Abiotic stress breeding

1. Course: Breeding for Biotic Stress
Title: Review on Breeding Approach for MLN disease
Resistance of Maize Crop: Status and Research Progress in
Ethiopia
By
1.Tefera Kumsa
2.Mohammed Abdurazek
3.Rameto Roba
Instructor: Dr. Zelalem Bekeko
Feb, 2022
Haramaya, Ethiopia

2. Presentation Out Lines
Introduction
Literature Review
Current distributional status of MLN in Ethiopia
Research progress on MLN in Ethiopia
Response of Maize against MLN
Convectional breeding approach for MLN resistance
Molecular breeding approach for MLN resistance
Conclusion

3. 1.Introduction
Maize is the most important cereal crops in terms of production in
Ethiopia and considered as food security crops.
Its production was threatened by several factors such as biotic and abiotic
stress in general.
The factors cause maize yield loss is not limited to diseases, insect pests,
and weeds
However , the current national average of maize was 4.2 tone/hek.
(CSA,2019).
Maize Lethal Necrotic (MLN) is a disease that emerged as the single most
important component of constraint amongst maize diseases in Ethiopia
(Demissie et al., 2016).

4. Cont’d
MLN is an emerged maize serious disease that further threatens maize
production in the country.
MLN disease that is caused by double infection of Maize chlorotic mottle
virus (MCMV) along with Sugarcane mosaic virus (SCMV) is known to
commonly occur on maize and first reported in Ethiopia (Mahuku et al.,
2015).
MLN is a viral disease caused by combined infection of maize with Maize
Chlorotic Mottle Virus (MCMV) and any of the Potyviruses infecting
cereals (e.g., SCMV, MDMV or WSMV)
MLN was first reported in Ethiopia in 2014 and maize plants exhibiting
severe yellowing and chlorotic mottle symptoms were observed in the
upper awash Valley of Ethiopia (Mahuku et al .,2015).
De novo of outbreak MLN disease was distributed in major maize
production areas of Ethiopia, especially in central, western, southern and
southwestern parts of the country (World Atlas, 2021).

5. Cont’d
According to the report of (Demissie et al., 2016) indicated that it's
supposed to cause yield loss up to 65% in the region of the survey
conducted.
Singular infection with either MCMV or SCMV produce milder symptoms.
The MLN causal agent MCMV can be experimentally transmitted by thrips
and beetles while SCMV is vectored by aphids (Cabanas et al. 2013).
Leaf Beetles, aphids, Leaf grass hopper and corn root worms have also been
found to transmit this virus
Thrips

6. Cont’d
Overall mean seed to the seedling transmission rate of MLN disease was 0.073%
with a range of 0 to 0.17% (Bayissa et al.,2021).
Present study showed that MLN infested soil and infected maize residue play an
important role in the survival, inoculum source and spread of MCMV and SCMV,
the major component of MLN (Bayissa et al., 2021).
Therefore , MLN develops in favor of the following factors.

7. Cont’d
The wide spread of MLN in Ethiopia maize belt area were aggravated
since the outbreak of it.
Therefore knowing the current status and research progress of this
emerged dead virus is the core point this paper with the objective :
Reviewing the breeding approach , status and research progress of
MLN disease in Ethiopia.

8. 2.Literature Review
2.1 Current Distributional Status of MLN in Ethiopia
Maize lethal necrosis (MLN) was first reported in Ethiopia in 2014.
Almost all of the currently grown, commercial varieties in East Africa
including Ethiopia are susceptible to MLN (Mahuku et al 2015).
Characteristic symptoms include chlorotic mottling on leaves, stunted
growth and shortened internode distance, dead heart, leaf necrosis, tassel
sterility, and poor seed set.
No MLN resistant / tolerant varieties released in Ethiopia , however maize
inbreed lines were screened and identified.
According to the survey result revealed that MLN disease spread to the
Rift valley, plus Central and Western maize growing areas of Ethiopia
including Oromia, SNNP, Amhara. Tigria and Benushangul Gumuz
(Demisse et al.,2016 and Bayissa et al.,2020) .

9. Cont’d
The Temporal analysis of (Habtamu Tefera and Getacho Gudero, 2019)
revealed that the 2017 cropping season disease severity of MLN is
lowered by 7.92% as compared to 2016 in the southern region of
Ethiopia, where the highest MLN incidence and severity recorded.
De Groot et al. (2016) noted a progressive decrease of disease outbreak
towards highland areas.

10. 2.2. Research Progress On MLN In Ethiopia
Maize lethal necrosis is a complex challenge in maize production and
productivity that must be addressed through a multipronged approach.
To tackle the fast distortion of the epidemic over the last few years,
research efforts are underway in Ethiopia to develop integrated
management options which used as user guide.
In Ethiopia, most of the research studies of MLN concern on
identification, characterization of causal agent , its economic importance
and geographic distribution (Fantahun et al.,2019 ).
Thereby experimental study (Bayisa et al.,2021) revealed that a total of
306 maize genotypes (275 inbred lines and 31 commercial varieties)
collected from various research centers and seed companies were
evaluated in the greenhouse under artificial MLN inoculation.
Seed transmission study was conducted to determine the potential of seed
transmission and its role in the spread of MLN, therefore, Overall mean
seed to the seedling transmission rate of 0.073% with a range of 0 to
0.17% was confirmed (Bayissa et al.,2021) .

