presences in the field is very damaging and could result in a total crop failure Howard et al.,(2005).Response of how much loses this found Sudan,

2.2.4.2.2 Fungal diseases of faba bean:

A number of fungal diseases were known to infect the Faba beans. In general, those, diseases were divided in two groups, according to the plant parts that they infect. The fungal diseases that infect the subterranean parts and their causal agents includes; Black Root Rot (Thielaviopsis basicola) Fusarium Root Rot (Fusarium oxysporum ) Fusarium Wilt (Verticillium spp.) Pythium disease ( Pythium aphanidermatum ) Rhizoctonia Root Rot ( Rhizoctonia solani ) Alternaria leaf and pod spot (Alternaria brassicae and Alternaria alternate ) Anthracnose (Colletotrichum species ) Cercospora Leaf Spot and Blotch (Cercospora beticola) and (Stagonospora nodorum) Downymildew(Pseudoperonospora humuli) Powdery mildew(Blumeria graminis f. sp. hordei ) Rust; which caused bygenus Cronartium. White Leaf Spot(Cercospora brassicicola) Howard et al., (2005).

2.2.4.2.3 Viral diseases of faba bean:

2.2.4.2.3.1Bean Leaf roll virus (BLRV)

Bean Leaf roll virus (BLRV) is the most important virus known to affect faba bean cultivation in many areas. It was first reported in Germany from Pisums ativum and Vicia faba,by Quantz and Volk (1954). (BLRV) is transmitted by different species of aphids in a persistent manner and it gives rise to a range of different symptoms, depending on the species that it infects. The main symptoms of the Bean leaf roll virus (BLRV), infection, appear as interveinal chlorosis, yellowing, stunting, and leaf rolling, reddening and thickening of the leaves. In some cases, it results in suppression of the flowering and pod setting of the infected plants. Moreover, the virus gives a characteristic leathery appearance of the older leaves in the infected faba bean plants Makkouk et al., (2003a).

2.2.4.2.3.2Bean yellow mosaic virus (BYMV)

Bean yellow mosaic virus (BYMV) was first reported in Phaseolus vulgaris by Doolittle and Jones (1925) from U.S.A. and the Netherlands. The virus is transmitted by over 20 species of aphids in a non-persistent manner. Seed transmission of the

12 virus was also recorded at less than 4% Barnett et al., (1987). (BYMV) infection gave rise to variable symptoms according to the plant species and the virus strain. Aggressive strains of (BYMV), can result in stem and tip necrosis and early death of the infected plants, while the general symptoms of the other strains appear as mosaic, mottling, chlorosis and green vein banding Makkouk et al., (2003a).

2.2.4.2.3.3 Beet western yellows virus (BWYV)

Beet western yellows virus (BWYV) was first reported by Duffus (1960 and 1961) in USA from Beta vulgaris, Lactuca sativa, Spinacia oleracea and Raphanus sativus .The virus was transmitted in a persistent manner by a number of different aphid species.(BWYV)has a very wide host range and it can infect more than 150 plant species in 23 dicotyledonous families; the symptoms can seasonally vary from mild chlorotic spotting, yellowing, and thickening to brittleness of the older leaves Johnstone and Duffus, (1984).

2.2.4.3.3.4 Broad bean mottle virus(BBMV)

Broad bean mottle virus ( BBMV) was reported early in UK from V. Faba plant by Bawden et al., (1951). It was transmitted by some insects belonging to order Coleopterana, like Acalymma trivittata,Colaspis flavida,Diabrotica undecimpunctat and Scroll lineate Brunt et al.,(1996) (BBMV) infection gives rise to symptoms of mottling, marbling or diffuse mosaic which is sometimes accompanied by malformation and stunting Makkouk et al., (2003

2.2.4.3.3.4.5 Faba bean necrotic yellow virus (FBNYV) Faba bean necrotic yellow virus (FBNYV) was first reported in Syria by Makkouk et al.,(1991) from V. faba plants. The virus was transmitted by different species of aphids in a persistent manner. The symptoms of yellowing, stunting, rosetting and leaf necrosis are very common with (FBNYV) infection at various species accompanced by premature death when the plant coinfected with Bean leaf roll virus (BLRV).

2. 2.4.2.3.6.The Genus Ilarvirus:

The GenusIlarvirusType species Tobacco streak virus (TSV) The virions consist of a non enveloped capsid, isometric to bacilliform, 19-36 nm in diameter, with a length of

20-55 nm. The genome is linear, positive-sense, single-stranded RNA, segmented; tripartite, 86 Kb long. The viral genome encodes structural proteins and non-structural proteins. Virions consist of 1 structural protein(s) located in the capsid, the capsid protein has a molecular mass of 19-25.2-30 KDa, which constitute about 76-84.56- 88% of the particle weight. The Virus is transmitted by arthropods and by insects of the order Thysanoptera. Ilarviruses are divided into seven subgroups or clusters based on serological relationships ICTVdB Management, (2006).

