Application of molecular and biotechnological techniques in plant disease management: A review

Plant diseases are a major challenge in crop production. They are caused by nematodes, bacteria, fungi, viruses as well as plant nutritional factors. Diseases interfere with the normal physiological and metabolic processes of plants. This results in various effects including wilting, stunting, yellowing and death of plant tissues and organs. Crop losses due to diseases manifest in form of reduced yield, poor quality produce, and reduced post-harvest storage. Past research has brought to the limelight the continuous capacity of pathogens to revert to new pathotypes and strains, some that break resistant varieties or are less sensitive to chemical control products. Currently, farmers are advised to combine several plant disease management practices, a strategy known as integrated plant disease management. Such strategies include crop rotation, use of disease free planting materials, field sanitation, and chemical methods as well as use of resistant varieties. However, some of these methods are expensive and substantially increase the cost of production. Development in molecular biology and biotechnology found application in plant disease management. This ranges from identification, diagnosis to control through gene transfer, mutation breeding and RNA interference, among others. In this paper, the current developments in the application of molecular techniques and biotechnology to manage plant diseases, outlining their possible future application and potential for enhanced plant disease management.

The impact of pathogens on agricultural crops has been wide.Pathogens release enzymes, growth regulators, toxins and other substances which manifests in the affected plants through a myriad of symptoms.The symptoms may be in form of destroyed vascular bundles, yellowing and drying of leaves, witling, and necrosis among others.These developments inhibit absorption and movement of water and minerals from the soil to plant tissues, leading to reduced photosynthesis and death of plants (De Werra et al., 2015;Prince et al., 2015).For the case of cassava brown streak virus, there is a development of necrotic spots on the roots and rotting, which has a direct impact on the yields (Patil et al., 2015;Legg et al., 2015;Anjanappa et al., 2017).Maize lethal necrosis leads to stunted plants that could turn yellow and die causing 40% or higher loss of yields.The cyst nematodes in potato (Solanum tuberosum) growing areas attack tubers, which results in discoloration and sometimes rotting, which directly affect yields (Adams et al., 2014;Thorpe et al., 2014;Mahuku et al., 2015).In tomatoes, Ralstonia solanacerum attacks cause accumulation of bacterial mucilage and exudates in the vascular bundles that blocks flow of water and minerals to other parts of the plant (Sarkar and Chaudhuri, 2016).Furthermore, sometimes the bacteria act synergistically with Fusarium species as well as the root knot nematodes in an infection complex.In such a case the wilting is severe and impacts adversely on total yields (Lamichhane and Venturi, 2015).Some fungal pathogens lead to development of spots on leaves or fruits which substantially decrease their market value (Hayes et al., 2014).
The common plant disease management strategies can be categorized into cultural, mechanical, biological and chemical approaches.In cultural methods, there is maintenance of good hygiene at the field through removal and destruction of diseased plants, selection and use of clean planting materials and planting resistant crops (Katan, 2000(Katan, , 2010;;Mehta, 2014).Mechanical methods include heat treatments to kill pathogens in planting materials, in the soils borne pathogens (Bruez et al., 2017) and in reducing postharvest pathogens (Wisniewski et al., 2016).Chemicals are used widely in soil fumigation as well as in control of foliar pathogens and their vectors (Li et al., 2016;Gao et al., 2016).Biological methods involve use of viral, bacterial and fungal organisms as biopesticides in control of plant disease causing microorganisms (Tjamos et al., 2013;Mach, 2016).
Integrated plant disease management combines several of the aforementioned methods to control particular plant diseases or the pathogen vectors (Fry, 1982).To complement the approaches described earlier, molecular tools have lately found wide applications in the study, diagnosis and management of plant diseases.This paper reviews the application of current molecular tools as well as some tissue cultures techniques in the management of plant diseases.

