GENETIC BASIS OF Striga hermonthica (Del.) BENTH RESISTANCE IN WILD SORGHUM ACCESSIONS
Dorothy Annah Mbuvi (BSc)
I56/28368/2014
A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Masters of Science (Biotechnology) in the School of Pure and Applied Science of Kenyatta University
June, 2017 ii
DECLARATION
This thesis is my original work and has not been presented for a degree or other awards in any other university.
Dorothy Annah Mbuvi
Signature…………………… Date…………………
Supervisors:
We confirm that the candidate under our supervision carried out the work reported in this thesis.
Dr. Steven Runo Department of Biochemistry and Biotechnology Kenyatta University P.O Box 43844-00100 Nairobi, Kenya
Signature……………………. Date ………………………
Dr. Mark Wamalwa Department of Biochemistry and Biotechnology Kenyatta University P.O Box 43844-00100 Nairobi, Kenya
Signature ……………………. Date……………………… iii
DEDICATION
I dedicate this thesis to my dear parents Paul and Margaret for their perseverance, prayers, comfort and encouragement accorded to me during my studies. iv
ACKNOWLEDGMENTS
I greatly appreciate my supervisors Dr. Steven Runo and Dr. Mark Wamalwa for the guidance they provided as I undertook my project. Your valuable time, ideas, productive discussions, instructive guidance and unlimited support during the research work are highly appreciated.
To my dear parents, Paul and Margaret, my brother George and my sisters Mary and Ruth, I appreciate you for realizing the academic potential in me and encouraging me to further my studies. I am proud of you and forever grateful. Dad, the financial support you gave to me throughout the study will never be forgotten. I thank you mum for your encouragement when things were not working the way I wished to.
Last but not least, to all my colleagues at the Plant Transformation Laboratory, Kenyatta University for their support during the study; Immaculate Mueni Mwangangi, Eric kuria, Joel Masanga, Sylvia Mbula Mutinda, Paul Karanja and Joshua Muli. May God bless you. v
TABLE OF CONTENTS
DECLARATION ...... ii DEDICATION ...... iii ACKNOWLEDGMENTS ...... iv TABLE OF CONTENTS ...... v LIST OF TABLES ...... viii LIST OF FIGURES ...... ix LIST OF APPENDICES ...... xi ABBREVIATION AND ACRONYMS ...... xii ABSTRACT ...... xiv
CHAPTER ONE ...... 1 INTRODUCTION ...... 1 1.1 Background of the study ...... 1 1.2 Problem Statement ...... 5 1.3 Justification ...... 6 1.4 Null hypotheses ...... 8 1.5 Objectives ...... 8 1.5.1 General objective...... 8 1.5.2 Specific objective ...... 8
CHARPTER TWO...... 9 LITERATURE REVIEW ...... 9 2.1 Origin, classification, distribution and infestation of Striga ...... 9 2.2 Striga biology...... 11 2.2.1 Striga Germination ...... 11 2.2.2 Haustorium development ...... 12 2.2.3 Establishment of Striga parasitism and completion of the life cycle ...... 13 vi
2.3 Economic importance of Striga weeds and constrains of sorghum production due to Striga infestation ...... 15 2.4 Domestication and taxonomic classification of Sorghum bicolor ...... 16 2.6 Control measures to Striga infestation in SSA ...... 19 2.7.1 Low germination stimulant production ...... 22 2.7.2 Low production of the haustorial initiation ...... 23 2.7.3 Genetic basis for Striga resistance ...... 23 2.8 Host plant cellular aspects against Striga parasitism ...... 25 2.9 Retrieving global arrays of gene expression in resistance ...... 27 2.11 Functions of pathogenesis related proteins ...... 31 2.12 Wild sorghum as source of natural resistance to Striga parasitism ...... 32
CHAPTER THREE ...... 34 MATERIALS AND METHODS ...... 34 3.1 Plant materials ...... 34 3.1.1 Study area ...... 35 3.1.2 Experimental design ...... 35 3.1.3 Germination of Striga seed ...... 35 3.1.4 Germination and growing of sorghum plants in rhizotrons ...... 36 3.1.6 Quantification of post-attachment resistance ...... 37 3.1.7 Microscopic screening of Striga resistance ...... 37 3.2.1 Identification of Striga resistance genes from RNA-sequencing ...... 38 3.2.2 Quality control and processing of RNA-Seq data ...... 38 3.2.3 Reference mapping of the reads to Sorghum bicolor genome ...... 39 3.2.4 Functional annotation ...... 40 3.2.5 Primer design...... 40 3.2.6 RNA extraction and qualityfication ...... 41 3.2.7 Complementary DNA synthesis (cDNA) and Polymerase Chain Reaction (PCR) ...... 42 vii
3.3 Statistical data analysis ...... 43
CHAPTER FOUR ...... 44 RESULTS ...... 44 4.1 Assessments of Post-attachment Striga Resistance ...... 44 4.1.1 Germination of S. hermonthica Seeds ...... 44 4.1.2 Growth of sorghum seedlings in Rhizotrons ...... 45 4.1.3 Macroscopic screening of Striga resistance ...... 45 4.1.4 Evaluation of resistance response of sorghum accession to Striga parasitism ...... 47 4.1.5 Microscopic screening for Striga resistance ...... 52 4.2.1 Gene ontology ...... 56 4.2.2 RT- PCR analysis of isolated PR genes ...... 58
CHAPTER FIVE ...... 59 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ...... 59 5.1 Discussion ...... 59 5.1.1 Evaluation of wild sorghum accessions in response to Striga infestation 59 5.1.2 Expression of pathogenesis related proteins in wild sorghum ...... 63 5.2 Conclusion ...... 67 5.3 Recommendations ...... 68 5.4 Areas of Future Research ...... 68
REFERENCES ...... 69 APPENDICES ...... 83
viii
LIST OF TABLES
Table 2.1: Sorghum production in SSA by 2016 ...... 18
Table 2.2: Recognized and proposed families of pathogenesis-related proteins . 30
Table 3.1: Sorghum accession used in the study and source countries...... 34
Table 3.2: Primer sequences used for amplification of pathogenesis related genes ...... 41
ix
LIST OF FIGURES
Figure 2.1: Striga hermonthica, Striga asiatica and Striga gesnerioides showing their variations in morphology...... 14
Figure 4.1: In vitro germinated S. hermonthica seeds 18 hours after introduction of the artificial germination stimulant (GR24)...... 44
Figure 4.2:Profile on different development stages of sorghum...... 45
Figure 4.3:Profile of wild sorghum accession infected with Striga……………………………………………………………………. 46
Figure 4.4:Profile of differential Striga hermonthica attachments in wild sorghum accessions...... 46
Figure 4.5 (a):Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica attachment on wild sorghum accessions using three ecotypes of Striga...... 49
Figure 4.5 (b):Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica biomass on wild sorghum accessions using three ecotypes of Striga...... 50
Figure 4.5 (c):Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica length on wild sorghum accessions using three ecotypes of Striga...... 51
Figure 4.6: Levels of Striga resistance in sorghum accessions based on Striga attachments.………………………………………………… ...... 52
Figure 4.7: Profile of different sorghum resistance mechanism to S.hermonthica… ...... 54 x
Figure 4.8: Venn diagrams of differentially expressed genes in WSE-1 and N13……………………...... 56
Figure 4.9: Combined Venn diagram for differentially expressed genes in both early and late stage of Striga infection for both N13 and WSE-1...... 56
Figure 4.10: Gene Ontology analysis of the 448 genes aligned to the GO database ...... 57
Figure 4.11: RT-PCR product for isolated pathogenesis related genes (PR-2) and (PR-4) on a 1% agarose gel...... 58
xi
LIST OF APPENDICES
Appendices 1: ANOVA table for S.hermonthica ecotype from Kibos...... 83
Appendices 2: ANOVA table for S.hermonthica ecotype from Mbita...... 84
Appendices 3: ANOVA table for S.hermonthica ecotype from Alupe...... 85
xii
ABBREVIATION AND ACRONYMS
AATF Africa Agricultural Technology Foundation
AM Arbuscular Mycorriza
ANOVA Analysis of Variance
ARC Agricultural Research Center
B Billion
BLAST Basic Local Alignment Search Tool
Bp Base Pairs cDNA Complementary Deoxyribonucleic Acid
DMBQ 2, 6-Dimethoxy-P-Benzoquinone
ETI Effector Triggered Immunity
EtOH Ethanol Absolute
FAO Food and Agriculture Organization
GO Gene Ontology
HGT Horizontal Gene Transfer
HIF Haustorial Initiation Factors
HR Hypersensitive Response
ICRISAT International Crops Research Institute for the Semi-Arid
Tropics
JA Jasmonic Acid
LB Luria Bertani
LGS Low Germination Stimulant xiii
LRR Leucine Rich Repeats
M Million
MAMP Microbial Associated Molecular Patterns mRNA Messenger RNA
NBS Nucleotide Binding Site
PAMPS Pathogen-associated Molecular Pattern
PCD Programmed Cell Death
PCR Polymerase Chain Reaction
PRR Pathogen Recognition Receptors
PRs Pathogenesis Related Proteins
PTI Pattern Triggered Immunity
R Resistance Genes
RPKM Reads Per Kilobase Per Million
SAR Systematic Acquired Resistance
SOAP Short Oligonucleotide Analysis Package
SSA Sub-Saharan Africa
USD United States Dollar
xiv
ABSTRACT
Striga weeds are root parasitic plants that affect production of food in sub- Saharan Africa. Striga genus includes species; Striga hermonthica, S. gesnerioides, S. aspera, S. asiatica and S. forbesii with S. hermonthica being the most widely spread in the semi-arid tropical African zones. The most effective Striga control strategy is to exploit natural resistance. Wild grasses contain a resistance mechanism that enables them to resist Striga infection resulting in low Striga quantity in feral grasslands as compared to cultivated cereals. In this study, seven wild sorghums accessions (WSE-1,WSA-1,WSA-2,WSA-3,WSD-1,WSD-2 and WSD-3) were evaluated for post-attachment Striga resistance against three common ecotypes of S. hermonthica weeds in Kenya (Alupe, Kibos and Mbita). Cultivated race N13(resistant) for post Striga germination served as control. The resistant accessions were the WSE-1, WSA-1 and WSA-2 which had lowest mean count (<30), mean length (< 0.243cm) and mean biomass (< 9.233mg) of S. hermonthica plantlets growing on their roots as compared to N13 which had mean count (<44), mean length (<0.329 cm) and mean biomass (<23.733mg) of S. hermonthica plantlets while the highly susceptible accessions were WSD-1,WSD- 2, WSD-3 and WSA-3, which had the highest mean count (> 80), mean length (> 0.651 cm) and mean biomass (> 36mg) of S. hermonthica plantlets. According to Tukey’s Honest significant Difference test, there were significant differences in the mean counts, mean biomass and mean lengths (P˂ 0.05) of Striga growing on the roots of sorghum accessions. Therefore it was established that resistance response against S. hermonthica among sorghum varied widely. Subsequent to determination of resistance response in wild sorghum accessions, RNA- sequencing was done on WSE-1 infected with Striga seeds at two different stages. This led to identification of two different pathogenesis resistance genes (PR-2 and PR-4). Gene ontology annotation revealed these to be anti-fungal agents involved in cell wall loosening and chitinase, as well as a cell wall degrading enzyme respectively. When the two genes will be transformed in cultivated sorghums, they will act in signaling induction of defensive response and also they will act in defensive mechanism, stabilizing cellular structures against further pathogen or Striga infection. Therefore, these genes will provide a platform for enhancing Striga resistance in sorghum and develop a durable, broad-spectrum resistance in cultivated sorghums.