2.2.4.2.3.7 Type species Tobacco streak virus (TSV)

TSV was first reported in Wisconsin, USA from N.tabacum by Johnson (1936), followed by Brazil in 1940 Costa, (1945), then it was known to infect several cash crops such as cotton (Gossypium hirsutum L.), tomato (Lycopersicon esculentum), soybean (Glycine max), peanut (Arachis hypogaea L.), sunflower (Helianthus annuus L.) beside some weeds Costa and Carvalho, (1961). In 1955 the association between the soybean bud blight and TSV was clarified, Costa et al., (1955). TSV was detected in different soybean cultivars in soybean fields of Santa Fe and Cordoba provinces, where diseased plants showed mosaic and malformation, shortening of internodes and apical shoots, and necrosis, as a result of which the axillary buds proliferated. Al cultivars imported into Scotland from the USA and three distinct isolates of the virus were recorded Jones et al., (2001). TSV was detected in two groundnut plants at two different localities during different seasons in the North-West Province of South Africa. The symptoms appeared as a reduction in size of the young leaves with malformation and chlorotic patches, while the older leaves showed a very mild chlorotic ringspots, Cook et al., (1999). TSV was found to infect a wide range of hosts in India Prasada et al., (2003). In the year 2002 TSV was observed in the majority of the Blackgram growing areas of India with symptoms that vary between brown necrotic lesions on young leaves to brown streaks on petioles and stems, however, severe infection kill the plants Ladhalakshmi et al., (2005). Recently, TSV was found to cause an epidemic in peanut (A. hypogaea) crops in India Reddy et al., (2002).The virus was found in many weed plants such as Parthenium hysterophorus, and other crop species of H. annuus (Sunflower) and Tagetes patula (Marigold) that may act as an inoculum resorvoir. TSV was also found to infect cucumber (Cucumis sativus L.) and gherkin (Cucumis anguria L.) in India and causing characteristic

14 symptoms of necrotic lesions on leaves, followed by a general leaf and stem necrosis extending to mid veins, petioles, flower buds and tip, eventually resulting in dieback of vines Krishnareddy et al., (2003). Natural infection of chilli pepper by TSV occurred in some parts of northern India, the comparative amino acid sequence analysis revealed that the virus infecting chilli shared very high levels of similarity both at nucleotide (98-99%) and amino acid (98%) levels with the corresponding region of TSV isolates originating from multiple hosts and locations Bhat et al., (2002b), thus suggestions was made in-order to illustrate that the virus infecting chilli is a strain of TSV and should be designated as TSV-CH, Jain et al., (2005).most the small sheet appeared misshaped and useless, while the seeds possess dark brown spots without definite edges Truol et al.,(1987). Natural infection with TSV in Vaccinium species was detected for the first time in cranberry

2.2.4.2.3.8 The morphology of TSV

TSV is the type species of the Ilarvirus, with an isometric non enveloped virions, 27- 35 nm in diameter; rounded in profile; without a conspicuous capsomere arrangement. The virions contain 14 % nucleic acid; 86 % protein and 0 % lipid. TSV has a tripartite single-stranded messenger-sense RNA genome, with a total genome size of 7.915 Kb , the largest genome part of about 2.94 kb; the 2nd largest 2.77 kb; the 3rd largest 2.205 kb. Genomic nucleic acid was isolated by Van Vloten-Doting (1975). He found that base composition 25 % G; 25 % A; 20 % C; 30 % U. 5´ terminus of RNA has a methylated nucleotide cap Van Vloten-Doting, (1981), and the size of the virus coat protein is 28.5-30 KDa.

2.2.4.2.3.9 The symptomatology and host range of TSV

TSV has a host range of more than 140 genera, including members of the Solanaceae and Leguminosae Kaiser, (1982) and Hull, (2002); TSV usually produces systemic symptoms that persist and kills the plants. TSV gives variable symptoms with different plants, and it can also affect different parts of the plants, in the leaves TSV can cause leaf mottling, leaf vein yellowing or systemic necrosis, while other

15 symptoms could be in the form of reddening of nodes, bud blight or stunting, however, TSV can be symptomless in some plant species Rabedeaux et al. , (2005).

2.2.4.2.3.10 The transmission of TSV :

TSV was found to occur as an endemic in specific regions with high populations of the Thrips tabacci Linderman and Frankliniella occidentalis Pergande Kaiser et al., (1982), along with the presence of some known weeds that host the virus (Ambrosia polystachia L.) Costa and Carvalho, (1961). Recently, Microcephalothrips abdominalis Crawford was reported as a vector, Greber et al., (1991). However, thetransmission by thrips was firstly demonstrated by Costa and Lima Neto (1976). Recently, in India three thrips species mainley, Megalurothripsusitatus, Frankliniella schultzei and Scirtothrips dorsalis were found to be associated with the virus transmission from the infected pollens, Prasada et al., (2003). TSV was reported to be seed transmitted in many plants like bean, Brunt, (1969), Glycine max, V. unguiculataKaiser et al.,(1982), black raspberry (Converse and Lister, 1969), and tomato Sdoodee and Teakle, (1987).

2.2.4.2.3.11 Relations of TSV with Cells and Tissues:

TSV particles usually aggregates in the cytoplasm and the nucleus of their hosts Edwardson and Purcifull, (1974), although in soybean, TSV was obtained only from the embryos of mature seeds but was present in both the embryos and coats of immature seeds Ghanekar and Schwenk, (1980). In cotton TSV appeared systemically only when it occurs in combination with an anthocyanosis virus Costa, (1969).

2-2.4.2.3.12 the family Luteoviridae:

The family Luteoviridae is composed of three genera: Enamovirus (one species), Luteovirusspecies), Polerovirus (13 species) and unassigned viruses (8species). Luteoviruses and Poleroviruses are exclusively transmitted by aphids in a persistent (circulative) and nonpropagative manner Brault et al., (2011); Raccah and Fereres ( 2009).Luteovirus is one of three genera in the luteoviridae family whose primary hosts are plants. The geographical distribution of luteovirus is widespread, with the virus primarily infecting plants via transmission by aphid vectors. The name luteovirus arises from the Latinluteus, which is translated as yellow. Luteovirus was

16 given this name due to the symptomatic yellowing of the plant that occurs as a result of infection (http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/39010000.htm). Luteovirus is a group IV virus according to the Baltimore classification of viruses. Each virion contains a single strand of (+) senseRNA. The genome is non-segmented, linear and between 5300 and 5900 nucleotides long. Notably, luteovirus has two 5'open reading frames (ORFs) which are located upstream of the coat protein. One such ORF encodes an RNA-dependent RNA polymerase. Several other ORFs are present at the 3' terminus and are expressed from subgenomic RNA. Luteovirus also lacks polyadenylation at the 3' terminus.Virions are non-enveloped, icosahedral structures, ranging from 25–30 nm in diameter with 32 capsomeres in each nucleocapsid. The nucleic acid content is around 28% [(http://www.dpvweb.net/notes/showgenus.phpgenus=Luteovirus].