Gene transfer
The initial work by Flor (1955) that gave rise to the genefor-gene concept has been very instrumental in the study of resistance as well as spearheading development of disease resistant plants (Jones and Dangl, 2006).Genetically, this requires a virulence (Avr) gene in the parasite and a resistance gene (R-gene) in the host plant.Several R genes have been identified to date and characterization of the Arabidopsis resistance gene complement managed to provide better understanding of the r genes structure (Meyers, 2003).Identification of resistant plants initially provided sources for resistance genes; this has always been quickly followed by breeding efforts aimed at introgression of the resistance genes to cultivated crops.However, normal classical breeding faced many drawbacks including low success and carryover of unwanted traits to crops (Miklas et al., 2006;Fry, 1982).
Over time, new more precise molecular approaches came into play.These include proteomics, metabolomics, transcriptomics, plant tissue culture and genetic engineering.Genetic engineering involves gene transfer, gene silencing, mutation breeding, and regulation of transcriptional factors (Sankaran et al., 2010;Ocsoy et al., 2013;Mahlein, 2016).Gene transfer method of developing disease resistant plants has been used for several decades.Some of the earlier achievements include Agrobacterium mediated introduction of rice chitinase gene in strawberry (Fragaria ananasa), which led to resistance to powdery mildew, done using the CaMV 35S viral promoter.Expression of rice chitinase-3 gene in transgenic peanut through Agrobacterium mediated gene transfer was also found to be effective in conferring resistance against many soil borne fungal pathogens.This transformation involved Agrobacterium tumefaciens strain LB4404 having the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker (Iqbal et al., 2012).Studies on chitinase genes have led to their wide utilization against many other fungal pathogens (Jabeen et al., 2015;Richa et al., 2016Richa et al., , 2017;;Munir et al., 2016).
Embryogenic tissue transformation through particles bombardments using tungsten particles coated with DNA of the PRSV HA 5-1 coat protein gene was able to produce ringspot virus resistance in pawpaw.Polyethylene glycol (PEG) method was also successfully used to transfer Stilbene synthase gene to rice to increase resistance to blast caused by Pyricularia oryzae (Stark-Lorenzen et al., 1997).
CRISPR/Cas9 is a new technique in genome editing that has enabled manipulation of plants allowing study of resistance genes, and has been used in mutational breeding in rice (Zhou et al., 2014;Lu et al., 2017), tomato (Brooks et al., 2014) and tobacco (Gao et al., 2015).In Arabidopsis, CRISPR/cas9 was used to introduce mutation on elF (iso) 4E locus enabling acquisition of resistance against Turnip mosaic virus (Pyott et al., 2016).CRISPR/cas9 was also used to modify elF4E gene in cucumber thereby creating resistance to a number of viral diseases including cucumber yellowing virus, zucchini yellow virus and papaya ring spot virus (Chandrasekaran et al., 2016).The technique has also been used against fungal diseases (Wang et al., 2016).Currently, gene transfer is being widely applied in crop improvement techniques to introduce resistance mechanisms to plants (Vleeshouwers and Oliver, 2014).

RNA interference
RNA interference (RNAi) is a molecular technology that uses gene down regulation principle via transcriptional gene silencing (TGS) or posttranscriptional gene silencing.Three types of RNA silencing have been investigated in plants.These are cytoplasmic small interfering RNA (SiRNAs), micro RNA (miRNAs) in down regulating endogenous mRNA and DNA methylationsuppression of transcription.In these different RNA interference pathways, long double stranded RNA (dsRNA) precursors are cleaved by DICER enzyme (DCL2 or DCL3) into approximately short 21-nt length of SiRNAs and miRNAs (Bernstein et al., 20101).Once constructed, based on the fact that large and small RNA molecules are mobile between organisms especially in plant-pathogen interactions (Castel and Martienssen, 2013;Kim et al., 2014), SiRNAs and miRNAs will down regulate plant pathogen mRNA and chromatin modification.RNAi has been investigated as a powerful approach in developing disease-resistant crops.It has been used in combating plant fungi, for example Sclerotinia sclerotiorum, the causal agent of white mould, a devasting plant disease that causes up to 100% yield loss. RNAi approach has been reported to be more efficient against white mould as compared to conventional methods.Transgenic tobacco plants were used to construct a hairpin RNA in order to down regulate Chsgene, the gene controlling chitin synthesis in the fungus.A reduction of 55.5 to 80% in disease severity was observed as compared to non-transgenic tobacco (Andrade et al., 2016).
Fusarium oxysporum is a soil-borne fungus responsible for significant economic damages in potato, bean, wheat and bananas, among other crops.Studies have shown that Fox can cause 30 to 70% yield losses in different host crops.Host-induced RNAi has been used in silencing the pathogenicity genes (FOW2, FRP1, and OPR) that allow F. oxysporum to counteract its host resistance mechanism (Hu et al., 2015).RNA silencing has also been used in protecting crops from viral infection such as tomato leaf curl virus, potato virus X (PVX) and citrus tristeza virus (CTV) (Soliman et al., 2008;Praveen et al., 2010;Soler et al., 2011).Despite being a powerful method for switching off expression of pathogen genes during infection, RNAi has some drawbacks to the plant as well as in the environment.RNA silencing could result in host genome modification which might interfere with gene flow between plants and their relatives leading to biodiversity reduction.In addition, RNAi construction is difficult for some plant species (Rodrigues et al., 2009).