1
CHAPTER ONE
INTRODUCTION
1.1 Background of the study
Sorghum is an economically important cereal grain which is grown as a food crop by small-scale farmers in sub-Saharan Africa (SSA) (Faostat, 2016). Sorghum serves as source of food, feed, and energy to humankind in marginal areas of
SSA, where soils are poor, rains are erratic and other crops do not do well
(Huligol, 2003; Kimber et al., 2013). The crop is highly productive, drought tolerant and has fibrous root structure that can infiltrate up to 8 feet inside the soil to obtain essential nutrients and water. The genus sorghum, originated from a diverse array of African environments hence, it shows an unlimited range of phenotypic diversity and varied resistance to biotic and abiotic stresses (Kimber et al., 2013). Production of cereal crops including sorghum in SSA, is highly constrained by parasitic weed known as Striga (Rich and Ejeta, 2008), that attaches to roots of cereal crops causing severe stunting, necrosis and sometimes cause death of crops resulting to loss of yield (Ejeta and Gressel, 2007).
The genus Striga belongs to family Orobanchaceae and comprises 30 to 35 species and over 80% of these species are found in Africa while the rest occur in
Asia (Westwood et al., 2012). Striga asiatica and S. hermonthica infect cereals including sorghum, maize, upland rice and wheat while S. gesnerioides infects tobacco and cowpea (Ejeta and Gressel, 2007). Among the Striga species S. 2
hermonthica is the most widely spread (Parker, 2009) and this is caused by increase of areas cultivated with susceptible host crops, suitable climate, mono- cropping and reduced soil fertility (Gressel, 2009). In addition to commonly known Striga species, S. forbesii and S. aspera are known to have random effects on infected cereal crops within their habitats (Westwood et al., 2012).
In SSA, the geographical spread and accumulation level of Striga weeds is gradually increasing (Ejeta and Gressel, 2007). The percentage of crop yield loss due to Striga infestations depends on amount of Striga seeds in the soil, distribution of rainfall, soil fertility and variety of cereal species grown (Ejeta,
2007a). Striga weed infests 40% of cereals producing areas resulting into an estimated loss of USD 1B per annum and affects about 100 million people livelihood’s (Labrada and Officer, 2008; Waruru, 2013). Farmers in SSA are known to lose 20-80% of plants infested with Striga while yield loss of up to
100% have been reported in Striga susceptible varieties (Atera et al., 2011). In
Kenya, a survey done in western province revealed that Striga parasitizes about
217,000 ha causing crop loss of USD 53M per annum and also revealed that fields cultivated with cereal crops are mostly infested with S. hermonthica (Woomer et al., 2009) ; a clear indication that Striga is an agriculturally and economically important pest in those areas.
3
In Africa, food security is highly affected by Striga weeds infestation and therefore control measures must be implemented to mitigate the negative effects
(Atera et al., 2012). Management of Striga weeds is difficult as each Striga flower spike produces huge number of seeds (Gethi et al., 2005) and these seeds can remain dormant for over 14 years in the soil, germinating only in response to chemical cues expressed by host roots (Bouwmeester et al., 2003). Control methods for Striga include; crop rotation, intercropping, transplanting, soil and water management, application of fertilizers, use of herbicide’s (Oxyfluorfeen, 2-
4 D and Imazapyr), hand weeding and push and pull (Cook et al., 2006). All these methods are based on reduction of Striga seeds density already in soil (Parker,
2014), but they are not effective because they are expensive, time consuming, labour intensive and cereals are overcome by emerging races and outcrossing of
Striga species (Ejeta, 2007b).
Plants do not harbor immune system like animals yet existence of pathogens in plants has enabled an intricate relationship leading to exchange of molecules among species (Benhamou and Bélanger, 1998; Benhamou and Nicole, 1999).
Based on this, plants have developed defensive mechanisms against invading pathogen while pathogens have developed strategies to overcome these defensive mechanisms expressed by plants. The time taken by plant to recognize and induce defensive mechanism against the pathogen, differentiates between success and failure of plant immunity (Tayeh et al., 2013). When plants induce defense 4
system a wide range of proteins are expressed including pathogenesis related proteins (PRs) (Van Loon et al., 2006) and they functions to protect against invasion of pathogens.
However technique such as next-generation sequencing have recently been used to understand the molecular basis of Striga parasitism in host plants (Yoder and
Scholes, 2010). These in silico techniques will enable identification of Striga resistance genes in wild sorghum accession and provide valuable data for selection of genes responsible for Striga resistance in wild sorghum accessions.
This techniques are used to study association of genes in parasite and host interactions during resistant and susceptible interactions. During incompatible interactions of Tagetes erecta and S. asiatica, 23 genes were identified whose expression were up-regulated in the roots after infection (Gowda et al., 1999). In cowpea cultivars, during infection with S. gesnerioides, it revealed that a PR-5 transcript was highly expressed in roots of the host plants, which confers resistance to S. gesnerioides as compared to susceptible genotypes (Li and Timko,
2009).
Cultivated cereals do not express complete resistance to Striga infestation, however, Striga species distribution in a natural ecosystem is usually scarce but abundant in agro-ecosystem (Raynal-roques and Pare, 1998). The wild grasses contain a resistance mechanism that enables them to resist Striga infection 5
resulting in low amount of these Striga species in wild grassland as compared to cultivated cereals (Hearne, 2009). In Africa, a study on Savanna grasses showed resistance to S. hermonthica by displaying impaired normal parasite development
(Kuiper et al., 1998). There is a possibility for wild relatives of the cultivated sorghum to provide genetic basis for resistance or tolerance to Striga infection and may be of enormous value for the development of Striga resistant crops. It is against this background that this study was designed to identify specific genes in wild sorghums that enable them to confer post-attachment resistance to Striga infestations.
1.2 Problem Statement
Striga species, commonly distributed in SSA, cause severe losses in cereal production. The parasites infestation covers approximately 40% of cereals producing areas, affecting more than 300 million livelihoods annually (Parker,
2009). Cereals are of economic value to the farmers for consumption and as animal feed (Parker, 2009). Sorghum is widely distributed in S. hermonthica prone regions of Africa hence its productivity in Africa is constrained by this root parasitic weed (Parker, 2009).
Cultivated germplasm have limited sources of resistance to Striga infection and also they are overcome by emerging races and outcrossing of Striga species 6
(Scholes and Press, 2008), however wild relatives of these cultivated cereals show potential sources of Striga resistance (Hearne, 2009). The current methods used to control Striga, which involve use of trap crops, crop rotation, use of fertilizers, herbicide hand weeding and push and pull are inefficient because they are time consuming, expensive and labour intensive (Hearne, 2009). However technique such as next-generation sequencing have recently been used to understand the molecular basis of Striga parasitism in host plants (Yoder and Scholes, 2010).
These in silico techniques will enable identification of Striga resistance genes in wild sorghum accession and provide valuable data for selection of genes responsible for Striga resistance in these accessions. Therefore, there is a need to find sources of Striga resistance from wild sorghum accession for introduction in farmers’ preferred cultivated sorghum for long-term Striga hermonthica resistance.
1.3 Justification
Development of sorghum germ-plasm that is resistant to Striga weed infestation seems to be elusive (Hearne, 2009).Currently there is no effective method for
Striga control, yet sorghum production in SSA is highly affected by Striga weeds leading to 45% in grain yield loss (Showemimo, 2010). Cultivated sorghums have limited sources of Striga resistance in additions the cultivated varieties which showed tolerance and resistance to Striga now are overcome by emerging 7
races of Striga weeds (Parker, 2009). Therefore, there is a need to find additional sources of Striga resistance for introduction in farmers’ preferred cultivated races for long-term resistance.
However, wild relatives of these cultivated sorghums shows Striga resistance in native grasslands and therefore wild sorghum provides a potential source of Striga resistance for developing host derived resistance (Gurney et al., 2002). Screening a wide range of wild relatives of sorghum and identifying key resistant genes from differentially expressed ones, between Striga susceptible cultivated varieties and Striga resistant wild sorghum via intensive transcriptomics approaches will provide valuable data to select genes responsible for Striga resistance. This will be of fundamental importance in developing durable and broad based resistance against Striga and has far-reaching implications in developing resistance in other important cereal crops in SSA.
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1.4 Null hypotheses
i. Wild sorghum accessions do not show post attachment Striga resistance
ii. Wild sorghum accessions do not provide sources for Striga resistance
genes
1.5 Objectives
1.5.1 General objective
To identify Striga resistance genes from wild sorghum accession suitable for development of durable and broad-spectrum resistance in cultivated sorghum.
1.5.2 Specific objective
i. To determine wild sorghum accessions for post attachment Striga
resistance
ii. To identify Striga resistance genes in wild sorghum accessions 9
CHARPTER TWO
LITERATURE REVIEW
2.1 Origin, classification, distribution and infestation of Striga
Striga weed is believed to have originated between Nubian hills of Sudan and
Semien Mountains of Ethiopia (Atera et al., 2011). The genus Striga comprises of obligate root hemi-parasites, which are serious pests to agriculture (Parker, 2009).
Striga belongs to Orobanchea family, which has high numbers of parasitic species (Bennett and Mathews, 2006). Among the Striga genus, 30 species have been described to parasitize grass species (poaceae) and one species, which parasitize legumes (Mohamed and Musselman, 2008). Currently, Striga spp of economic value are S. hermonthica, followed by S. asiatica, S. gesnerioides and less extent, S. forbesi and S. aspera (Parker, 2009).
Striga species are classified into two major groups, autogamous and obligate allogamy. Striga asiatica is classified as autogamous species; does not require pollinators while S. hermonthica and S. aspera are both allogamy; requires insects for pollination (Mohamed and Musselman, 2008). Genetic variation in sub population of S. hermonthica is contributed by its cross breeding nature (Berner et al., 1997). Morphologically, S. gesneriodes is different from other species of
Striga (Estep et al., 2012) in that, haustoria of S. gesnerioides has branched vascular system and lack hyaline body.
10
Striga weeds are extensively distributed all over the world however; they are generally innate in tropical and semi-arid areas of Africa (Ejeta and Gressel,
2007). Striga curviflora, S. multiflora, and S. parviflora are Striga spp native to
Australia while S. asiatica was innate in tropical parts of Africa and Asia but now days is found in Carolina in United States of America (Mohamed and Musselman,
2008). Striga gesnerioides is inborn in Asia, Africa, and Arabia but now days is found in United States (Mohamed and Musselman, 2008) while S. hermonthica dominates semi-arid areas of Northern Tropical Africa, the Democratic Republic of Congo, South West Arabia and Southern tropical Africa (Parker and Riches,
1993). In Africa, 25 countries had been reported to be infested with Striga by year
2005 (Groote et al., 2008).