2.2.4.2.3.13 Pepper yellow leaf curl virus (PYLCV) and Pepper vein yellows virus (PeVYV):

The disease symptoms include shortening of stem internodes, inter-veinal yellowing, upward curling of the leaf blade and small, discolored fruit. The disease-causing agent was tentatively named Pepper yellow leaf curl virus (PYLCV) Antignus et al.,(2001); Antignus et al.,(2003) and Dombrovsky et al.,(2010). PYLCV is transmitted by grafting and is also transmitted in a persistentmanner by two aphid vectors, Aphis gossypii and MyzuspersicaeAntignus et al., (2001); Antignus et al., (2003) and Dombrovsky et al.,(2010). Reports from Turkey and Tunisia Buzkan etal., (2o12) and morerecently from India, Indonesia, Mali, Philippines, Taiwan, andThailand Knierim et al., (2013) inform of pepper crops showing symptoms similarto those found in PYLCV-infected pepper crops. Serological analysis, the morphology of the virions, disease symptoms and the partial sequences of the coat protein and movement protein indicate that PYLCV is a putative member of the genusPolerovirus (family: Luteoviridae) (Antignus et al., (2001); Antignuset al., (2003) and Dombrovsky et al., (2010).Another pepper-infectingpolerovirus, Pepper yellows virus (PepYV), have been reported in Australia and Japan, respectively Yonaha et al., (1995).Both of these viruses were assigned to the genus Luteovirus,based on particle morphology, serology and aphidtransmission, but not on any sequence information. Recently,the complete

17 genomic sequence of PeVYV was determined and examination of this sequence led to PeVYV being classified as a new polerovirusMurakami et al., (2011).

2.2.4.2.3.14The family The Potyviridae:

The Potyviridae are a family that encompass more than 30% of known plant virusesRiechmann et al.,(1992) and Berger et al.,(2005) many of which are of great agricultural significance. They are flexuous filamentous rod-shaped particles. Their genome is composed of positive-sense RNA which is surrounded by a protein coat made up of a single viral encoded protein called a capsid. All induce the formation of virus inclusion bodies called cylindrical inclusions (pinwheels) in their hosts. These are composed of a single protein (~70 kDa) made in their hosts from a single viral genome product. Based on the amino acid sequences of their coat proteins, the Potyiviridea are now divided into eight genera. All but the Bymovirus genus are single stranded particles. The eight genera are: Genus Brambyvirus:Genus Bymovirus; type species: Barley yellow mosaic virus ;Genus Ipomovirus; type species: Sweet potato mild mottle virus; Genus Macluravirus; type species: Maclura mosaic virus; Genus Poacevirus; Genus Potyvirus; type species: Potato virus Y ; Genus Rymovirus; type species: Ryegrass mosaic virus and the Genus Tritimovirus; type species: Wheat streak mosaic virus.The largest genus in the Family Potyviridea Riechmann et al., (1992) and Berger et al.,(2005) is the potyviruses. There are more than 100 known species in this genus. These viruses are 720-850 nm in length and are transmitted by aphids. They can also be easily transmitted by mechanical means. Potyviruses infect plants and belong to the family Potyviridae. The genus is named after the type virus - Potato virus Y.Potyviruses, like begomoviruses, account for ~30% of the currently known plant viruses and have at least 180 definitive and possible members. Members of this genus may cause significant losses in agricultural, pastoral, horticultural and ornamental crops. There are 73 described genera of plant viruses. More than 200 species of aphids spread potyviruses and most are from the subfamily Aphidinae (genera Macrosiphum and Myzus). The potyviruses evolved between 6,600 to 7,250 years ago Gibbs et al., (2008) Gibbs and Ohshima 2010).They appear to have evolved in southwest Eurasia or north Africa. The estimated mutation rate is about 1.15×10−4 nucleotide substitutions/site/year. The virion is non-enveloped with a filamentous nucleocapsid, 680 to 900 nanometers (nm)

18 long and is 11-20 nm in diameter. The nucleocapsid contains ~2000 copies of the capsid protein. The symmetry of the nucleocapsid is helical with a pitch of 3.4 nm.The genome is a linear positive sense ssRNA ranging in size from 9000- 12000bp. A number of species have a bipartate genome. The base composition is: 21- 23.51-26% G; 23-30.15-44% A; 14.9-22.41-28% C; 15.6-24.41-30.9% U.In the species with a single genome, at the 5' end a protein is covalently linked (the Vg protein). It encodes a single open reading frame (ORF) expressed as a 350kDa polyprotein precursor. This is processed into seven smaller proteins: P1, helper component (HC), P3, cylindrical inclusion (CI), nuclear inclusion A (NIa), nuclear inclusion B (NIb), capsid protein (CP) and two small putative proteins known as 6K1 and 6K2. The P3 protein also encodes asecond protein - P3N-PIPO - which is generated by a +2 frameshift Chung et al.,(2008).