Transcriptomics as an approach of managing plant diseases
Transcriptomics entails the study of RNA transcripts produced by the genome within a specific cell using high throughput approaches such as Illumina sequencing.Improved understanding of the cell genome has enabled various techniques such as genome editing which plays a vital role in plant disease elimination, besides improving plant immunity.Through transcriptomics, many disease resistance genes have been identified leading to significant breakthrough in the management of plant diseases (Horgan et al., 2011;Lowe et al., 2017).
Transcriptomics has been used successfully in the management of Xanthomonas oryzae on rice (Cheng et al., 2016).Genome editing technologies have been used successfully in enhancing plant resistance to phytopathogens (Andolfo et al., 2016).The advent of this technology has envisioned the use of RNA-sequencing for transcripts or genes expression profile in the management of various plant diseases (Prabha et al., 2013).Furthermore, manipulation of the key plant immunity modulators such as the R-genes can boost the generation of disease free plants.This technique has also improved understanding of the interaction of various diseases and the plant host such as Phytophthora nicotianae infecting Nicotiana tabacum (Yang et al., 2017).Understanding plant response to infections is important in the development of effective plant disease control measures.Investigation of gene expression profiles during viral infections would shed more light in ascertaining significant components of the resistance alleyways (Yang et al., 2017).

Proteomics
Various proteins and their functions as well as their interaction in an organism can be studied through the mean of proteomics analysis (Zulkarnain et al., 2015).This can be useful in determining the pattern and the specificity of a particular protein released in plants when there is a pathogenic stress.Common techniques used in proteomics analysis are Two-dimensional Electrophoresis (2DE), Fluorescence 2D Difference Gel Electrophoresis (2D-DGE), Mass Spectrometry (MS) and Multidimensional Protein Identification Technology (MudPIT) also known as ‗'shotgun'' approach (Chandramouli and Qian, 2009).
About 1,500 proteins were identified in rice during bacterial leaf streak (BLS) infection with 23 up-regulated proteins that were potentially associated with BLS resistance in rice (Li et al., 2012).Brown root honey caused by Monilinia laxa proteins was investigated from apple and apricot, up to 800 proteins were expressed and around 10 proteins isolated from apple showed potential use in developing M. laxa host specific diagnostic marker (Bregar et al., 2012).In order to understand the host response mechanism against Alternaria alternate infection, both resistant and susceptible apple varieties were subjected to comparative proteomics analysis using Two-Dimension (2-DE) and Mass Spectrometry (MS).A total of 43 differentially expressed proteins were detected which included pathogenesis-related proteins beta-1,3 glucanase, mald 1 and ascorbate perioxidase.The pattern of mald1 in resistant, as well as in susceptible apple contributed to understanding the mechanism underlying A. alternata resistance (Zhang et al., 2015).Likewise, 2-DE and MS were used in identifying different proteins expressed during Liberibacter asiaticus (Las) infection on Citrus.The management of L. asiaticus disease commonly known as Citrus Huanglongbing (HBL) has been successful using heat treatment.Through comparative proteomics study, 107 Las/heat-induced proteins such as HSP70-like proteins, ribulose-1, 5bisulphate and carboxylase were identified.They were up-regulated due to heat treatment, which gives an insight on the underlying heat-induced host defense mechanism (Nwugo et al., 2016).Proteomics as an approach in controlling plant disease is more efficient than conventional methods; however, it has some limitations in dynamic resolution for large-scale proteomes analysis as well as in quantifying proteomes (using Mass Spectrometry).Furthermore, separation, visualization and identification of hydrophobic proteins can be a challenge (Van Wijk, 2001).