11. Cont’d
In Oromia and SNNP in the year 2016 and 2017 main cropping season
alternate hos for MLN were assessed.
Accordingly, Cyperus rotundus, Cyperus cyperoides, Snowdenia polystachya,
Digitaria sanguinalis, Echinochloa colona, Oplismenus hirtellus, Pennisetum
purpureum and Phalaris paradoxa were identified as alternate hosts for
MCMV. Sorghum bicolor and Saccharum officinarum were identified as
alternate hosts for SCMV (Regassa et al., 2021).
Research on detection of the viruses from soil and maize parts was assayed
serologically using DAS-ELISA for the presence of MCMV and SCMV were
conducted in Ethiopia.

12. 2.3 Maize Response to MLN
Plants are a rich source of nutrients for many organisms including bacteria,
fungi, protozoa, virus, insects, and vertebrate.
This is why plant are surrounded by disease causing organisms that make
plant to develop physical and chemical barrier to defense them self from
disease (Jennifer et al., 2005) .
Viral diseases are also fairly common in maize and they can cause
sporadic but very damaging epidemics.
Plant are develop an array of structural, chemical and protein-based
defenses designed to detect invading organisms. These response are
carried out through the following mechanisms
 Receptors in the cells respond to molecules from the pathogens, or to
chemicals produced when the plant cell wall is attacked.
 This stimulates the release of signaling molecules that appear to switch on
genes in the nucleus.
 This in turn triggers cellular responses, which include: Chemical, sending
a signal and strengthen cell wall (Michael Hanh,1996).

13. Cont’d
The diverse plant defense mechanisms induced by elicitors include the
synthesis and accumulation of antimicrobial (phytoalexins) , the induction
of cell death (hypersensitive necrosis), the synthesis of proteins that inhibit
degradative enzymes produced by pathogens (Ebel,1996)
Proteomics of maize-virus interactions is a relatively new research area ,
addressing the effect of viruses on maize protein expressions (Wu et
al.,2013).
Pathogenesis- related proteins represented the largest and most diverse
group of proteins associated with maize resistance
Generally, PRs are inducible by a variety of pathogens via salicylic acid,
jasmonic acid, or ethylene signaling, which leads into their accumulation
at the site of the attack but also systemically at distant parts of the
plant(Van Loon et al.,2006).

14. Plant defense mechanism
Some molecules from the
pathogen are recognized
directly by plant cell
Some defensive
molecules directly
attack the pathogen
When pathogenic
enzymes break down
the cell wall the
breakdown products
are recognized
Signal molecules
alert nucleus to on
attack
Polysaccharides(callose
and lignin made to
strengthen the cell walls
Defense chemicals
gives alarm to other
cells before they are
attacked

15. Cont’d
Physical and biochemical base of plants to protect pathogen entrance
which makes passive the invention.
Plant defense
Physical barriers
Chemical barriers

16. Cont’d
Diversity of plant cell wall structure. (A) primary, and (B)-(C) secondary
cell walls (Taiz and Zeiger , 2010).
Cell wall structure , biogenesis and expansion.
 Some plants have very thick cell walls and/or cuticles, and bark which can
all provide a better barrier to infection.
 Callose blocks sieve plates in the phloem, sealing off the infected part and
preventing the infection from spreading (Lang et al., 2003).

17. summarized plant defenses against pathogens
Source: Ethan et al.,2018

18. 2.4.Conventional breeding for MLN
The methodology of breeding for disease resistance is the same as that
used in breeding for any other trait.
The discovery of Mendel’s work lighted the way for producing them
scientifically (Curtis, 1973).
Availability of suitable sources of resistance is a basic prerequisite for
successful resistance breeding.
sources of resistance were to be selected among cultivated crops or their
wild relatives.
After adaptation or introduction of the resistance line the next step is
choose of breeding methodology.
The basic breeding methodologies are Recurrent selection, back crossing ,
inbreeding and hybridization.

19. Cont’d
1.Recurrent selection scheme
 This method consists of two phases : (a) Population formation:
Susceptible selected germplasm is crossed with the source of resistance
and balanced bulk and salfed to produce resistance genotypes
 Deliberate efforts should be made to utilize plants combining virus
resistance with agronomical desirable traits.
(b) Population improvement: After formation, the populations are
subjected to recurrent selection (Beddehagen,1978, SoTo et al, 1982)
2. 2. Backcross
 The availability of relative simple, though multigenic, heritable source of
resistance and the ease of identifying resistant plants in segregating
populations, suggest that a backcross program can be used to convert
virus susceptible cultivar to resistant varieties (Eeron et al.1989).