2.2.4.2.3.15 Pea seed-borne mosaic virus (PSBMV):

Is an economically damaging viral pathogen of field peas and lentils that can cause significant losses in seed yield and quality, especially when infections occur before or during bloom. PSbMV is seed-transmitted and spread between plants by aphids. When aphid populations are high, planting even low levels of infected seed can result in severe epidemics. Infested seed and the movement of aphids from infested crops are important contributors to the local spread of PSbMV (Pea Seed-borne Mosaic Virus (PSbMV) in Field Peas and Lentils, (2014) .www.ag.ndsu.edu).Symptoms In peas, PSbMV causes stunting, reduced internode lengths and malformation, and often results in the formation of malformed terminal rosettes . The virus can delay plant maturity, leading to uneven crop maturation . Infected leaves can exhibit clearing and swelling of veins, slight downward curling of leaf margins, chlorosis and/ or a mottled or mosaic discoloration . Pods often are deformed , and seeds produced from infected plants can exhibit pronounced discoloration , splitting of seed coats, shriveling and reduced size (Pea Seed-borne Mosaic Virus (PSbMV) in Field Peas and Lentils,2014 •www.ag.ndsu.edu). Disease Cycle Transmission PSbMV is seed-borne and seed- transmitted, and it is introduced into new regions through the movement of virus- infected seed. Seed transmission rates as high as 100 percent have been reported for PSbMV in field peas, with transmission rates as high as 30 percent commonly observed. Seed transmission rates as high as 44 percent have been reported for

PSbMV in lentils, although the efficiency of seed transmission of PSbMV in lentils may differ by the strain of the virus. Secondary spread of PSbMV is facilitated by aphids.Aphids transmit the virus from diseased to healthy plants within fields, and they can spread the virus to neighboring fields. When aphid populations are high, even low levels of infected seed can result in severe PSbMV epidemics. More than 20 aphid species, including the pea aphid , are known to transmit PSbMV. Aphids transmit the virus in a nonpersistent manner, obtaining and transmitting the virus in short feeding periods. The virus also can be introduced to fields from infected volunteers and other infected hosts. PSbMV infects and causes disease in chickpeas, faba beans and vetches (Pea Seed-borne Mosaic Virus (PSbMV) in Field Peas and Lentils,2014 • www.ag.ndsu.edu)

2.2.5 faba bean (phyllody):

Phyllody is the abnormal development of floral parts into leafy structures. It is generally caused by phytoplasma or virus infections, though it may be also because of environmental factors that result in an imbalance in plant hormones. Phyllody causes the affected plant to become partially or entirely sterile, and unable to normally produce flowers. The condition is also known as phyllomorphy or frondescence; though the latter may sometimes refer more generically to foliage, leafiness, or the process of leaf growth. Phyllody is usually differentiated from floral virescence, wherein the flowers merely turn green in color, but sometimes retain their normal structure. However, floral virescence and phyllody (along with witch's broom and other growth abnormalities), commonly occur together as symptoms of the same diseases.The term chloranthy is also often used for phyllody (particularly flowers exhibiting complete phyllody, which resembles leaf buds more than flowers), though in some cases it may refer to as floral virescence (Lee et al., 2000; Bertaccini and Duduk 2009).

CHAPTER THREE

3. MATERIALS AND METHODS

3.1 Field experiment: Afield experiment was conducted at the experimental farm - Faculty of Agricultural Sciences ,University of Gezira , Wad Madani - Sudan at the altitude of (14-22N -33- 39 E) and 407m above sea level, for two consecutive seasons 2010/2011 and2011/2012 in randomized complete block design with three replicates . In the first season, the sowing date was started at the third week of November and at the first week of November in the second season. The land was prepared by deep ploughing , harrowing, then leveling followed by ridging. Four local varieties were used namely Hudeiba, Saalim, Dameer, BB7/2009 and twelve international varieties were kindly provided by ICARDA, namely, C.98/2 , C.98/8 , C.1/02 ,C.4/02 , C.8/02 ,C.9/02 , C14/02 , C.15/02 , C.19/02 , C.28/02 , BB7 ,H.93. Seeds of these varieties were treated with Methyl bromide fungicide at a rate of 32g/ml/4hours.Three to four seeds were sown in each hole on the top of the ridge at spacing of 25cm between holes and 80 cm between ridges. Plots were immediately irrigated after sowing and then followed by 7 to10days’ intervals, depending on the plant's needs. Plant received one dose of fertilizer (Urea) six weeks after sowing. Expression of symptoms appearance and developments were observed every week and recorded throughout the growing season according to following formula : Disease incidence (plant leaf for natural infection observation are recorded any well and duster infection use assessed accord to

= No. of diseased plants/per plot ×100 Total No. of plants

3.2Green house experiments: Experimental where conducted glass house cannot result 3.3 Mechanical transmission: The plant sap was extracted from naturally by which whether infected faba bean fresh materials by the addition of 1:10 w/v inoculation buffer , pH 7.0 (0.05M) Na/K

Phosphate ; 1mM EDTA; 5mM Na DECA and 5mM Thioglycolic acid ) together with a small amount of celita and charcoal From each faba bean two plants each variety/line were used for this test. For each plant three leaves were inoculated by soaking a cotton swab on the extracted sap, then rubbed on to the leaf surface starting from the petiole and ending at the leaf tip, the leaf was supported by a paper towel. Following the inoculation, the leaves were washed with water in-order to remove the excessive of sap, and the inoculated plants were kept under where condition daily observations in the green house for symptoms development . 3.2.Seed testing (for which room): Ten seeds used in this test and selected randomly from all the faba bean varieties/lines under the study. The seeds were disinfected before sawing and kept under observation in the greenhouse for the appearance of symptoms.

3.3 Laboratory tests: 3.3.1 Serological test: Faba bean plants showing symptoms suggest to viral infect such as chlorosis, interveinal yellowing, and white areas at the margin of the leaves, leaf curling and stunting were tested against different known yellowing causing viruses antisera (ELISA) includes: Alfalfa mosaic virus (AMV, genus Alfamovirus), Bean common mosaic virus (BCMV, genus Potyvirus), Bean leaf roll virus (BLRV, genus Luteovirus), Bean yellow mosaic virus (BYMV, genus Potyvirus), Beet western yellows virus (BWYV, genus Polerovirus), Chickpea chlorotic stunt virus (CpCSV, genus Polerovirus), Cucumber mosaic virus (CMV, genus Cucumovirus) and Tomato spotted wilt virus (TSWV, genus Tospovirus), beside a general potyvirus and luteoviruse antisera.Elisa proceeded, Field samples showing different types of symptoms were collected in a polythene bags, labeled according to the date of collection; type of the observed symptoms; plot and location rep number. The samples were then transferred to ELISA bags and weighed. The bags were kept at up to40C seven days prior to processing.