Metabolomics
Plant-pathogen interaction could be better understood Dayou et al. 941 based on the identification and quantification of small molecules called metabolites (Rojas et al., 2014).A number of techniques have been used in the past in metabolomics analysis.To-date the commonly used are high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS) as well as nuclear magnetic resonance spectroscopy (NMRS) (Kasture et al., 2012).Using GC-MS approach, Warth et al. (2015) showed that wheat metabolome is modified by deoxynivalenol (DOIN) secreted by Fusarium graminearum that causes Fusarium Head Blight (FBH) disease.Earlier, Levenfors et al. (2008) investigated biological management of snow pink mould (Micrcodochium nivale) on wheat and rye using Pseudomonas brassicacearum MA250.The study found a significant biocontrol effect of P. brassicacearum on M. nivale.Later, Anderson (2012) discovered that the biocontrol activity of P. brassicacearum on M. nivale was associated with the secondary metabolites Piliferolide A and SB0253514.Parker et al. (2009) had hinted on the possibility that metabolites control pathogenesis, when Magnaporthe grisea was observed to counteract rice, barley and Brachypodium distachyon responses by reprogramming its hosts through secretion of different patterns of metabolites.
Known for their devastating effect, Botrytis cinerea (Bot) and Pseudomonas syringae pv tomato (Psd) are two major pathogens affecting tomato production.Analysis of altered metabolites isolated from both B. cinerea and P. syringae infected tomatoes revealed that the host resistance is associated with metabolomics reprograming in the host allowing biochemical changes in tomatoes (Camañes et al., 2015).Melatonin-mediated innate immunity against host specific bacteria in Arabidopsis has been determined to be reinforced by sugars and glycerol increases.

Link between quorum sensing (bacterial communication system) and plant disease resistance
Quorum sensing (QS) is a cell-to-cell communication mechanism in bacteria allowing them to control their local population density and virulence factors (Bouayed et al., 2016).Through this mechanism small signaling molecules are secreted and detected by bacteria enabling them to assess their population.A number of signaling molecules are involved in QS.These include oligopeptides in Grampositive bacteria and N-acylhomoserine lactones (AHL) in Gram-negative bacteria and some auto inducers (AI-2) in both Gram-positive and Gram-negative bacteria.AHL is the best characterized amongst QS molecules.Studies have shown the positive effect of AHL in priming induced resistance in plants against phytopathogens.It has been demonstrated that AHL-derived from Serratia plymuthica can induce systemic resistance in bean and tomato against Pythium aphanidermatum (Pang et al., 2009).Oxo-C14-HSL has been reported to induce resistance against P. syringae in Arabidopsis thaliana.The induced resistance is due to accumulation of callose, phenolic compounds and lignification in plant cell wall (Schenk et al., 2014).Similarly, Oxo-C14-HSL derived from Ensifer melioloti, a rhizobium of root nodulation in legume, which has been found to fortify host response mechanism in plants.This has been reported for Phytopthora infestans, Blumeria graminis and Puccinia graminis resistance in tomato, barley and wheat, respectively.This Oxo-C14-HSL induced systemic resistance was later confirmed in Arabidopsis and barley against B. graminis and Golovinomyces orontii (Schikora et al., 2011).

TISSUE CULTURE AS AN APPROACH TO MANAGING PLANT DISEASES
Haberlandt (1969) published a paper which envisioned the idea of tissue culture procedures and provided a paradigm for many scientists to delve deeper into the aseptic production of plant cells, tissues and organs in culture (Akin-Idowu et al., 2009).In plant tissue culture, plant cells, tissues, and organs are propagated in vitro under aseptic conditions on artificial medium (Hussain et al., 2012).Plant tissue culture has gained popularity in the recent past, and it has been of great importance in plant disease elimination, large scale plant multiplication and plant improvement (Ogero et al., 2012) as well as in the production of secondary metabolites.Subsequently, the application of tissue culture in managing plant diseases is elucidated.