Striga infests important staple crops including sorghum, maize, wheat, rice, sugarcane and cowpea which are of social and economic important to local farmers in areas affected (Atera et al., 2011). Plants infested by Striga weeds display severe symptoms characterised by chlorosis, leaf lesions, leaf desiccations, stunted growth and necrosis (Berner et al., 1997). Striga in SSA has been estimated to affect the lifestyle of 300 million people per year and economic damage of about 7B USD (Waruru et al., 2013).
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2.2 Striga biology
Parasitic plants have developed traits, which enable them to adapt to host life cycle. The critical life stage of parasitic plants includes;
1. Presence of suitable host for germination and growth of seedlings
2. Formation of structure called haustorium to enable access of water and
nutrients from host for its survival.
3. Parasite establishment and maintainace to the host plant until the parasite
seeds are set.
2.2.1 Striga Germination
Striga seeds remain viable in soil for over 14 years before they germinate (Atera et al., 2011) and only germinate in presence of suitable host; since its germination is induced by stimulant, which is produced by the suitable host roots. Striga endosperm can only sustain the growth for only 3 to 7 days after germination
(Berner et al., 1997) and therefore, Striga must develop a good relationship with host. For Striga seeds to respond to germination stimulant they undergo a period of conditioning; under higher temperatures and wet conditions, they then fall into secondary dormancy if there is no germination stimulant perceived during this period of conditioning (Cardoso et al., 2011).
12
Strigolactones, Sesquiterpene, Kinetin, Ethylene, Fungal metabolites,
Dihydrosorogoleone, Coumarin and Jasmonate are the classes of germination stimulants which have been isolated (Cardoso et al., 2011) but the best studied stimulants are strigolactones . Strigolactones is plant hormone known to prevent branching of shoots and roots and also is known to induce growth of Arbuscular mycorriza (Am) fungi in less nutrient environments and also promote its hyphal branching (Xie et al., 2010). Strigolactones were first discovered in non-host plants, cotton roots exudates (Cook et al., 1966) and are known to induce Striga seeds germination at low concentrations of 10-16 picomolars (Musselman, 1980).
Host plants that produce low amounts of strigolactones reduce the number of
Striga germination and infestation too (Umehara et al., 2010). In sorghum, low germination stimulant (lgs) is a gene, which controls secretions of germination stimulant (Satish et al., 2012). Sorghum lines with low germination stimulants are tolerant to S. hermonthica but this tolerance is not reliable in areas with higher density of Striga seeds (Atera et al., 2011).
2.2.2 Haustorium development
After germination of Striga seeds, the root radicle grows chemotropically towards the host roots. When the radicle contacts the host roots, it stops to grow and forms a haustorium. In S. asiatica root meristem re-organization is initiated by haustoria- inducing factors (HIF) within 12 hours of attachment (Spallek et al., 13
2013). The mode of action on which HIF acts as been studied by following 2, 6- dimethoxy-p-benzoquinone (DMBQ) (Chang and Lynn, 1986).In order to produce morphologically normal haustorium thigmotropic responses is required (Wolf and
Timko, 1991). Elongation of distal cells promotes penetration to the host epidermis and underlying ground tissues. It takes 48-72 hours for a parasite to completely penetrate the host roots (Hood et al., 1998). The cotyledon of Striga enlarges and breaks from seed coat within 24 hours, once xylem-to-xylem connection has been established (Hood et al., 1998).
2.2.3 Establishment of Striga parasitism and completion of the life cycle
When Striga xylem connects to the host xylem vessels, parasitism establishment occurs and the parasite shoot emerge which results in production of adventitious roots system. Secondary haustorium are formed from these adventitious roots system and are believed to be evolutionary older than primary haustoria and can infect the same or other host plants (Westwood et al., 2010). In ordinary conditions, the host plants are infested by huge numbers of Striga weeds and this parasitism makes Striga weed to become metabolic sink for photo assimilates and nutrients (Knutson, 2012).
Cultivated sorghum plants affected by S. hermonthica display reduction in photosynthetic parameters including electron transport rate through photochemical quenching and photosystem (Rodenburg and Bastiaans, 2011). 14
Also elevation of plant hormones such as abscisic acid and cytokinins levels in
sorghum plants due to parasitism by S. hermonthica have shown to have negative
effects on photosynthesis (Frost et al., 1997). Once Striga weed emerges above
the ground, it starts to photosynthesize, but its leaves are characterized by less
number of chloroplasts per cell and a degenerated palisade cell layer (Ranjan et
al., 2014). After emergence above the soil, S. hermonthica and S. asiatica flowers
after four weeks while S. gesnerioides flowers after two weeks (Berner et al.,
1995). The flower colour varies between and within species, S. hermonthica and
S. gesnerioides varies from blue and pink to white while S. asiatica varies from
yellow to red (Ejeta, 2007) (Figure 2.2).
Figure 2.1: Striga hermonthica, Striga asiatica and Striga gesnerioides showing their variations in morphology. Photo: (Schmidt et al., 2009).
Striga seed matures within four weeks after pollination in seedpods and after
bursting of seedpods, the seeds spread above the ground resulting in increases of
Striga seeds density in the soil (Berner et al., 1997). 15
2.3 Economic importance of Striga weeds and constrains of sorghum production due to Striga infestation
Striga has become the major biological constraints for farmers growing maize, sorghum, millet and rice in SSA (Parker, 2009) causing devastating losses in yield resulting in shortage in food supply in developing countries (Joel et al., 2007).
Farmers in areas infested with Striga weeds report losses of between 20 and 80%
(Atera et al., 2011) and in susceptible cultivars, grain yield losses can end up to
100% under drought conditions and higher infestation (Haussmann and Mauck,
2008). In Africa Striga causes economic loss comparable to USD 1 billion yearly
(Labrada and Officer, 2008); and this depends on Striga seed density, soil fertility, distribution of rainfall and variety of cereal species grown (Teka, 2014).
In sorghum, Striga hermonthica infestation affect post flowering stages, resulting to 45% in grain yield loss (Showemimo, 2010).
Potential yields of sorghum in SSA remains low because of factors such as poor agronomic practices, lack of commercialization of the crop which has resulted in low usage of productivity and Striga weeds problem which affects sorghum growing areas (Machado et al., 2002). Striga infection causes disruption of host plant development resulting in retarded growth, necrosis and general drought-like prone like before it appears above the ground. The severiarity of Striga infection 16
depends on several factors including host genotype, infection time and level, the nitrogen content in soil and Striga species and ecotypes (Gurney et al., 2006).
2.4 Domestication and taxonomic classification of Sorghum bicolor
It has been evidenced by anthropology that gatherers and hunters consumed sorghum in late 8000 BC (Kimber, 2000). Sorghum was first domesticated in
Ethiopia and intermediate countries around 3000 and 4000 BC. Many varieties of sorghum were generated through selection of a particular character in a sorghum population (Doggett et al., 1970). The selection was based on farmer’s choice on cultivated varieties and natural selection within wild sorghums and both selection led to improvement of wild type, intermediate types and sorghum types (Doggett et al., 1970). People movement during trading in Middle east, India and Africa led to spread of these improved sorghums in these areas around 400AD and later
1900s sorghum was introduced into America.
Sorghum genetic diversity was improved through geographical selection, disruptive selection and creation of new varieties and races based on movement of people (Dillon et al., 2007).The genus sorghum belongs to the family poaceae, tribe Andropogoneae (Clayton and Renvoize, 1986) and has been classified into five sub genus; Parasorghum, Stiposorghum, Heterosorghum, Chaetosorghum and Sorghum (Snowden, 1936). Under sub-genus sorghum, three species are known, S. propinquum (L) Kunth, S. bicolor (L) moench and S. halepense (L) 17
Pers. Cultivated sorghum (Subsp. Bicolor) is classified into five races; Caudatum,
Guinea, Kafir, Durra and Bicolor, (Harlan and De Wet, 1972) on the basis of glume shape, panicle type and grain size. Verticilliflorum, Arundinaceum,
Virgatum and Aethiopicum are four wild races of cultivated sorghum (S. bicolor) and now placed in subspecies verticilliflorum (Harlan and De Wet, 1972). In addition, crossbreeds between bicolor and wild sorghum occur and are classified as drummondii (Paterson and Tang, 2012).
2.5 Production and economic importance of sorghum in SSA
Sorghum is a major source of food in marginal areas of SSA where other cereals do not thrive (FAO, 2011). As human feed, sorghum has many uses and includes products such as pilau, ugali, ginger biscuits, bread, porridge and beverage
(Mohamed et al., 2001). In brewing industries, when sorghum is compared to malted barley, it provides extract at less cost and also is available in abundance
(Ogbeide, 2011). In bio-industries sorghum is used in production of products such bio plastics and also in production of ethanol (Massoud and El-Razek, 2011). By this year 2017, the United States Department of Agriculture (USDA) estimates that the world sorghum production to be 62.5 million metric tonnes in world.
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Table 2.1: Sorghum production in SSA by 2016
Country Production (Tonnes) Nigeria 7 081000 Sudan 4 470000 Ethiopia 1 538000 Burkina Faso 1 372000 Egypt 862000 Tanzania 736000 Niger 656000 Chad 497000 Cameroon 450000 Uganda 423000 Mozambique 314000 Ghana 280000 Rwanda 175000 Benin 165000 Senegal 147000 Kenya 133000 Zimbabwe 103000 Somalia 100000
Source: Food and Agriculture Organization of the United Nations. FAOSTAT 2016. http://faostat3.fao.org.
Sorghum is typically grown in United States of America, South America, Asia and Africa, based on production sorghum remains the filth major crop and second most important after maize, rice, wheat and barley (FAO, 2000). Sorghum production in Africa has steadily increased in the past 40 years from 10 to 26 million per tonnes (FAO, 2000). Production of sorghum in SSA is highly influenced by abiotic stresses (heat, low soil fertility and lack of rainfall) and biotic (weeds, insects and diseases) (Wortmann et al., 2007). Low soil fertility and lack of rainfall shortages accounted for approximately 1.8M loss per year, 19
while loss of sorghum due to insects such as stalk borer was estimated to be more than 1.1M per year (Faostat, 2013). Important weeds such as Striga is one of the most important constraint in sorghum production in Kenya, and the second most important in Ethiopia and Uganda (Faostat, 2013). Quelea species, other bird species, shoofly (Kamala et al., 2009), and phosphorus deficiency each caused more than 0.5M loss per year in production. Of great interest, researchers have identified different landraces and wild varieties of sorghum, which have resistance mechanisms against these abiotic and biotic stress (Faostat, 2013).
2.6 Control measures to Striga infestation in SSA
Parasitic plants are difficult to control because by the time they emerge above the ground, they have done greater damage to crops (Ransom et al., 2012). In addition, the parasite produces huge numbers of seeds of about 20000-600000
(Yoder and Musselman, 2006). To manage Striga, small-scale farmers have engaged in different control approaches with the aim of reducing the quantity of
Striga seed in the soil.