3.3.2 Different buffers and steps for applying Double antibody sandwich ELISA (DAS-ELISA) test:

The buffers used: Coating buffer (pH 9.6) 1.59g sodium carbonate (Na2CO3) 2.93g sodium bicarbonate (NaHCO3) 0.20g sodium azide (NaN3) Dissolve in 900ml water, adjust the pH to 9.6 with HCL acid and make up to 1L. Phosphate buffer saline (PBS pH 7.4): 8.0g sodium chloride (HCL) 0.2g monobasic potassium phosphate (KH2PO4) 1.15g dibasic sodium phosphate (Na2HPO4) 0.2g potassium chloride (KCL) 0.2g sodium azide (NaN3) Dissolve in 900ml water, adjust the pH to 7.4 with NaOH or HCL and make up to 1L. Washing buffer -PBS-Tween (PBST): PBS +0.5ml Tween 20 per liter Sample extraction buffer (pH 7.4) PBST + 2% PVP (sigma PVP-40 polyvinyl pyrrolidone) Conjugate buffer: PBST + 2%PVP + 0.2%egg albumin (Sigma A-5253) Substrate buffer: 97ml diethanolamine 600ml H2O 0.2g sodium azide (NaN3) Adjust the pH to 9.8 with HCL and make up to 1L Steps for ELISA: 1 Purified Immunoglobulin G (IgG) was diluted in coating buffer (recommended dilution see delivery note and tube). 200 µl was pipetied in to each well of the microtitre plate. Incubated at 370C for 2-4 hours Plates were washed with PBS-tween for 2-3 times by washingbottle. For a few minutes (3-5 min) and the plate was dried by using tissue paper.

200 µl aliquots of each sample was added of duplicate wells. (Sampls were prepared by grinding in extraction buffer). Three wells of each healthy and diseased control were also included in the test. The plate was Incubated overnight at 40C Plates were washed and dried as the plate was as mentioned in step 200µl of anti-virus conjugate was added to each well. The plates were incubatedat370C for 4 hours. The plate was washed and dried as . 10- 200µl of were freshly prepared substrate (10mg of p-nitrophenyl phosphate {sigma 104-105}dissolved in 10ml of substrate buffer) was added to each well and incubated at room temperature for 30-60min or as long as necessary to obtain clear reaction. 11- The result swere assessed by: A) Visual observation. b) Spectrophotometric measurement of absorbance at 405nm

3.3.3 Molecular tests: faba bea Leaf showed chlorosis, interveinal yellowing, and white areas at the margin of the leaves, leaf curling and/or stunting were dried between news papers to be further studded at the Grupo de Virología. Instituto Agroforestal Mediterráneo. Universidad Politécnica de Valencia (IAM-UPV). Camino de Vera s/n. 46022 Valencia, Spain. The leaf samples were analyzed for different viruses that commonly affect legume crops by molecular techniques.

3.3.4 Molecular analysis : This was done for the samples (C.98/8),(C14/02),(C.28/02) different yellow- inducing viruses which were 'tested serologically before and for phytoplasma infected samples. Total RNA extraction was performed using the silica capture protocol MacKenzie et al., (1997) and analysed by two step RT-PCR with degenerate primers for amplifying different viral species among genus Potyvirus Zheng et al., (2010), family LuteoviridaeAbraham et al,. (2006), genus Tymovirus Sabanadzovic et al., (2000), and genus Rhabdovirus Lamprecht et al., (2009). Moreover, one step RT-PCR was performed with specific primers for Alfalfa mosaic virus (AMV) Martínez-Priego et

24 al., (2004), Bean Leaf roll Virus (BLRV) Ortiz et al., (2005), Cytomegalovirus (CMV) Herrera-Vásquezet et al.,(2009) and Tomato spotted wilt virus (TSWV) Mumford et al., (1994). Total DNA was extracted with the commercial kit E.Z.N.A® Plant DNA Miniprep Kit (OMEGA Biotech, Doraville, USA) and analysed by PCR using degenerated primers for detecting species belonging to the genus Begomovirus Font-San-Ambrosio, (2003).RT-PCR and PCR obtained products were analyzed in 1.2% agarose in TAE buffer, stained with ethidium bromide and visualized under UV light, and fragment sizes were compared with 100 bp DNA standard marker (GeneRulerTM DNA Ladder Plus, MBI Fermentas). Amplified PCR products were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and directly sequenced. Faba bean plants showing yellowing, general stunting and phyllody symptoms were collected together with some asymptomatic plants, dried on calcium chloride and send to Spain for molecular testing.Total DNAwas extracted from the plant material using the cetyltri- methynlammonium bromide (CTAB) buffer and the DNeasy Plant Mini Kit (Qiagen, Valencia,CA) as described by Green and Thompson (1999). A nested-PCR was performed using the universal phytoplasma primers P1/P7 (Deng and Hiruki1991and Schneider et al; 1995) in the first amplification, followed by R16F2n/R16R2 Gundersen and Lee (1996) in the second amplification. Total DNA from the reference phytoplasmas of the different groups belonging to the Virology Group’s (IAM-UPV) collection of isolates were used in this study as positive controls. These positive controls belonged to subgroups 16SrIA (tomato big bud, BB), 16SrI-B (Aster yellows, AY), 16SrIII-A (x-disease, CX), 16SVI-A (potato witches’ broom, PWB), 16SrX-A (apple proliferation, AP) and 16SrXII-A (stolbur, STOL). PCR products were analysed in 1.2 % agarose gels, stained in ethidium bromide and visualised with a UV transilluminator. Fragments with the expected size of 1.2 kb were amplified by nested PCR.To confirm the identity of the phytoplasmas detected, the 1.2 kb amplified products, were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and directly sequenced.