Meristem-tip culture and meristem heat therapy
Organized apex of the shoot from a selected donor plant can be subsequently cultured in vitro (Grout, 1990).The cultures are established from axillary buds or from shoot tips, after excision the explants are inoculated into a culture medium that allows the explant to propagate into shoot.The explant of meristem culture may either be the apical dome (apical meristem) or the apical dome plus a few leaf primordia.Studies have shown that larger explants are desirable as they are easier to dissect and have much higher survival growth rate than the smaller ones.The excised apical meristem tip often measures 0.1 mm in diameter and 0.25 to 3.0 mm in length and is done under sterile condition.The significant importance of using meristem tip cultures is that small explants are paramount for excluding devastating pathogens present in the donor plant (Grout, 1990).Besides, axillary shoot proliferation offers lower risks of genetic instability and is easily achievable in most plant species.This technique has been used to eliminate virus infection in sweet potato (Frison and Ng, 1981).Smith (2013) reported that meristem culture technique had made it possible to save many vegetative propagated plants from viruses.Earlier, Ogero et al. (2012) demonstrated an optimized tissue culture approach for disease-free sweet potato seedlings production in Kenya.Furthermore, this technique can be optimized by combining with heat treatment prior to meristem culture, hence contributing vastly to production of healthy plants.Mwangangi et al. (2014) combined thermotherapy at 38°C with meristem tip culture to eliminate brown streak virus from infected cassava.Meristems excised from plants subjected to thermotherapy had enhanced CBSV eradication as compared to the control resulting in 68.8% plant survival with 84% of the plants surviving being virus-free.These findings confirm previous reports (Acedo, 2006) and elimination of sweet potato fatherly mottle virus (Mashilo et al., 2013).Application of meristem culture combined with thermotherapy at 35°C is reported to increase the survival rate of in vitro explants (Manganaris et al., 2003;Mashilo et al., 2013).This is because larger tips can be obtained from heat-treated plants while ensuring virus-free plant production.
In some cases, it is problematic to eliminate viruses from meristem tip culture; hence, thermotherapy coupled with meristem culture has been proposed.Thermotherapy is applied before in vitro meristem-tip culture and has been shown to be effective against potato virus S (PVS) and PVX (López-Delgado et al., 2004).Chatenet et al. (2001) and Fitch et al. (2001) proved apical meristem culture to be effective in eliminating sugarcane yellow leaf virus (SCYLV).What is more, a combination of meristem culture, heat therapy, and cryotherapy has been used successfully in the elimination of various plant diseases.Thermotherapy coupled with meristem-tip culture has enabled the elimination of bean yellow mosaic virus (BYMV) from infected corms, hence leading to production of BYMV free plants (Sharifi Nezamabad et al., 2015).

In vitro shoot grafting and callus culture
In vitro shoot-tip grafting has also been applied successfully in the elimination of viruses in some woody plants.It entails grafting of apical meristem on young root stock seedling.According to Navarro (1992), this technique has been applied successfully in the elimination of approximately 16 diseases in citrus plants, including Psorosis (Navarro et al., 1980).
Calli is a group of unorganized proliferative cells produced by subjecting explants to suspension culture.During culturing, some cells may escape from viral infections due to the high rate of cell proliferation and attain viral resistance due to mutation.In callus culture derived from infected cells, it is evident that not all calli uniformly contain the viral infections.Studies showed that approximately 40% of calli derived from tobacco infected with tobacco mosaic virus (TMV) contained the virus (Hansen and Hildebrandt, 1966).The main reason for the escape from this devastating virus is the high rate of cell proliferation hence the virus is unable to keep pace with the high rate of cell multiplication, and acquisition of resistance by some cells through mutagenesis (Warren et al., 1992).
Through somaclonal variation, an array of disease resistant plants has been developed.Out of 370 Solanum lycopersicum plants propagated from callus cultures, six showed enhanced resistance to TMV.Similarly, late blight (P.infestans) resistant potato plants and calli resistant to bacterial blight of rice have been developed.Different pathogens produce different secondary metabolites which can be used to screen different calli for disease resistance.Resistant calli can survive in the presence of toxins, hence generation of disease resistant plants.Through this technique, different disease resistant plants have been developed such as rice resistant to the brown spot pathogen Helminthosporium oryzae (Mwendo et al., 2017).Similarly, TMV resistant plants, Helminthosporium maydlis toxin resistant Zea mays plants and Helminthosporium sacchari resistant sugarcane have been generated.Besides, meristem callus culture has been used effectively in the eradication of PVX.Experiments on potato used culture media made up with culture filtrates of different P. infestans pathotypes to successfully isolate resistant lines.Embryo culture is the other technique used in tissue culture in embryo rescue in wide crosses, monoploid production and overcoming seed dormancy.It proved to be a very effective tool for transfer of Alternaria blight tolerance in oilseed brassicas (Yadav et al., 1991;Aneja and Agnihotri, 2016).