The recent control approaches for Striga comprises of cultural and mechanical practices, planting resistant crops, biological control practices, use of nitrogen fertilizers and chemicals/herbicides (Jamil et al., 2011). Cultural practices recommended for Striga control include; crop rotation, intercropping, 20
transplanting, soil and water management, application of fertilizers and hand weeding (Ransom et al., 2012). These methods are based on reduction of Striga seeds density already in soil (Reda and Verkleij, 2007). These practices are of less success for small-scale farmers due to financial and socio-economic limits.
However, planting resistant crops is much preferred because they are friendly to environment, easy to handle, affordable and cheaper to local farmers in SSA
(Yoder and Scholes, 2010).
2.7 Mechanisms of plants resistance to pathogen
The most widely accepted model on how host plants respond to parasitic weeds is
―zigzag‖ model (Jones and Dangl, 2006). This model suggests that plants respond to parasitic plants through two-levels of innate immune response system. The first level responds to slowly evolving molecules referred as microbe associated molecular patterns (MAMPS) or pathogen associated molecular pattern (PAMPS)
(Boller and He, 2009). The host defense response known as pathogen triggered immunity (PTI) is activated when host sensor referred as pattern recognition receptors, recognize MAMPS and PAMPS (Boller and Felix, 2009).
Pathogenic plants have evolved effector and virulence factors that enable them to invade host defense mechanisms by suppressing PTI. These effectors and 21
virulence factors enter the host cell via discrete secretion machinery (Martin,
2012). In attempt to remain safe from these effectors, plants have developed a second level of defense mechanism known as effector-triggered immunity (ETI) that enables them to disable such effectors. Effector-triggered immunity involves second class of receptor proteins containing nucleotide‐binding site (NBS) and
Leucine‐rich repeat (LRR) domain (Prihatna, 2009). NBS_LRR proteins commonly referred as resistance proteins (R) are encode by plant to protect against pathogens.
When plants are infected with biotic (weeds, insects and pathogens and abiotic
(heat, low soil fertility and lack of rainfall) inducers results to induction resistant proteins which result to expression of systemic resistance at the point of infection and the phenomenon is referred as systematic acquired resistance (SAR). Major role of SAR is systemic and rapid accumulation of pathogenesis related (PR) proteins. In sorghum Striga parasitism leads to induction of two path ways, salicylic acid (SA) responsive genes and jasmonic acid (JA) responsive genes
(Hiraoka and Sugimoto, 2008). Bio-trophic pathogens are known to induce activation of SA pathway which is responsible for initiation of hypersensitive response, production of pathogenesis related proteins and production of phytoalexin all which are involved in protection against further infection with pathogen (Hiraoka and Sugimoto, 2008). Jasmonic acid (JA) is always activated 22
in response to neurotropic pathogens. Treatment of sorghum cultivars with SA led to inhibition of Striga development in those sorghum roots and this led to conclusion that SA responsive genes are involved in host resistance against
Striga (Hiraoka and Sugimoto, 2008).
2.7.1 Low germination stimulant production
Sorghum genotypes with low germination stimulant (LGS) produce low amounts of the exudates essential for stimulation of germination of pre-conditioned Striga seeds (Hauck et al., 1992). These genotypes producing insufficient germination stimulants are known to resist to Striga infestation. Sorghum genotypes susceptible to Striga are known to produce high amounts of germination stimulants although not all sorghum genotypes which are resistant to Striga do produce low germination stimulants (Ejeta and Gressel, 2007).
There are several classes of chemical signals for Striga seed germination, but the most common and important one is strigolactones (Hauck et al., 1992). In sorghum low germination stimulant (LGS) trait which is known to have high addictive gene action is transferred to off-springs as a single, nuclear, recessive gene. Sorghum cultivars with this mechanism are Framida, SRN 39, 555, SAR lines, IS 15401 and IS 9830 (Haussmann and Mauck, 2008). 23
2.7.2 Low production of the haustorial initiation
Germinated Striga seeds near the roots of sorghum genotypes with low production of the haustorial initiation factor (HIF) do not form haustoria and therefore die from their inability to attach to their potential host (Gurney et al.,
2006). Signals for Striga seeds germination have been identified unlike signals for haustoria induction which have not yet been identified, but a large number of phenolic compounds have shown to function as haustoria initiators in Striga. A simple Quinone, 2, 6 dimethoxy1, 4-benzo quinine (DMBQ), though not present in host root exudates has been shown to act as a strong haustorial initiating factor
(Lenny et al., 1997). Extended agar gel assay (EAGA) is used to study low production of haustorial initiation factors by distinguishing host genotypes qualitatively on their ability to induce haustorial formation.
2.7.3 Genetic basis for Striga resistance
Genes for Striga resistance in wild relatives of many agronomically important cultivated cereals including sorghum, maize and rice have been studied using F1,
F2 and progressive population generated by crossing susceptible and resistant phenotypes (Hess, et al., 2004). In these wild relatives, resistance to Striga spp appears polygenic involving both major and minor genes with large genotypes depending on environment (Scholes and Press, 2008). The genetic basis of 24
resistance present in these grasses is weak and breaks down under a new geographic or when infested by specialized forms of the parasite.
Many wild sorghum varieties exhibit partial post-attachment Striga resistance when compared to cultivated ones (Gurney et al., 2006). Resistant phenotypes associated with low stimulant production are controlled by single nuclear recessive gene (LGS) with highly additive gene action (Reddy et al., 2006).
Inheritance of trait responsible for production of low haustorial initiation factor is governed by dominant allele of single gene (Ejeta and Butler, 1993).Riedel et al.,
(2006) described that sorghum populations that show resistance to S. hermonthica were derived from SRN-39 (a resistant line). The resistance in this line is controlled by single nuclear recessive gene. In wild sorghum Striga resistance is controlled by major recessive and some minor genes (Haussmann and Mauck,
2008). When wild sorghum species, arundinaceum which exhibited hypersensitive response (HR) was crossed with two cultivated varieties, it led to discovery that, the resistance trait is controlled by two nuclear genes HR1 and HR2; all in genetic linkage map being associated with different markers (Mohamed et al., 2003) .
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2.8 Host plant cellular aspects against Striga parasitism
There are two essential stages for Striga survival during host parasitism; successful breaching of host root system and subsequent formation of vascular connection (Timko et al., 2012). During these developmental stages, the host can mount resistance response that can block parasite development (Timko et al.,
2012). Three broad categories of resistance response against parasitism have been revealed using histological analysis (Hood et al., 1998). Susceptible Striga parasitism is observed when the host plant fails to respond to the penetrating haustorial peg or inability to activate cascade responsible for turning on defense response (Gurney et al., 2006). On the other hand, resistance response can either delay or be immediate during various developmental stages of parasite (Timko et al., 2012).
Several mechanisms are known involved for inducing immediate resistance response against Striga parasitism (Spallek et al.,2013). After parasitic attachment
(within 24-72 hours), phenolic compounds accumulate at the interface of parasite- host and this has been reported in S. hermonthica and sorghum (Jamison-
McClung, 2003). The accumulation of these phenolic compounds leads to build up of physical barriers including; thickening of cell walls as result of lignin, cellulose or suberin deposition at attachment site of parasite to host root (Lane et al., 1997). Hypersensitive response (HR) characterized by browning and localized 26
host cell death has been observed in cowpea cultivars resistance to S. gesnerioides
(Lane et al., 1997) also in accessions of wild sorghum and some sorghum cultivars against S. asiatica parasitism (Mohamed et al., 2003).
Host root endodermis has been reported to confer resistance response in many cereals including sorghum against S. asiatica parasitism (Haussmann and Mauck,
2008) and rice against S. hermonthica parasitism (Gurney et al., 2006). After successful parasitism, resistant response can also occur, taking a number of forms.
In one form, host vessels can be sealed by gel or gum-like substance in order to block the flow of nutrients and water to parasite from the host plant. In another form, the parasitized plant can transfer toxic compounds to the parasite through its haustorium (Scholes and Press, 2008).
The observed resistance mechanisms against parasitic plants can broadly be categorized into post-attachment resistance and pre-attachment (Rodenburg and
Bastiaans, 2011). The pre-attachment resistance mechanisms comprises all mechanisms that enable potential host to evade parasitism; inhibition of germination of Striga seeds, reduced production germination stimulant, haustorium formation inhibition, haustorium development inhibition and thickening in cell walls of host roots preventing the haustorium from penetrating host roots (Rodenburg and Bastiaans, 2011). Parasite will try to breach the host root vascular system once the haustorium forms. During these parasite 27
developmental stages, host could induce different post-attachment resistance mechanisms including; cell wall lignification, programmed cell death (PCD) as result of necrosis at the location of parasite attachment to host root and abiosis release cytotoxic compounds within infected host cells (Rodenburg and Bastiaans,
2011).
2.9 Retrieving global arrays of gene expression in resistance
Techniques such as next-generation have recently been used to understand the molecular basis of Striga parasitism (Yoder and Scholes, 2010). These techniques are used to study association of genes in parasite and host interactions during resistant and susceptible interactions. During incompatible interactions of Tagetes erecta and S. asiatica, 23 genes were identified whose expression were up- regulated in the roots after infection (Gowda et al., 1999). The up regulated genes included disease resistance proteins which have been isolated in some plants. In cowpea cultivars, during infection with S. gesnerioides, it revealed that a PR-5 transcript was highly expressed in roots of the host plants, which confers resistance to S. gesnerioides as compared to susceptible genotypes (Li and Timko,
2009).
In sorghum in response to S. hermonthica infections, 30 genes have been identified to be up regulated as result of S. hermonthica infection (Hiraoka and 28
Sugimoto, 2008). A highly susceptible sorghum variety to Striga has been found to activate jasmonic acid (JA) and suppress sacylic acid (SA) which are responsive genes for resistance, revealing that susceptible cultivars identify Striga parasitism as wounding stress rather than microbial stress (Timko et al., 2012). In contrast, less susceptible sorghum recognizes Striga parasitism as both microbial and wound stress and induces both jasmonic acid and salicylic acid genes respectively. Sacylic acid pathway is frequently triggered against bio trophic attacks, fungal pathogens resulting to production of pathogenesis related (PR) proteins where else JA pathway triggered against neurotropic viruses and insects
(Swarbrick et al., 2008).
Microarray technology has been used in identification of genes expression patterns during Nipponbare infection against S. hermonthica parasitism
(Swarbrick et al., 2008). Pathogenesis related proteins including Thaumatin-like protein (PR-5), Glucanases (PR-2) and endochtinases (PR-3) are among genes which are up-regulated during Striga resistant rice (Nipponbare) interaction
(Swarbrick et al., 2008). Pathogenesis related genes are produced by plants in responses to microbial or pathogen attack and are induced systematically.
Pathogenesis related proteins are associated with development of systematic acquired resistance (SAR) to protect against further infection by virus, bacteria 29
and fungi by accumulating around infected tissues (Van Ast and Bastiaans,
2006).