CHAPTER FOUR

RESULTS AND DISSCUSION

4.1 Field experiment:

From the two growing seasons of faba bean some symptoms were observe the more prevalence such as yellowing, blackening, stunting, and in a descend phyllody (photo.1.). The intensity of these symptoms varied season 2010/2011 there was a dominance of blackening, stunting by yellowing and phyllody (Table.1.). On the other hand in season, 2011/2012 there was a dominance of only phyllody followed by yellowing and blackening compared to low incidence of stunting throughout the season (Table.2.). The different types symptoms started to develop within four to five weeks after sowing except for the leaf blackening which start early in less than two weeks. In the first season (2010/2011), the variety BB7 showed the highest percentage incidence of blackening up to (20%) while variety Hudeiba recorded the lowest disease incidence (7%). Concerning the yellowing incidence, the three varieties (C.98/8), (C.14/02) and (C.28/02) showed the highest yellowing incidence (11%). variety (C.1/02) recorded a lowest incidence (3%). Varieties while varieties Saalim, Damer, Hudeiba, (C.4/02), and (C.9/02) revealed some of tolerance to the yellowing infection and exhibited yellowing degree symptoms. With regard to the stunting incidence, the two varieties (BB7/2009) and (H.93) recorded the highest incidence (17%), and the varieties (C.1/02) and (C.8/02) gave a lowest incidence (5%). No incidence of stunting was observed in variety (C.9/02). Concerning phyllody, highest incidenc was recorded from the variety (C.98/2) (14%) versus the lowest ( 4%) was recorded in variety(BB7/2009). The varieties Saalim, Damer, Hudeiba,(C.98/8),(C14/02), (BB7) and (H.93) showed some degree of resistance and showed no apparent symptoms in season (2010/2011) (Table .1, figure .1). In the second season 2011/2012, variety (H.93) recorded higher incidence of phyllody (31%) and variety C.15/02, recorded higher incidence of yellowing and blackeing incidence both (29%). Concerning the yellowing incidence the variety (C.28/02) showed the highest incidence (29%) while variety Hudeiba exhibitedthe lowest percentage incidence (6%).Three varieties (C.98/2), (C.98/8)and (C.9/02) showed some degree of resistance no showed no apparent symptom. The highest incidence of

26 stunting was observed in variety, (C.28/02) (14%) and the lowest incidence on the variety (C.8/02), (5%) and eleven varieties Saalim ,BB7/2009, Daamer, Hudeiba, (C.98/2), (C.98/8), (C.1/02), (C.4/02), (C.9/02), (C.19/02), BB7, provoked some degree of resistance with . In case of the pholldy, the highest incidence was recorded from the variety (H.93) ( 31%) versant the owest incidence (10%) variety C.98/2 no incidence of phyllody was noticed on the variety (C.28/02) which gave some degree of resistance (Table.2, figure.2). According to the results obtained from the two successive growing seasons of faba bean in Gezira area, four types of viral symptoms were found to be dominant in the fields with different severity levels. Three of which symptoms were caused by three groups of viruses, luteoviruses, potyviruses and illarviruses while the fourth was caused by a phytoplasma Out of the different luteoviruses tested only Pepper yellows leaf curl virus and Pepper vein yellows virus were detected , from the potyviruses Pea seed-borne mosaic virus was detected , from the ilarvirues tobacco streak virus was detected and finally the phyllody phytoplasm.TSV and phyllody were considered the major constraints to faba bean production in the Gezira area, and to a lower extent Pepper yellows leaf curl virus, Peppervein yellows virus and Pea seed-borne mosaic virus Generally, none of the faba bean lines/varieties screened showed resistance to both TSV or phyllody infection. In the first growing season more than six lines/varies showed some degree of tolerance to phyllody infection and only one variety was found tolerant to TSV infection, however, the reverse was noticed the following growing season where four lines/varieties showed resistance to TSV infection and only one line showed tolerance to the phyllody. The variety Hudeiba showed resistance /tolerance against yellowing and phyllody in the first season and to blackening and stuting in the second season whereas The line C.9/02 showed resistsnce tolerance to yellowing and stunting in both season. Thus, the variety Hudeiba and the line C.9/02 were considered promising and could be incorporated in breeding programs aiming at controlling most of the faba bean viral disease as they showed consistent levels of tolerance or resistance. The virus which was detected and isolated from faba beans fields in the Gezira area (central Sudan ),was found to be a TSV isolate that infects the faba bean.

Table.1.The incidence percentage of Blackening, Yellowing, Stunting and Phyllody symptoms observed on different faba bean varieties during the growing season 2010/2011.

Different type of symptom%

No. Variety Blackening(TSV) Yellowing Stuntin Phyllod

1 Saalim 0 0 15 0

2 BB7/2009 8 8 17 4

3 Damer 10 0 15 0

4 Hudeiba 7 0 15 0

5 (C.98/2) 14 5 11 14

6 (C.98/8) 13 11 11 0

7 (C.1/02) 12 3 5 7

8 (C.4/02) 13 0 8 9

9 (C.8/02) 10 6 5 13

10 (C.9/02) 17 0 0 13

11 (C14/02) 17 11 15 0

12 (C.15/02) 14 8 15 10

13 (C.19/02) 13 10 14 9

14 (C.28 /02) 15 11 15 11

15 (BB7) 20 8 13 10

16 (H.93) 12 7 17 0

12 Incidence % Blackening(TSV) 10 Incidence % Yellowing 8 Incidence % Stunting 6 Incidence % Phyllod

(BB7)

Selem

(H.93)

DAMER

(C.98/2) (C.98/8) (C.1/02) (C.4/02) (C.8/02) (C.9/02)

(C14/02)

(C.19/02) (C.15/02)

BB7/2009 (C.28/02) (HUDEIBA)

Figure.1.The incidence percentage of Blackening,Yellowing, Stunting and Phyllody Symptoms observed on different faba bean varieties during the growing season 2010/2011.

Table.2.The incidence percentage of Blackening,Yellowing, Stunting and Phyllody symptoms observed on different faba bean varieties during the growing season 2011/2012.