Somatic embryogenesis
Somatic embryogenesis refers to the in vitro development of embryo like structures from somatic cells rather than from combination of male and female gametes.According to D'onghia et al. (2001), somatic embryogenesis has been applied successfully in the management of devastating citrus psorosis virus (CPsV) from three different Citrus species, namely, Dweet tangor, Common Mandarin and sweet orange.Psorosis virus-free citrus can be propagated via heat therapy, and shoot grafting or combination of both techniques (Calavan et al., 1972;Navarro et al., 1980;D'onghia et al., 2001).However, enhanced competence during the sanitation procedure is necessary because virus eradication differs between isolates and hardly exceeded 70 to 80% (Roistacher, 1993).Somatic embryogenesis obtained by culture of style and stigma has been used successfully in the management of CPsV, hence a promising technique in the propagation of healthy citrus plants (D'onghia et al., 2001).

Protoplast fusion and somaclonal variation
The variability generated from in vitro cultured somatic Dayou et al. 943 cells, may be due to genetic, epigenetic or physiological causes.Somaclonal variation has been observed in economically important crop species such as wheat, rice, sugarcane, oats, potato, tobacco, among many other plant species with numerous traits, for example resistance to viruses, bacterial and fungal infections.This technique has been used for developing disease resistance in wheat and production of dihaploids through wheat × maize hybrids (Mehta and Angra, 2000) and also for resistance to Verticillium dahliae in potato (S. tuberosum var L.) plants regenerated from callus (Sebastiani et al., 1994).Protoplast fusion is a vital technique for the generation of hybrid plants among different incompatible species and incorporation of an alien genetic factor for pathogen resistance (Larkin and Scowcroft, 1981).In various cases, vital gene variability in the cultivated germplasm may be missing, and use of protoplast fusion can curb this.Hassan (2014) showed that protoplast fusion of two fungicide tolerant mutants of Trichoderma harzianum and Trichoderma viride enhances β-glucanase, chitinase and protease enzyme activity in fusant strains (fusion of two different species of fungus) as compared to the parental strains and they had a powerful antagonistic activity against grapevine pathogens Macrophomina phaseolina, Pythium ultimum and Sclerotium rolfsii.Plant fusion can also cause hypovirulence in other phytopathogenic fungi (Lee et al., 2011).

Haploid and polyploid plants
Haploid plants have been generated from anther and ovule culture.The production of homozygous lines within a span of a short period has paved the way for many types of research.Also, haploid plants are highly useful for research related to plant breeding and genetics.Furthermore, they provide convenient systems for induction of mutations and selection of plants with desired traits.Through these techniques, mutants that are resistant to various pathogens have been developed.For example haploids were used to produce melon with resistance to multiple virus diseases (Lotfi et al., 2003).
Polyploidy usually occurs from one generation to another and it results from variation that alters the number of chromosomes in the cells.Different methods describe the origin of polyploidy but they mainly occur due to doubling of somatic cells in mitosis, non-reduction in meiosis yielding gametes that are unreduced, polyspermy and endo-replication (Bharadwaj, 2015).Due to increase in number of chromosomes of related gene dose in polyploids, the gene expression and some secondary metabolites production can be enhanced, thus, boosting host plant resistance mechanisms.According to Van (1975), Lolium, an autotetraploid has extra structural carbohydrate and good resistance to diseases than the diploids due to relationship changes in dose of genes, silencing of gene and secondary metabolites expression.
By applying polyploidy, one can produce allopolyploids from parent plants having multiple endogenic chemicals of protection and secondary metabolites which usually provide all metabolites and biocatalysts of the two fused parent plants, thus, successfully promoting the resistance to pest characteristic (resistance thus is more in horizontal form).This strategy can also strengthen tolerance to specific stresses of the environment (Bharadwaj, 2015).