2.10 Classification of pathogenesis related proteins (PRs)
Pathogenesis related proteins have been classified into different families based on the biological activity of the induced proteins, isoelectric points of protein, the shared sequence homology parameter and based on the migration in the native
PAGE (Van Loon and Van Strien, 1999). Pathogenesis related proteins have chitin-binding domain, which is mostly D helical and the catalytic domain contains two glutamates (Hong and Hwang, 2002).For a protein to be classified under new family of pathogenesis related proteins, it has to fall under certain criteria. First, the protein is expressed in tissues, that under normal condition do not express it. Secondly, the expression of induced protein must occur in at least into two different kinds of plants following pathogen interaction (Van Loon and
Van Strien, 1999).
In tomato and tobacco, eleven families of PRs were isolated (PR1-PR11) and classified as shown in table 2.1. In cucumber and parsley PR-8 and PR-10 were isolated respectively (Van Loon et al., 1994). Later PR-12 was isolated from
Arabidopsis, PR-13 isolated from radish, PR-14 were isolated in barley (Van
Loon and Van Strien, 1999) and PR-16, PR-15 were isolated from hot pepper 30
against viral attack (Parker, 2009). Striga parasitism’s induces JA-responsive genes and suppress SA-responsive genes in susceptible sorghum cultivars
(Hiraoka and Sugimoto, 2008). Thirty genes were up regulated in sorghum in resistance to S. hermonthica, among them pathogenesis related protein were identified including hypersensitive-related HSR 203J and chitin-inducible gibberellin (Hiraoka and Sugimoto, 2008).
Table 2.2: Recognized and proposed families of pathogenesis-related proteins
Family Type member Properties PR-1 Tobacco PR-1a Unknown PR-2 Tobacco PR-2 β-1,3-glucanase PR-3 Tobacco P,Q Chitinase type I,II,IV,V,VI,VII PR-4 Tobacco ―R‖ chitinase type I, II PR-5 Tobacco S Thaumatin-like PR-6 Tomato Inhibitor I proteinase-inhibitor PR-7 Tomato Endoproteinase PR-8 Cucumber chitinase chitinase type III PR-9 Tobacco ―lignin-forming Peroxidase peroxidase‖ PR-10 Parsley ―PR1‖ ―ribonuclease-like‖ PR-11 Tobacco class V chitinase Chitinase type 1 PR-12 RadishRs-AFP3 Defensin PR-13 Arabidopsis THI2.1 Thionin PR-14 Barley LTP4 Lipid-transfer protein PR-15 Barley, rice, corn, oat and rye Oxalate-oxidase and PR-16 PR-17 wheat Aminopeptidase’s Source: (Van Loon et al., 2006)
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2.11 Functions of pathogenesis related proteins
Pathogenesis related genes provides defensive function in plants, against pathogens and they are apparently validated by their expression in plant organs, seeds and also in their accumulation and their release into fungal structures invading plant cell (Abad et al., 1996). Identification of chitinase and β-1,3
Glucanases activity in PR-2 and PR-3 in tobacco challenged the assumption that
PRs are devoid of enzymatic activity (Van Loon et al., 2006).Antifungal activity is an important function in all pathogenesis related proteins (PRs) although others display nematicidal, antiviral, insecticidal and antibacterial activity (Van Loon and Van Strien, 1999). Proteinase-inhibitory and membrane-permeabilizing and hydrolytic activity of PRs explains their toxicity activity.
Thus β-1,3 Glucanases, Chitinase and Proteinase referred as hydrolytic enzymes, functions as tools for fading and decomposing fungal structures while PR-8 due to its lysozyme activity can disrupt the cell wall of gram negative bacteria (Van
Loon et al., 2006). Proteinase inhibitory protein, PR-6, exhibit anti-insecticidal and anti-nematicidal effects inactivating the proteins secreted by invading parasite to plant tissues. Pathogenesis related protein-5, -12, -13 and -14 due to their plasma membrane-permeabilizing activity, hinders growth and development of attacking pathogens by plasmolysis (Abad et al., 1996). Activity of PR-9 32
peroxidase contributes in strengthening and rigidification of plant cell walls as a result of pathogen attack (Lagrimini and Rothstein, 1987).
2.12 Wild sorghum as source of natural resistance to Striga parasitism
The only cereal cultivar which show partial resistance against Striga parasitism is sorghum (Arnaud et al., 1999). Host plants develops resistance mechanism against Striga parasitism only after a long period of parasitism. Striga species also occur in natural ecosystem such as savanna lands (Musselman, 1987) but the infestation is low with less dense populated in natural ecosystem as compared to agro-ecosystem (Cochrane and Press, 1997).This less dense infestation level of
Striga spp in natural grasslands may be due to resistance mechanisms in wild grasses to Striga which is not present in cultivated cereals (Timko et al., 2012).
Sorghum arundinaceum demonstrated resistance to Striga infestation when infected with S. aspera (Kuiper et al., 1998) while Sorghum drummondii demonstrated resistance when infested with S. asiatica (Ejeta, 2007). The resistance may be as result of unsuccessful attachment or early death of parasite as well parasite failing to penetrate the host endodermis to access water and minerals.
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Domestication inevitably leads to decreased genetic diversity in the selected crops
(Doebley et al., 2006). In some organism, the lost genetic diversity may represent the organism’s capacity to adopt to change in a dynamic ecosystem including pathogen resistance (Sakai and Itoh, 2010). Based on this assertion, most crop improvement programs are now utilizing genomics and molecular genetic technologies to reclaim lost genetic diversity specifically targeting genes responsible for pathogen resistance (Jones et al., 2014). Success in such programs hinges on the premise that wild relatives of crops can have valuable disease resistance (Brozynska et al.,2015). 34
CHAPTER THREE
MATERIALS AND METHODS
3.1 Plant materials
Striga hermonthica seeds were obtained from three different Striga infested sorghum-growing areas of Kibos, Mbita and Alupe located in Western Kenya.
Seven wild sorghum accessions used in this study were collected from fields in
Sudan and after identification through taxonomy were stored in agricultural research corporation (ARC) research institution and one cultivated sorghum,
N13 (resistant) used as control was collected from Kenya, as indicated in table
3.1.
Table 3.1: Sorghum accession used in the study and source countries
Sorghum races Class Accessions Source Aethiopicum Wild sorghum WSE-1 ARC in Sudan Arundinaceum Wild sorghum WSA-1,WSA-2 ARC in and WSA-3 Sudan Drummondii Sorghum×drummondii WSD-1,WSD-2 ARC in (Sudangrass) and WSD-3 Sudan N13 Cultivated ICRISAT in Kenya
ICRISAT- International Crops Research Institute for Semi-Arid Tropics, ARC- Agricultural Research Corporation WSD-wild sorghum Drumondii, WSE-wild sorghum Aethiopicum and WSA-wild sorghum Arundinaceum.
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3.1.1 Study area
The study was carried out at Plant Transformation Laboratory (PTL) located in Kenyatta University, Kenya.
3.1.2 Experimental design
Randomized complete design, using five sorghum plants per accession were used for screening for post-attachment Striga resistance and experiment repeated three times in all S. hermonthica ecotypes used (Kibos, Mbita and
Alupe).
3.1.3 Germination of Striga seed
Striga seeds were surface sterilized on sterile bench, using 10% (v/v) sodium hypochlorite for 10 min followed by thoroughly rinsing using 200ml of sterilized water. Approximately 0.25g of Striga seeds were spread on filter paper, placed in petri plates and wetted with 5ml of sterile water. The plates were sealed and wrapped with parafilm and aluminum foil respectively and then incubated at 30°C for 11 days. Prior to infection, pre-conditioned Striga seeds were treated with 5ml of 0.1-ppm solution of artificial germination stimulant,
GR-24 and germination frequency determined under stereomicroscope.
Standard germination frequency of above 70% was required for each Striga ecotype before infection.
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3.1.4 Germination and growing of sorghum plants in rhizotrons
Sorghums seeds were germinated between blocks of wet cotton wool lined with filter paper, day and night temperatures were set at 24 and 28°C, respectively, and the relative humidity was set at 60%. After eight days of germination, single sorghum plantlets were transferred onto 25cm x 25cm x 5cm, rhizotron filled with vermiculite and separated with white linen cloth to allow for ease monitoring of sorghum roots without destruction with top and bottom openings
(Gurney et al., 2006). The top opening allowed growth of sorghum shoot and bottom opening allowed drainage of nutrients. The rhizotrons were wrapped with aluminum foil to prevent light from reaching sorghum roots and supplied with 100ml of 40% Long Ashton nutrient solutions containing 2mM ammonium nitrate twice a day (Hudson, 1967). Plants were then grown in the greenhouse at
28°C and 60% relative humidity.
3.1.5 Infection of sorghum with Striga hermonthica seeds
Sorghum roots were infected with 0.25g of pre-germinated S. hermonthica seeds after 11 days of transfer to rhizotrons, by aligning along the roots using a fine paintbrush (Gurney et al., 2006). After infection with pre-germinated Striga seeds, rhizotrons were wrapped with foil and then returned to the green house and then supplied with 100ml of 40% Long Ashton nutrient solutions containing
2mM ammonium nitrate twice a day (Hudson, 1967).
37
3.1.6 Quantification of post-attachment resistance
Striga plantlets from each rhizotrons were harvested after 21 days of infection from sorghum roots, then placed in petri dishes and photographed using a
Canon EOS 300D digital camera. The number and length of S. hermonthica plants from each sorghum plant were determined from the petri plate photographs using Image analysis software (Image J, Media Cybernetics)
(http://rsb.info.nih.gov/ij/). To determine the amount of dry biomass per host sorghum, Striga plantlets were dried at 26°C for four days in an incubator and weighed using analytical weighing balance.
3.1.7 Microscopic screening of Striga resistance
In order to identify the extent of parasite penetration into host roots, small sections of Striga-sorghum root attachments were dissected from sorghum roots at third and ninth day after infection for sectioning (Cissoko et al., 2011). The collected tissues were first fixed using Carnoys fixative (made up of 60 ml of
Ethanol, 30 ml of chloroform and 10 ml of glacial acetic acid), then pre- infiltrated in ethanol and Technovit solution 7100 (Haraeus Kulzer GmbH,
Germany) in ratio of 1:1 for 15 minutes and then infiltrated into fresh 100%
Technovit solution overnight. The tissues were placed into eppendorf lids for embedding using Technovit 7100 kit according to manufacturer’s instructions
(Haraeus Kulzer GmbH, Germany). To ensure complete drying of the upper part of resin, the lids were covered with foil and incubated at 37°C for 10 min. The 38
tissues were mounted onto histoblocs using Technovit 3040 mounting medium kit according to manufacturer’s instructions (Haraeus Kulzer GmbH, Germany).
Tissues were sectioned using a Leica RM 2145 microtome and small sections of 5µm were transferred using a fine needle onto microscope slides. Sections were then stained with 0.1% toluidine blue O (BHD Poole, UK) in 100mM phosphate buffer (pH7) for 2 min, rinsed with sterile water, dried at 65°C for 30 min then mounted with DePex (BDH, Poole, UK) observed at 100X magnification (viewing power at 10X and power of the objective at 10X) and photographed using the Olympus BX51 microscope.