Different type of symptoms%

No. Variety Blackenin Yellowin Stuntin Phyllody

(TSV)

1 Saalim 7 7 0 14

2 BB7/2009 0 23 0 23

3 DAMER 7 14 0 28

4 Hudeiba 0 6 0 19

5 (C.98/2) 16 0 0 10

6 (C.98/8) 22 0 0 17

7 (C.1/02) 0 13 0 20

8 (C.4/02) 9 13 0 16

9 (C.8/02) 5 10 5 21

10 (C.9/02) 10 0 0 12

11 (C14/02) 13 7 7 27

12 (C.15/02) 29 7 0 13

13 (C.19/02) 7 20 0 13

14 (C.28 /02) 7 29 14 0

15 (BB7) 8 20 0 20

16 (H.93) 8 23 8 31

20 Incidence % Blackening 15 Incidence % Yellowing

10 Incidence % Stunting Incidence % Phyllody 5

Figure.2.The incidence percentage of Blackening,Yellowing, Stunting and Phyllody symptoms observed on different faba bean varieties during the growing season 2011/2012

4.2 Serological test:

No results were obtained from the tested samples with all antisera used in the DAS- ELISA test except for the TSV antisera.

4.3 Molecular analyses:

4.3.1 First season

The results of the molecular analyses performed to assess the incidence frequency of the most common yellow-inducing viruses and the Identification and molecular characterization of the main virus (es) infect Faba bean and represent serious constraints to its cultivation and production in Sudan, also Screening of some Faba bean cultivars to select resistant lines. The 15 samples collected from different areas of Gezira state Sudan, found to be positive to AMV, BRLV, TSWV, CMV and BnYDV when analyzed by RT-PCR or PCR with specific primers for those viruses. All samples tested were also found to be negative when two-step RT-PCR or PCR were performed with degenerated primers for detection of different species within the genus Rhabdovirus, Tymovirus or Begomovirus. However, three samples C.98/2, C.98/8, C.4/02 were gave positive result when two steps RT-PCR was performed for detecting different species within the family Luteoviridae and the genus Potyvirus respectively. The amplified fragments of the faba bean sample C.98/8 showed 97% identity with the CP of the newly described Polerovirus (Pepper yellow leaf curl virus) (PYLCV; under Gen Bank Acc. No. HM439608). Nevertheless, The two nucleotide sequences amplified from faba bean samples C.98/8 and C.4/02 with generic primers for Potyvirus were 93-95% identical with Pea seed-borne mosaic virus under Gen Bank (Acc. No. AJ252242).

4.3.2 Second season

The molecular testing of the faba bean samples that showed yellowing, stunting and phyllody symptoms revealed that the tested samples for phytoplasmas belonged to the group 16SrII. Phylogenetic analyses of the 16S rRNA gene of the obtained sequences indicated that the faba bean phytoplasmas from Sudan were more closely

33 related to the phytoplasmas subgroup 16SrII-D. The sequences obtained from a three faba bean samples (SUD-Fb1, SUD-Fb3, and SUD-Fb4) were deposited in the Gen Bank data base under accession numbers JN233801, JN233803 and JN233804, respectively.

DISCUSSION

The observed symptoms were similar to the symptoms described by Ali et al., (2008), who stated that the chlorotic mottling and/or mosaic symptoms which was accompanied by black streaks on stems and leaf petioles, in some faba bean varieties, severe leaf chlorosis which subsequently developed into browning and blackening of the entire leaves , resulting in plant death was caused by anew isolate of TSV. This isolate showed close similarities to other TSV isolates described by Jones et al., (2001); Prasada et al., (2003); Arun Kumar et al., (2008) and a slightly distant similarities with TSV from cucumber in India Krishnareddy et al., (2003) and TSV Chickpea in Queensland Shepherd, (2007). The appearance of the TSV symptoms within two weeks of sawing suggest the seed transmission of the virus a finding which was also agreed with Ali et al., (2008) who stated that a highest degree of infection was detected in most of the faba bean seeds tested (73% and in some cases 90%), and moreover the seed transmission of TSV was also illustrated by a number of scientist like Fulton, (1985) and Bruntet al., (1996). Although seed transmission of TSV was not detected in naturally infected alfalfa from USA (Central Ferry - Washington) and black gram in India Kaiser et al., (1991) and Ladhalakshmi, et al., (2005).During the past few years the occurrence of TSV increased significantly. In India, a severe sunflower disease, which is the sunflower necrosis disease, was first attributed to an infection with a Tospovirus Jain et al., (2000) but later it was discovered that TSV was the causal agent of this disease Ravi et al., (2001). Following sunflower, TSV was then found to infect groundnut, okra and even cotton, among several other crops, causing serious disease problems in several regions of the Indian subcontinent Reddy et al., (2002); Bhat et al., (2002b) and Krishnareddy et al., (2003). In Sudan, the disease caused by TSV in faba bean warrants that TSV infections deserve high attention and priority since it is one of the most consumed crops. Faba beans are considered the most popular stable food in Sudan, consumed on daily bases specially among the low income people. Thus, any reduction in the yield of this crop will create a nutrition gap and consequently the price will be unaffordable for low incomepeople. As far as TSV was proven to be seed transmitted in faba bean this will indicates a real risk of disease dissemination not only to other faba bean fields but to other