FUTURE PROSPECTS OF APPLYING MOLECULAR TECHNIQUES IN PLANT DISEASE MANAGEMENT
Pathogens have the potential to develop resistance against the mechanisms employed to manage them.The use of only one type of R genes is quite a temporary disease solution.For example, the Brassica resistance mediated in Rim1 collapsed in 5 years of use (Sprague et al., 2006).As done in S. tuberosum (Kim et al., 2012;Vossen et al., 2014), integrating several R genes may be the way out to make sure that whenever mutations occur in the pathogen to surpass one of the R genes, additional sources of resistance will persist.R genes have to be prolonged by studying the complex of resistance of the evolution of R gene.Grzeskowiak et al. (2014) studied the mechanism of resistance in tomato focusing on Pto/Fen/Prf resistance complex.Some studies have shown that studying non-host resistance (NHR) may lead to and devise mechanisms of resistance that are long lasting and independent of recognition of R protein.
According to Singh et al. (2013), non-host resistance emerges when the whole species of a plant is pathogen resistance and compared to resistance mediated by R gene, it is more persistent, thus being a new method for enhancement of crops.
There are two promising tools to exploit for genome editing; the first one is the system of nuclease (Christian et al., 2010;Bogdanove and Voytas, 2011;Schornack et al., 2013) which uses effectors of TAL from Xanthomonas species a pathogenic plant to bind DNA regions that are short in a way that is specific to sequence a method described by Boch et al. (2009).The second method is referred to as modulate gene expression and the system of CRISPR which is a technique that enables creation points of gene mutations in absence of placing additional unwanted DNA that is foreign (Belhaj et al., 2013).
Various techniques have emerged that permit hasty cloning and gathering of standard constructs.For instance, fusion of USER that uses cloning based on excision of uracil (Geu-Flores et al., 2007) and golden gate with its associated organization like golden braid (Engler et al., 2008;Sarrion-Perdigones et al., 2013).Tool kits of molecular studies are also available and permit cloning to be prompt and adjustable as discussed by Engler et al. (2014) and Binder et al. (2014).Such techniques may be utilized both in favor of the prevailing immunity mediated by R gene through assembling of R genes in cassettes of resistance and in recent constructed biology advances.Constructed biology methods have thrilling possibility of forming inductive resistance of diseases in plants.According to Hou et al. (2012), one can direct constructed promoters that are sensible to stress in Arabidopsis.This upcoming technology may aid in dealing with larger problems related to crops inclusive of genome of polyploids (Galletta and Maas, 1990).As described by Wang et al. (2014), three homoalleles of the susceptible gene of powdery mildew Mlo was fruitfully mutated from wheat that was hexaploid for prevention of growth of pathogen.

CONCLUSION
In this work, how plant diseases are being managed through application of molecular techniques was discussed.However, plants are still being affected by diseases thus, there is need for sustained innovation in this area of science to identify more effective strategies.The use of ecologically safe and environmental friendly methods of protecting crops from diseases is gaining importance.Among the various methods of control of plant diseases, resistance of host is still the method of preference despite the fact that shortfall of persistence has been a repeated limitation.Advancement in transfer of genes systems in crops is perhaps the most difficult form of research in plants.Presently, the preferred two methods are the biolistic-mediated and Agrobacteriummediated DNA delivery systems.The evolution of nanoparticles for delivery of DNA cells of plants is coming up and the probabilities of incorporating the success of Agrobacterium and biolistic mediated systems is looming.The use of Agrobacterium in transforming plants will continue to have consideration since one avoids the step of tissue culture during regeneration of plants.Genomics has proven to be a potential tool in plant disease management either by targeting virulence of pathogen or by genetic manipulation of the host plant.The most important advantage of utilizing transcriptomics techniques is the capability of being able to carry out detailed studies at transcriptional level of interactions between host plant-pathogens interplay.Some of the other molecular methods are associated with limitations for example.Polymerase chain reaction (PCR) can only be used to find sequences that are known in some details due to primer requirement, but is still a useful tool in crop disease management.Tissue culture on the other hand can be associated with problems in maintaining uniformity and stability of clonally propagated plants and can lead to loss of morphogenetic capacity.Luckily, new advancements will arrive through combination of the available techniques that will enable gene transfer that is accessible and easy to apply plant species in large numbers.