3.2.1 Identification of Striga resistance genes from RNA-sequencing
RNA was extracted from WSE-1 and N13 infected with S. hermonthica seeds at two differential stages (3 and 9 days after infection) in three biological replicates and sequenced using Illumina HiSeq 2000 platform (Runo unpublished).
3.2.2 Quality control and processing of RNA-Seq data
FASTQC v0.11.2 tool was used to provide quality control checks and normalization of High-throughput mRNA sequencing (RNA-seq). Specifically, the 5’-ends of the reads were trimmed so that the abundance of each base
(A,C,T,G) per position was within two standard deviations of the average across 39
all cycles. All bases with a paired quality score of less than 20 were trimmed off at the 3’-ends. Reads that were shorter than 100 bases or had a median quality score below 20 were excluded.
3.2.3 Reference mapping of the reads to Sorghum bicolor genome
One billion clean reads of 150bp of RNA-sequence (RNA-seq) were mapped to the reference Sorghum bicolor genome (accession number:
NZ_ABXC00000000) using Bowtie2 which is in-built in TopHat
(http://tophat.cbcb.umd.edu/) to ensure strand-specific processing of the reads for each condition. TopHat attaches meta-data to each alignment, which
Cufflinks and Cuffdiff software (http://cufflinks.cbcb.umd.edu/) can use for accurate assembly and quantification respectively. The mapped reads were provided to Cufflinks to generate a transcriptome assembly for each race and condition and quantified as reads per kilobase per million (RPKM) using
Cuffdiff. These transcriptome assemblies were then merged together using the
Cuffmerge (http://cufflinks.cbcb.umd.edu/), which provides uniform basis for calculating gene and transcripts expression in each condition. Differentially expressed transcripts and genes were identified using DESeq2 package (Love et al., 2014).
40
3.2.4 Functional annotation
Differentially expressed transcripts/genes (Bonferroni correction P<0.01) were compared https://phytozome.jgi.doe.gov/pz/portal.html S.bicolor and gene ontology (GO) databases for functional annotation of the encoded proteins. A
BLAST search was carried on the differentially expressed genes on panther software (https://www.pantherdb.org/), which classified the differentially expressed genes in wild sorghum based on their functions.
3.2.5 Primer design
The gene sequences for the two known pathogenesis related proteins
(Sobic.004G323300.1 and Sobic.005G169200.1) were downloaded from the https://phytozome.jgi.doe.gov/pz/portal.html S. bicolor database and used for designing primers (Table 3.2). These primers were then used to amplify PR genes from cDNA synthesized from WSE-1 roots that were infected with Striga seeds.
41
Table 3.2: Primer sequences used for amplification of pathogenesis related genes Gene Primer ID Primer sequence Temp Fragment size
Sobic.004G323300.1 Sobic 3300 R 5’-TACCTCCCGTGGAGT 55.3°C 450bp TCTTC-3’ Sobic 3300 F 5’-ATGGAGGGCACCTCA 55.6°C AGAAG-3’ Sobic.005G169200.1 Sobic 9200 R 5’TACCCCCCTACTGTCG 56.4°C 456bp -3’ Sobic 9200 F 5’ATGGGGGGATGACAG 55.6°C C-3’
Temp – annealing temperature
3.2.6 RNA extraction and qualityfication
Total RNA was extracted from WSE-1 roots infected with pre-germinated S.
hermonthica seeds using RNeasy plant mini kit according to the manufactures
instructions (Qiagen, Valencia, Spain). The quality of sorghum-Striga total
RNA was determined by running it on a 1% (w/v) agarose gel. Five microliters
of extracted genomic RNA was mixed with 1 µl of loading dye (New England
Bio Labs Company, England) and 1 µl of SYBR(R) green (New England Bio
Labs Company, England). RNA was electrophoresed alongside 5 µl of 1 kb
ladder (Gene Ruler 1kb DNA Ladder, Thermo Scientific®). The gel was run at
80 volts for 45 min and then visualized using an ultra-violet trans-illuminator
(Bioline USA Inc. USA).
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3.2.7 Complementary DNA synthesis (cDNA) and Polymerase Chain Reaction (PCR)
The quantified RNA was converted into complementary DNA (cDNA) using
Tetro cDNA synthesis kit according to manufacturer’s instruction (Bioline,
USA Inc. USA). Reaction mixture of 20 µl was added into sterile RNase free tube on ice containing 4 µl of 5×RT Buffer, 1mm of primer: Oligo (dT)18, 1 µl of 10Mm dNTP mix, 1 µl of Ribosafe inhibitor, 1 µl of Tetro reverse transcriptase, 5 µg of template RNA and top up with DEPC-treated water.
PCR was performed in a 20 µl final reaction mixture containing 10 µl of 5×
MyTaq reaction Buffer containing 5mM dNTPs, 15mM MgCl2, stabilizers and enhancers, 1 µl of each of forward and reverse primer, 0.125 µl of MyTaq DNA polymerase (New England Bio Labs Company, England), 2 µl of cDNA and top of with 12.875 µl of water. The PCR amplification reaction was performed in an
Eppendorf master cycler (Hamburg, German) under the following parameters; for 450bp,thermo cycle were, initial denaturation at 94°C for 3 min, 40 cycles comprising of denaturation at 94°C for 1 min, annealing 56°C for 1 min and extension of 72°C for 1 min and final elongation time of 10 min at 72°C. For
456bp initial denaturation at 94°C for 3 min, 40 cycles comprising of denaturation at 94°C for 1 min, annealing at 59°C for 1 min and extension of
72°C for 1 min and final elongation time of 10 min at 72°C.
43
PCR product were visualized on 1 % (w/v) agarose gel. Five microliters of
PCR product was mixed with 1 µl of loading dye (New England Bio Labs
Company USA) (NEB) and 1 µl of SYBR(R) green (LTC). The product was electrophoresed alongside 5 µl of 1 kb ladder (Bioline USA Inc. USA). The gel was run at 80 volts for 45 min and then visualized using an ultra-violet trans illuminator (Bio view) and photographed.
3.3 Statistical data analysis
The statistical package SAS (version 9.1) was used for statistical analysis of
Striga post-attachment resistance data among sorghum accession. Analysis of variance (ANOVA) was performed to compare the means of length, biomass and number of infecting Striga and to further fit a factorial analysis of variance for each replicate across the eight races. In addition, Tukey’s honest significant difference (HSD) test was performed to calculate mean separation. All values ≤
0.05 were considered statistically significant. This data was presented as relative mean ± SE of biomass, length and number of infecting Striga among the sorghum races in form of graph pad prism version 6
(http://www.graphpad.com). In addition, number of infecting Striga from the nine sorghum races against three Striga ecotypes was clustered and significant value (p-value ≤ 0.05) were visualized as heat map using a custom hierarchical clustering R script.
44
CHAPTER FOUR
RESULTS
4.1 Assessments of Post-attachment Striga Resistance
4.1.1 Germination of S. hermonthica Seeds
The pre-conditioned Striga seeds germinated 18 hours after introduction of artificial germination stimulant (GR24) and were considered to have germinated after production of protruding radicle at the seed coats (Figure 4.1). The number of Striga seeds germinated ranged between 70 and 80% across all the three
Striga ecotypes used in the study.
Figure 4.1 In vitro germinated S. hermonthica seeds 18 hours after introduction of the artificial germination stimulant (GR24).
45
4.1.2 Growth of sorghum seedlings in Rhizotrons
Sorghum seeds germinated four days after sowing and were ready for transfer into rhizotrons at eight days after germination (Figure 4.2a). After transfer to the rhizotrons, 11 days sorghum plants developed enough roots ready for infection with the pre-germinated Striga seeds (Figure 4.2 b).
a b
Figure 4.2 Profile on different development stages of sorghum.(a) Sorghum seedlings after eight days of germination (b) sorghum roots 11 days after transfer to rhizotrons.
4.1.3 Macroscopic screening of Striga resistance
Striga seeds attached to sorghum roots by the third day after infection. After interaction, Striga attachment was characterized by swelling of the Striga radicle at the point of attachment (Figure 4.3a). The visible resistance response was as a result of intense necrosis at the site of Striga attachment to the host root resulting to the death of Striga after third to sixth date after infection
(Figure 4.3b). As a result of successful Striga parasitism, Striga shoot emerged 46
as shown in figure 4.3c. Successful Striga parasitism on sorghum roots was characterized by multiple fast growing Striga attachments. On the other hand resistance to parasitism on host roots was characterized by few attachments and
small Striga plants (Figure 4.4).
a b c
Figure 4.3: Profile of wild sorghum accession infected with Striga.(a) Successful attachment of Striga in WSD-1 susceptible accession (b) resistance response in WSE-1 resistant accession with hypersensitive response (HR) (c) a well-developed germinating Striga plant in WSD-1 susceptible accession.
Figure 4.4: Profile of differential Striga hermonthica attachments in wild sorghum accessions: WSD-1, Ochuti, WSA-1, N13, and WSE-1 21 days following infection of host roots with Striga seeds collected from Kibos. Red arrows indicate attachment points to the host by the parasite. Susceptible accessions represented here by WSD-1 and Ochuti showed a high number of Striga attachments compared to the control N13. Resistant accessions representedhere by WSA-1 and WSE-1 showed few Striga attachments. 47
4.1.4 Evaluation of resistance response of sorghum accession to Striga parasitism
Based on the resistance parameters used in this study, there was a significant difference at p< 0.05 in resistance among wild sorghum accessions in reference to N13 (control) (Figure 4.5). WSE-1, WSA-1 and WSA-2 were the highly resistant accession, because their resistance was higher compared to N13 with the lowest number of Striga attachment (Figure 4.5 a), least Striga mean length
(Figure 4.5 b) and the lowest Striga biomass (Figure 4.5c). WSD-1 emerged as the most susceptible wild sorghum accession with the highest Striga attachments (100.6±7.36) (Figure 4.5a), highest Striga mean length
(0.651cm±0.05) (Figure 4.5b) and the highest Striga biomass (5.5mg±0.18)
(Figure 4.5c) across all the Striga ecotypes. There was no significance difference in length and the number of Striga attachments among the three most resistant wild sorghum accession across three Striga ecotypes but there was significant difference observed in Striga biomass when the wild sorghum accession were infected with Alupe ecotype (Figure 4.5).
All the drummondii sorghums accession namely WSD-2, WSD-3 and WSD-1 had significantly lower resistance than N13 based on all resistance parameters across the Striga ecotypes (Figure 4.5). Between WSD-2 and WSD-3 accessions, there was no significant difference observed in mean length, biomass and the number of attached Striga, while WSD-1 had the lowest 48
significant resistance as compared to all other accession. In reference to Striga virulence, there were significant differences observed at p-value < 0.05 among three Striga ecotypes used in the study namely, Kibos, Alupe and Mbita as indicated in (Appendix). The most virulent Striga ecotype was from Kibos
(Appendix 1.1), which induced the highest mean attachments, highest mean length and highest mean biomass, followed by Alupe (Appendix 1.2) and Mbita
(Appendix 1.3) respectively.