35 cultivated cash crops in the country, like cotton, groundnuts and sunflower.In Sudan Phyllody disease had been sporadic during the 1950s Nour, (1962) and 1970 Hussein, (1976). In the years (1982/83) up to 20 % infection was recorded in some crops in the Gezira Jones et al., (1984). Since then, the impact of the disease thus seems to increase and losses can be considerable since infected plants do not produce seeds. In 1962Nour succeeded in transmitting the pathogen through grafting but not by sap inoculation or with the leafhopper Empoasca lybica Berg, and in 1976, 1978 Hssein described the disease on several varieties grown in the northern part of Sudan and reported that the disease incidence was highest in early sown crops. Moreover, in 1984 and 1986 additional evidence of MLO etiology was provided by O JNES et al., Dfalla Aand Cousin respectively. Recently in 2008 Saeed and Cousin studied the genetic relationship between faba bean phyllody and other phytoplasma diseases by the amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using AluI restriction endonuclease. The restriction patterns produced by faba bean phyllody phytoplasma were similar to that of Crotalaria saltiana phyllody phytoplasma which persists throughout the year in the Sudan. From these and serological results it is concluded that C. saltiana is a reservoir of faba bean phyllody phytoplasma in the Sudan. Moreover, restriction patterns also showed that phytoplasma of other diseases have the same RFLP fragment pattern as faba bean phyllody phytoplasma, including C.juncea witches' broom (Thailand) and tomato big-bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, all from France). Fragment patterns also revealed the existence of genetically diverse phytoplasma strains in the Sudan. It is suggested that faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flavescence dorée of grapevine, plum leptonecrosis of Prunus saliciana, peach yellow leaf roll, sunnhemp phyllody from Thailand and blueberry witches' broom.In this study it was found that although some of the phytoplasm symptoms described earlier by the other scientists were also noticed i.e the proliferation of the axilary buds, reduction of leaf size and virescence and phyllody ,but it was also noticed that the cup-shape appearance of leaves which was thought to be a physiological disorder(Gasim Abdalla ,personal communication) was also associated with phytoplasma infection.Although Faba bean phyllody phytoplasma (FBP) was early identifiedas 16SrII-C subgroup as descriped by Jones and Cockbain 1984; Lee

36 et al. 1998; White et al.,1998; Bertaccini and Duduk 2009 .The phytoplasma identified infecting faba bean in this study belongs to the 16SrIID subgroup. Therefore, this is the first report of a phytoplasma of these subgroup associated with faba bean plants not only in Sudan but worldwide.The 340 bp fragment of the coat protein gene (CP) from the faba bean samples that showed both yellowing and stunting was amplified with the degenerated primers S2/AS3 for Luteoviridae. The amplified fragments of one sample showed 97% nt identity with the CP of the newly described Polerovirus Pepperyellows leaf curl virus (PYLCV; Acc. No. HM439608). Nevertheless, the nucleotide sequence amplified from another sample showed 98% nt identity with the other new Polerovirus named Pepper vein yellows virus PeVYV; Acc. No. AB594828). Although these two viruses was never detected on faba beans and just recently described on pepper plants in Israel and Japan, respectively (Dombrovsky et al., 2010; Murakami et al., 2011) but the symptoms they develop either on pepper or on faba bean are more or less similar including shortening of stem internodes, inter-veinal yellowing, upward curling of the leaf blade and small, discolored fruit. With regard to the transmission of these viruses; Pepper yellow leaf curl virus (PYLCV) was transmitted by two types of aphid vectors, Aphis gossypii and Myzus persicae Antignus et al., (2001); Antignus et al.,(2003) and Dombrovsky et al., 2010) while for the Pepper vein yellows virus PeVYV only Aphis gossypii Yonaha et al.,(1995) was reported to transmit the virus , and in Sudan these two vectors were very common specially during winter time which is the growing season of faba bean and legumes in general so it is possible that they cross the virus transmission between the pepper and the faba beans which were growing in a neighboring fields in most of the Gezira farms. The two nucleotide sequences amplified from faba bean samples with the generic primers for Potyvirus were 93-95% identical with Pea seed-borne mosaic virus (Acc. No. AJ252242). This virus has been already detected in faba bean in Sudan in previous studies by Makkouk et al.,(1993) who reported that the virus was transmitted efficiently in the non-persistent manner by five aphid species, especially Myzus persicae which is one of the common aphid species that prevalent in Sudan andatypical symptoms of systemic dark and light-green leaf mottle, leaf margins become upright and the leaf

37 blade is reduced in size.Allthough the incidence and prevalence of typical symptoms of Pepper yellows leaf curl virus,Peppervein yellows virus and Pea seed-borne mosaic virus was rather sporadic compared to the highest incidence of both the TSV and phyllody but as far as there are detected and beside being transmitted by a common types of vectors which is the aphids ,they need more attention before the buildup of the disease and become a devastating epidemic.

CHAPTER FIVE

CONCLUTIONS AND RECOMMENDATIONS

6.1 Conclusions:

 The application of both serological and molecular tests, confirm that, most of the faba bean yellowing symptomswere caused by three gropes of viruses namely: luteovirus(PYLCV/ PVYV),poty virus (PSBMV), ilarvirus (TSV) and phyllody.  The phytoplasma associated with the phyllody symptoms on faba bean were found to be belong to 16srD subgrop.  TSV and phyllody were considered the major constraints to faba bean production in the Gezira area, and to a lower extent Pepper yellows leaf curl virus ,Pepper vein yellows virus and Pea seed-borne mosaic virus.  Although the incidence and prevalence of typical symptoms of Pepper yellows leaf curl virus,Pepper vein yellows virus and Pea seed-borne mosaic virus was rather sporadic compared to the highest incidence of both the TSV and phyllody but as far as there are detected and beside being transmitted by a common types of vectors which is the aphids, they need more attention before the buildup of the disease and become a devastating epidemic.  The variety Hudeiba and the line C.9/02 were considered promising and could be incorporated in breeding programs aiming at controlling most of the faba bean viral disease as they showed consistent levels of tolerance or resistance.

5.2 Recommendations:  Further studies should be done for the identification and characterization of yellow-inducing viruses and their vectors.  More lines/varieties should be screened for resistance to yellow inducing viruses.  More attention should be given to the seed borne and seed transmitted viruses like TSV.

 Special considerations should be done for the phytoplasa diseases and their rapid increase in the country not only on faba bean but in other crops and ornamentals. REFERNCES

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