20. Schematic representation of conventional backcrossing
George Acquaah ,2007

21. 2.5Molecular breeding for MLN-resistant maize
MLN is a complex disease as the interaction between the two viruses
(SCMV and MCMV)
Genetic studies on MLN showed both are recessive (Jones et al .,2017)
and dominance (Gowda et al., 2018) type of inheritance in different
populations
Combining all the favorable alleles from diverse sources of MLN
resistance or tolerance into one hybrid combination is challenging(Wang
et al. 2014a)
Nevertheless, understanding the genetic differences and the type of gene
action involved in MLN resistance is useful in prioritizing inbred lines
The traditional breeding of disease-resistant varieties mainly depends on
the breeder’s experiences in phenotypic selection which is time-
consuming, inefficient and environment dependent.

22. Cont’d
Ali et al., 2013 Evidence of Multiple Disease Resistance (MDR) and
Implication of Meta-Analysis in Marker Assisted Selection

23. Cont’d
The combination of traditional breeding and marker-assisted selection
(MAS) has proven to be very efficient for developing elite resistant lines for
maize production
With the availability of elite disease resistance genes and their tagged molecular
markers, ,(Zhang et al. 2012b)
GS, transgenesis technique, and genome editing approaches combining
methods with
DH technology could greatly accelerate the molecular breeding process in maize
(Xu et al. 2014).
Schemes for molecular breeding of disease-resistant maize
Collection of various excellent germplasm resources
Cloning and identification of disease resistance genes(screening)
R genes can be used for MAS and transgenic disease resistance breeding
Genomic selection (GS) can speed up resistance breeding programs
Combining gene editing with DH technology

24. Cont’d
Combining various strategies is an excellent way to greatly accelerate
the maize breeding process (Mang et al., 2021 )

25. Marker-assisted selection
MAS is a method of selecting desirable individuals based on DNA
molecular marker.
Molecular marker is a fragment of DNA that is associated with a certain
location within the genome. That used to identify a particular sequence of
DNA in a pool of unknown DNA
MAS are a powerful tool to reduce maize diseases by using natural
resistance genes (Zhao et al. 2012a).
If a resistance gene with pleiotropic effects is selected for resistant breeding
program pyramiding of various resistance genes is an effective (Asea et al.
2012).
MAS combined with phenotypic selection is a highly efficient, resistance
breeding programs in maize (Yousef and Juvik ,2001).
MAS relies on the availability of markers linked to genes

26. Marker-assisted selection
Melese Lema, 2018 Comparison to Conventional Plant Breeding: Review Article

27. Genomic selection
Genomic selection (GS) is conducted by combining genotypic (markers) and
phenotypic data in a training population to estimate the breeding values of
lines (Meuwissen et al.,2001) .
It substantially increases the rate of genetic gain
Combining two different heterotic groups in a single training set can lead
to significantly and
more accurate prediction for both heterotic groups, (Technow et al. ,2013).
Transgenesis Technique
useful for breeding disease resistant maize
involve the direct introduction or modification of a target gene of interest
using biotechnology (Christou , 2013) .
Disease-resistant maize could be bred by overexpressing or knocking down a
gene of interest
Expressing hpRNA derived from the capsid protein genes of MDMV and
SCMV significantly enhanced maize resistance to MDMV and SCMV,
respectively (Gan et al. 2014).

28. Transgenesis Technique
Jhansi Rani et al., 2013.Transgenic plants

29. Genome editing by CRISPR-cas9
CRISPR-cas9 (Clustered regularly interspaced short palindromic repeat and
associated protein 9) (Hua et al.,2019).
System has revolutionized the genome editing due to its simplicity, flexibility,
consistency, and high efficiency and powerful tool for genetic analysis and
crop improvement
It is a naturally occurring enzyme in bacteria that allows scientists to edit DNA
with precision

30. Combining genome editing and double-haploid technology
Removal of the CRISP-Cas9 cassette requires several generations.
Double-haploid (DH) technology is a powerful tool to promote the
breeding efficiency by reducing the need for multiple generation
selection (Ren et al. 2017).
Combining CRISPR-Cas9 with DH technology represents an
excellent way to accelerate maize breeding

31. Combining genome editing and double-haploid technology

32. Summary and Conclusion
Maize is one of the most important cereal crops grown for its food and feed
values in Ethiopia.
Maize production and productivity is threatened by the two major stress
Among the biotic stress that infecting cereals the Potyviruses group called
MLN is the serious virus that bring much yield loss
MLN is typically caused by the synergistic interaction between maize
chlorotic mottle virus (MCMV) and any cereal Potyvirus e.g, sugar cane
mosaic virus (SCMV) or maize dwarf mosaic virus (MDMV).
Its prevalence is some what decreased especially in highland area.
Knowing physiological, biochemical and molecular base of plant defense
mechanism is prerequisite.
Back crossing the most compatible method for resistant variety development
MAS, genetic , DH-technology, Genomic selection are among the molecular
breeding methods used for the development of resistance variety.
Combining convectional with molecular breeding method is a very important
new strategy in developing improved genotypes that we conclude.

33. Acknowledgment
We would like to thanks our instructor Dr. Zelalem
Bekeko for teaching and directing us in writing this
review paper.
And also we thanks our University for its convenience
environment

34. Haramaya Univeristy