This was inferred from the data where the average Striga plantlet mean count on each wild sorghum accession, ranged from 15±0.81 (WSE-1) to 111±1.73
(WSD-1) in Alupe ecotype, 66.5±2.23 (WSA-2) to 153±5.4 (WSD-1) in Kibos ecotype and 5.3±0.06 (WSE-1) to 100.6±7.36 (WSD-1) in Mbita ecotype. The average Striga plantlet mean length ranged between 0.243cm±0.10 (WSE-1) to
0.766 cm±0.02 (WSD-1) in Alupe, 0.487cm±0.00 (WSA-1) to 0.651cm±0.05
(WSA-3) in Kibos and 0.205cm±0.01 (WSE-1) to 0.288cm ±0.00 (WSD-1) in
Mbita. The average Striga plant-let mean biomass ranged between 1.3mg ±31.3
(WSE-1) to 31.3mg ±0.01 (WSD-1) in Alupe, 21.5mg±0.75 (WSA-1) to
36.6mg ±0.43 (WSD-1) in Kibos and 0.08mg±0.02 (WSE-1) to 5.5mg±0.18
(WSD-1) in Mbita (Figure 4.5).
Three groups with varying resistance with respect to control (N13) arose with highly resistant group comprising WSE-1, WSA-1 and WSA-2; an intermediate 49
resistant group comprising WSA-3, WSD-2 and WSD-3; and a highly susceptible WSD-1 (Figure 4.6).
Figure 4.5:(a) Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica attachment on wild sorghum accessions using three ecotypes of Striga. Striga plants were harvested after 21 days of infection and analyzed using image analysis software. Red colour showing susceptible accession and blue colour showing resistant accession. Vertical bars indicate standard errors of the means while letters represent mean separations at P≤0.05.
50
Figure 4.5:(b) Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica biomass on wild sorghum accessions using three ecotypes of Striga. Striga plants were harvested after 21 days of infection and dried at 26 °C for four days then weighed. The red circle shows highly susceptible accession and blue colour shows highly resistant accession. Vertical bars indicate standard errors of the means while letters represent mean separations at P≤0.05.
51
Figure 4.5:(c) Levels of resistance response of sorghum accessions to S. hermonthica based on mean of S. hermonthica length on wild sorghum accessions using three ecotypes of Striga. Striga plants were harvested after 21 days of infection and analyzed using image analysis software. Red colour shows susceptible accession and blue colour showing resistance accession. Vertical bars indicate standard errors of the means while letters represent mean separations at P≤0.05.
52
Figure 4.6 Levels of Striga resistance in sorghum accessions based on Striga attachments. Heat map showing 3 levels of resistance in the seven wild sorghum accession infected with Striga seeds. The dark blue region (WSD- 1) represents the most susceptible accessions. The light blue region (WSD- 3, WSD-2 and WSA-3) represent the group with intermediate resistance while the grey and red region (WSA-1, WSA-2 and WSE-1) represents the most resistant group. Mbita has few number of Striga attachment followed by Alupe and Kibos having the highest attachments.
4.1.5 Microscopic screening for Striga resistance
Transverse sections through sorghum roots at the site of attachment on third and ninth day after infection revealed two phenotypes, susceptible and resistant.
Striga attachment to the host roots was characterised by swelling of Striga radical at the site of attachments on third day after infection on susceptible interaction (Figure 4.7 ai and bi). Susceptible interactions in WSD-1 and N13 53
did not mount any resistance mechanisms by the third day after infection and transverse section through such attachment shown that Striga haustorium had penetrated the host root system (cortex and endodermis) forming parasite-host xylem connections (Figure 4.7 aii and bii ).The most resistant wild sorghum accession, WSE-1, on the third day after infection, showed intense necrosis at the site of parasite attachment (Figure 4.7 ci). The transverse section on such attachment revealed that the parasite had penetrated the host root cortex but was unable to transverse the endodermis to form host-parasite xylem continuity resulting to growth of the parasite haustorium around the vascular cylinder
(Figure 4.7 cii). In some cases, most of Striga weeds that attached to the roots showing necrosis died after 3rd and 4th day of infection.
As the infection progressed on WSD-1 and N13, the parasite’s vegetative tissue grew vigorously (Figure 4.7ai and bi) and transverse section through this haustorium at ninth day after infection on WSD-1 and N13, showed that, haustorium had significantly expanded and it was well developed with hyaline body (Hb), vascular core (Vc) consisting of the xylem vessels and endophyte
(En) that entered the host cortex and endodermis (Figure 4.7 aii and bii). The wild sorghum accession, WSE-1 showed intense necrosis and poorly developed
Striga plant (Figure 4.7ci) at the ninth day post infection. A transverse section through this haustorium showed a deposition of dense staining and limited 54
vascular connections and hyaline body was greatly reduced as compared to N13 and WSD-1 (Figure 4.7 cii).
Figure 4.7: Profile of different sorghum resistance mechanism to S. hermonthica. (ai) successful attachment of Striga to WSD-1 sorghum root three days after infection. (aii) cross section of an embedded WSD-1 sorghum root tissue, nine days after infection with Striga showing penetration of the host root cortex and endodermis. (bi) growth of Striga plant on N13 sorghum root after three days of Striga infection. (bii) cross section of embedded tissue of N13 nine days after infection showing full colonization of Striga haustorium in host root. (ci) resistance response of WSA-1 three days after infection. (cii) cross section of an embedded WSE-1 root tissue after three days of infection showing penetration of the root cortex but not the endodermis. HY-hyaline body, H-host, HX- host xylem, HC- host cortex, En- endodermis, P- parasite, PX- parasite xylem and HX- PX- host xylem, parasite xylem connections.
55
4.2 Gene expression in early and late stages of Striga infection During third day after infection N13 differentially expressed 1117 genes while
WSE-1 differentially expressed 2823 genes (Figure 4.8a). There was an overlap of 1099 differentially expressed genes between the two sorghum. Nine days after infection N13 differentially expressed 364 genes while WSE-1 differentially expressed 1818 genes, and there was an overlap of 117 genes between the two sorghum (Figure 4.8b). After combination of the two venn diagrams for both N13 and WSE-1 at both stages of infection, 448 genes were only differentially expressed in WSE-1 while in N13 only 55 genes were differentially expressed (Figure 4.9).
Figure 4.8: Venn diagrams of differentially expressed genes in WSE-1 and N13.(a) differentially expressed genes during early stage of Striga infection. (b) differentially expressed genes during late stage of infection. Two-way ANOVA with Bonferroni correction (p < 0.01) was used to limit genes differentially expressed the early and late stage of Striga infection in both N13 and WSE-1. 56
Figure 4.9: Combined Venn diagram for differentially expressed genes in both early and late stage of Striga infection for both N13 and WSE-1. In WSE-1, 448 genes were differentially expressed in both conditions and in N13, 55 genes were differentially expressed in both infection conditions.
4.2.1 Gene ontology
Functional annotation using the gene ontology on 448 differentially expressed genes in WSE-1 at two conditions (3rd and 9th days after infection), 22 protein molecular families were identified, since the work involved defense/immunity protein three proteins namely; PR-2 β-1-3 Glucanases, PR-4 chitinase and PR-
5 Thaumatin like protein were identified (Figure 4.10). 57
Figure 4.10: Gene Ontology analysis of the 448 genes was aligned to the GO database and classified functionally into at least 22 protein molecular families 448 differentially expressed genes in WSE-1, 22 protein molecular familes. Analysis of defense /immunity proteins led to identification of PR-2 glucanase, PR-4 chitinase and PR-5, thaumatine like protein.
58
4.2.2 RT- PCR analysis of isolated PR genes
RT-PCR analysis generated two products; the PR-2 fragment (456bp) and the
PR-4 (450bp) (Figure 4.11) amplified from RNA extracted from WSE-1. The products were of expected base pair size 450bp and 456bps respectively.
Figure 4.11: RT-PCR product for isolated pathogenesis related genes (PR- 2) and (PR-4) on a 1% agarose gel. M ladder Bioline 1kb, lane 1, negative control (no atemplate), lane 2 and 3 PCR products resulting from primer set for PR-4 and lane 4 and 5 PCR products resulting from primer set for PR-2 59
CHAPTER FIVE
DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Discussion
5.1.1 Evaluation of wild sorghum accessions in response to Striga infestation
The current study has shown that, WSE-1,WSA-1 and WSA-2 accessions exhibited highest levels of post-attachment resistance and WSD-1,WSD-2 and
WSD-3 accessions exhibited lowest resistant based on resistant parameters used in this study in host sorghum, against three common Striga ecotypes in
Kenya as compared to control N13 the most resistant cultivated sorghum. This may be contributed by genetic variations among sorghum lines against S. hermonthica parasitism (Haussmann et al., 2000; Zheng et al., 2011). In cultivated sorghum complete resistance to Striga parasitism has not been observed yet (Gurney et al., 2002); in addition, in this current study there was no complete resistance identified in these wild sorghum accessions screened.
The highly susceptible wild sorghum accessions were, WSD-1,WSD-2 and
WSD-3 with the highest number of Striga weeds attachments and weeds grew faster an indication that they were getting enough nutrients and water from host sorghums. On the other hand, the highly resistant wild sorghum accessions were, WSE-1, WSA-1 and WSA-2 with fewer number of Striga weed attachments and the weeds had stunted growth indication that they were not 60
getting enough nutrients from host root. The wild sorghum accessions were then grouped as susceptible or resistant accessions based on mean of biomass, mean of length (cm) and mean of number of S. hermonthica parasites attached on the sorghum roots (Berner et al., 1995; Rebeka et al., 2013). Previous studies reported similar observations when cultivated and wild relatives were challenged with S. hermonthica (Mohamed et al., 2003; Rich et al., 2004; Yoder and Scholes, 2010).
In the present study, WSE-1, WSA-1 and WSA-2 emerged as the most resistant accession with the lowest number of mean of biomass, mean of length and mean of attached Striga to host roots when compared to sorghum variety N13. This variety has been shown to be the most Striga resistant cultivar because N13 confers mechanical barrier through host cell wall rigidification against Striga penetration (Haussmann et al., 2004). This implies that three wild sorghum accessions may provide the genetic potential for Striga resistance (Mutegi et al.,
2011). WSD-1 WSD-2 and WSD-3 were the most susceptible genotypes with the highest mean of biomass, mean of length and mean of the number of attached Striga to host roots. Similar findings as been reported in sorghum in response to S.hermonthica resistance (Ejeta, 2007; Mbuvi et al., 2017).
The resistant ranking within wild sorghum accessions screened, was similar across the Striga ecotypes used in the study and this indicated that resistance in 61
wild sorghum accessions is relatively broad spectrum and not specific to particular Striga ecotype and this provides opportunity to study genetic basis of resistance in wild sorghum accessions. This was ensured by; infection of sorghum roots with same amount of Striga seeds (0.25g) for each Striga ecotypes used and standard germination rate of above 70%. This guaranteed uniform Striga attachments and also eliminated any differences in post- attachment resistance as result of variations in production of germination stimulants among wild sorghum accessions (Jamil et al., 2011). Similar observation were reported in new rice of Africa (NERICA) when challenged with different ecotypes of S. asiatica and S. hermonthica (Cissoko et al., 2011).
The current study has revealed that the highly virulent Striga ecotype was from
Kibos as compared to Mbita and Alupe ecotypes. This may be as result of continued growth of sorghum in Striga infected region of Kibos and Striga has developed mechanism for evading the resistance in the sorghum. Scholes and
Press, (2008) reported that host plants forms resistance mechanism against
Striga infections only after a long duration of their co-existence. The virulence could as well be contributed by the genetic diversity of S. hermonthica seed in
Kibos due to its out-crossing nature over a period of time (Huang et al., 2012).
In this study WSD-1,WSD-2 and WSD-3 wild sorghum accessions had the lowest resistance level relative to the other accessions. This may be as a result 62
of drumondii being a hybrid of wild sorghum and cultivated sorghum (Ho,
2010), some of the resistance genes may have been lost during domestication of cultivated sorghum (Doebley et al., 2006). Similar resistant was demonstrated in sorghum drummondii when infected with S. asiatica (Ejeta, 2007).
Different numbers of attached Striga plants developed successfully in susceptible interaction and others died on interaction with resistant sorghum accession. The failures of Striga plant growth after attachment on resistant accession was correlated with the appearance of necrosis at the site of host attachment in the first three to four days resulting to the death of parasite.
Similar resistance response were reported in rice cultivars such as Nipponbare when infected by S. hermonthica (Gurney et al., 2006) and some sorghum cultivars after infection with S. asiatica (Mohamed et al., 2003). Furthermore, this mode of resistance has been reported in cowpea cultivars which are resistant to S. gesnerioides infestation (Li and Timko, 2009).
In this study, comparison of transverse sections through resistant wild sorghum accession, WSE-1 and control N13, revealed that in WSE-1, Striga endophyte had penetrated the host cortex by day three but was not able to penetrate the host root endodermis while in the control N13, the Striga endophyte had penetrated the host endodermis and formed host-parasite xylem connection.
Inability of parasite endophyte to invade host endodermis is related with failure 63
of Striga haustorium to differentiate fully and this may have been contributed by toxic compounds which are produced by host through vascular system to parasite through haustorium(Scholes and Press, 2008; Cissoko et al., 2011).
This was previously found common in Nipponbare a rice cultivar that showed resistant to S. hermonthica infestation (Gurney et al., 2006; Yoshida and
Shirasu, 2009). After nine days, WSA-1, the most resistant wild sorghum accession, WSE-1, Striga endophyte took longer to penetrate the host root endodermis as compared to control, N13. This was as result of dense necrosis, which was exhibited in the WSA-1. A similar response after sectioning was shown in sorghum and maize resistance responses to Orobanchea species
(Fern ndez-Aparicio et al., 2008; Pérez‐de‐Luque et al., 2008). Successful attachments which results to high reproductive output of the Striga weeds is reduced by resistant mechanisms in resistant sorghum genotypes (Cissoko et al.,
2011).
5.1.2 Expression of pathogenesis related proteins in wild sorghum
In this study RNA sequencing technique enabled identification of differentially expressed candidate Striga resistant genes in WSE-1 and N13 in response to S. hermonthica seeds. The technique provides opportunity to study expression of thousand genes at a time. RNA sequencing has also been used to study differentially expressed genes expression in cowpea during incompatible and compatible interaction with S. gesnerioides (Timko et al., 2008). Also the 64
technique has been used in the study gene expression in Nipponbare rice cultivar against S. hermonthica (Swarbrick et al., 2008). Among the genes expressed in cowpea included PR-proteins, HR protein homologs, enzymes in phenylpropanoid metabolisms and pleiotropic drug resistance. The PR-proteins expressed in Nipponbare rice included Thaumatin like protein (PR-5), endochtinases (PR-3) and Glucanases (PR-2) (Swarbrick et al., 2008).
In the current study, WSE-1 expressed 2826 genes in early periods of Striga infection as compared to the late stage of infection1818 genes. This may be due to changes of mechanism of resistance response and nature of gene expression during resistance and susceptible interaction (Timko et al., 2008). Also this may be due to more genetic diversity in wild sorghum as compared to cultivated sorghums (Mutegi et al., 2010). Similar observations have been reported in cowpea cultivars in response to S. gesneriodes parasitism where by many genes were expressed during the early stage of infection as compared to late stage of infection (Timko et al., 2008). Genes expressed during early parasitism included genes for abiotic stimuli, HRs, oxidative stress, component of ethylene signaling pathways and biotic stimuli. On other hand genes that were expressed at late stage of infection are involved in pattern formation, cell differentiation and turning on PCD (Timko et al., 2008).
65
This study has revealed that WSE-1 differentially expressed 448 genes as compared to N13 which only differentially expressed 55 gene after 3rd and 9th day after of infection. Hypersensitive response that was observed in WSE-1 against S. hermonthica parasitism, may govern this. Similar changes in gene expression were observed in rice cultivar Nipponbare in response to
S.hermonthica parasitism (Swarbrick et al., 2008). Resistance genes such as genes encoding for HR; protein homologue PrMC3 and another putative hypersensitive-induced response proteins pathogenesis related proteins and
WRKY transcription factors were all up regulated in cowpea cultivar in response to S. gesneriodes (Swarbrick et al., 2008). An HR-like phenotype has been observed in some sorghum cultivars in response to S asiatica (Mohamed et al., 2003) parasitism and some cowpea cultivars in response to S. gesneriodes parasitism (Timko et al., 2012).
In this study, gene ontology led to identification of two pathogenesis-related proteins, PR-2, β 1-3 Glucanases, antifungal hydrolyses agent and PR-4, chitinase; a cell wall degrading enzyme (Bellincampi et al., 2015). The function of these genes is similar to other pathogenesis related genes expressed in different plants as result of resistance against fungal and bacterial pathogens
(Table 2.1). These proteins are characterized by defense against fungal pathogens; PR-4, chitinase hydrolyses chitin and PR-2, β1-3 Glucanases hydrolyses chitin in bacterial cell wall respectively (Zhu et al., 2006: 66
Bellincampi et al., 2015). According to van loon (2006) when the PR proteins are expressed in host plant in absence of pathogenic stimuli they are involved in protection of host cellular structures’ by stabilizing its cellular membrane and also keep harmful microbes on check. So in this current study PR-2 and PR-4 might have been expressed for defensive mechanism but not as result of pathogen attack. Expression of pathogenesis related proteins are related with induction of salicylic and jasmonic acid pathway (Hiraoka and Sugimoto,
2008), where by the most susceptible sorghum recognized Striga infection as wounding resulting to induction of JA genes while the highly resistant sorghum recognized Striga as bio-trophic stress leading to expression of SA genes
(Timko et al., 2012).
In this current study, co-expression of PR-2 and PR-4 in WSE-1 during Striga infection increased the level of resistance, leading to induction of hypertensive response and this has been previously been observed in rice cultivar,
Nipponbare when infected with S. hermonthica (Kim et al., 2004). Constitutive expression of PR-2 and PR-4 in WSE-1 enhanced resistance to Striga infection.
Similar results were reported in transgenic potato plants overexpressing PR-4 and PR-2 against late blight (Kombrink and Schmelzer, 2001). Śliwka et al.,
(2012), reported comparable results on the efficiency of the co-expression of
PR-4 and PR-2 in diseases resistance in plants.
67
5.2 Conclusion
In this study wild sorghum accessions, WSA-1, WSE-1 and WSA-2 exhibited higher post-attachment Striga resistance than WSD-1 WSD-2 and WSD-3 hence they could provide valuable source of resistance to S. hermonthica.
Additionally resistance of these wild sorghum accessions is effective against three common Striga ecotypes in Kenya indicating that resistance in wild sorghum accessions is broad spectrum and not specific to a particular ecotype.
The resistance mechanism in WSE-1 was hypersensitive response (HR) which was as a result of necrosis at the site of Striga attachment, resulting to parasite death at 3rd and 4th days after infection. Two candidate pathogenesis-related genes PR-2 and PR-4 were isolated from WSE-1, the wild sorghum accessions which showed hypersensitive response at site of Striga attachment. These genes are responsible for defensive mechanisms against pathogens attack in the WSE-
1 and their co-expression may confer high resistance to Striga.
68
5.3 Recommendations
Recommendations based on this study include;
i. WSE-1, WSA-1 and WSA-2 to breeders for incorporation in
sorghum improvement programmes because they exhibited
highest levels of post attachment resistance against all three
common ecotype of Striga in Kenya.
ii. Incorporation of PR-4 and PR-2 genes in genetic engineering
strategies for sorghum improvement, because co-expression of
these genes could confer resistance to Striga.
5.4 Areas of Future Research
i. Screening many wild sorghums accessions including virgatum and
verticilliflorum (since they have not been screened in this work) for
post-attachment Striga resistance and determining the potential
resistance mechanism.
ii. Validation of PR-2 and PR-4 expression in susceptible and resistant
sorghum using real time PCR 69
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APPENDICES
Appendix 1: ANOVA table for S. hermonthica ecotype from Kibos.
(a) ANOVA table for mean of attachment of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 7 19024.20625 2717.74375 62.07 <.0001
Error 16 700.58000 43.78625
Corrected Total 23 19724.78625
(b) ANOVA table for mean of length of S. hermonthica on sorghum roots
DF SS MS F-Value P-Value
Source 7 0.16014117 0.02287731 10.39 <.0001
Error 16 0.03524154 0.00220260
Corrected Total 23 0.19538271
(c) ANOVA table for mean of biomass of S. hermonthica on sorghum roots
DF SS MS F-Value P-Value Source 7 0.00063249 0.00009036 226.36 <.0001 Error 16 0.00000639 0.00000040 84
Corrected Total 23 0.00063888
Appendix 2: ANOVA table for S.hermonthica ecotype from Mbita.
(a) ANOVA table for mean of attachment of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 7 24769.71625 3538.53089 210.02 <.0001
Error 16 0.08570575 0.00476143
Corrected Total 23 0.6918907
(b) ANOVA table for mean of length of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 8 0.01298608 0.00185515 4.58 <.0056
Error 16 0.00000174 0.00000010
Corrected Total 25 0.00255942
(c) ANOVA table for mean of biomass of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 7 0.00006917 0.00185515 116.48 <.0001
Error 16 275.54167 15.30787
Corrected Total 23 33006.79796 85
Appendix 3: ANOVA table for S.hermonthica ecotype from Alupe.
(a) ANOVA table for mean of attachment of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 7 3273.25630 4091.40704 267.27 <.0001
Error 16 269.57333 16.84833
Corrected Total 23 25039.28958
(b) ANOVA table for mean of biomass of S. hermonthica on sorghum
DF Sum of Squares Mean Squares F-Value P-Value source 7 0.60618505 0.07577313 15.91 <.0001
Error 16 0.00648432 0.00040527
Corrected Total 23 0.01947040
(c) ANOVA table for mean of length of S. hermonthica on sorghum
DF SS MS F-Value P-Value
Source 7 0.01298608 0.00185515 4.58 <.0056
Error 16 0.00648432 0.00040527
Corrected Total 23 0.01947040