Development Of Disease-resistant Cultivars Research Articles

Enhancing Crop Resilience through CRISPR/Cas9-Mediated Development of Disease-Resistant Cultivars

A major issue in agriculture is the protection of crops against diseases and pests. Plant breeding has been primarily responsible for the growth of disease-resistant cultivars. The use of gene editing techniques in plant breeding is essential for obtaining desired features. Clustered Regular Interspaced Palindromic Repeats (CRISPER)/Cas9 (CRISPR-related protein) is a new advancement in gene editing technology. It can be utilised in plant defence mechanisms against pathogen attack by recognising the bacterial immune system and destroying invasive pathogen genes. Advances in plant breeding through CRISPR/Cas9 integration have helped develop cultivars including hereditary resistance to bacterial and viral diseases. Future crop generations can acquire CRISPR/Cas9-mediated transgene resistance if the Cas9/sgRNA transgene has been isolated in the F1 generation. Cas9/sgRNA transgene separation makes CRISPR/Cas9 safe for use in plant breeding. Although CRISPR/Cas9 has proven to be a wonderful tool to revolutionize plant breeding and develop various disease resistant varieties, its effect on many plant physiological processes remains to be thoroughly investigated.

Complete Genome Assemblies of All Xanthomonas translucens Pathotype Strains Reveal Three Genetically Distinct Clades.

The Xanthomonas translucens species comprises phytopathogenic bacteria that can cause serious damage to cereals and to forage grasses. So far, the genomic resources for X. translucens were limited, which hindered further understanding of the host–pathogen interactions at the molecular level and the development of disease-resistant cultivars. To this end, we complemented the available complete genome sequence of the X. translucens pv. translucens pathotype strain DSM 18974 by sequencing the genomes of all the other 10 X. translucens pathotype strains using PacBio long-read technology and assembled complete genome sequences. Phylogeny based on average nucleotide identity (ANI) revealed three distinct clades within the species, which we propose to classify as clades Xt-I, Xt-II, and Xt-III. In addition to 2,181 core X. translucens genes, a total of 190, 588, and 168 genes were found to be exclusive to each clade, respectively. Moreover, 29 non-transcription activator-like effector (TALE) and 21 TALE type III effector classes were found, and clade- or strain-specific effectors were identified. Further investigation of these genes could help to identify genes that are critically involved in pathogenicity and/or host adaptation, setting the grounds for the development of new resistant cultivars.

​Morphological and Molecular Diversity of Botrytis cinerea Infecting Chickpea in India

Background: The Botrytis gray mold (BGM) caused by Botrytis cinerea Pers. ex Fr., has become a major threat for chickpea cultivation in India. In view of monetary significance of this disease and growing its spread into new regions through seed and soil (sclerotia) encouraged us to study variability of this pathogen from diverse regions. Methods: Botrytis cinerea infected chickpea samples were collected from Punjab and Uttarakhand states. Pathogen was isolated using chickpea dextrose agar media. Pathogenicity was proved by inoculation of spore suspension (3x105 spores’mL-1) of the fungus on susceptible cultivar. In vitro studies were conducted for observations of morphological and cultural variability of the fungus. The pathogenic fungus was also identified through molecular characterization using ITS primers. Result: The isolates were categorized into different groups based on growth rate of the pathogen on PDA media. Pathogenicity test proved that B. cinerea isolates from Punjab were showed less disease severity scale compared to Uttarakhand state isolates. The B. cinerea fungus prefers PDA for efficient growth and multiplication than Chickpea Dextrose Agar (CDA) media. There was no conspicuous difference in colour of the colony on PDA and CDA media. The colour of the mycelium were initially white or dirty white or greyish and sporulated culture shown to have as grey with profuse mass on the surface of the mycelium. The fungus B. fabae, a different species was also responsible for causing BGM disease which was confirmed through pathogenicity and ITS sequencing. Twenty-eight isolates were studied for diversity in cultural, morphological and molecular level. The diversity in colony colour, growth type and diameter of mycelial growth, sclerotia initiation, number and pattern of sclerotia formation were observed amongst the isolates. This study would help in designing breeding strategy for development of disease resistant cultivars.

Genetics of Fusarium head blight resistance in three wheat genotypes

Fusarium head blight (FHB) is global concern as recent outbreaks reported in Canada, Europe, Asia, Australia and South America. The disease has emerged as one of the most important plant diseases worldwide in 21st century. One of the major threats posed by FHB fungus is the mycotoxin production which is harmful to human and animal health. Development of disease resistant cultivars is the only effective method for managing the disease. Control of these pathogen / Fusarium spp. is also challenging due to limited sources of known resistance. The famous Chinese wheat cultivar Sumai 3 and Frontana are the main sources of resistance to this disease. For genetic analysis and incorporation of FHB resistance into recently released high yielding wheat cultivars, HD 2967 and DPW 621-50, crosses were made with Sumai 3, Frontana and Aldan. The F2 plants from the crosses HD 2967/Frontana (140), HD 2967/Aldan (150), HD 2967/Sumai 3 (169) and DPW 621-50/Sumai 3 (182) were screened for resistance under controlled conditions. Disease score was recorded to identify resistant, moderately resistant and susceptible plants. The genetic ratios for resistance to FHB indicated a complex nature of resistance in all the three donors.

Use of Crispr/Cas9 for Development of Disease Resistant Cultivars in Plant Breeding

Crop protection against pests and diseases is a major challenge in agriculture. Plant breeding is a key solution for the development of disease resistant cultivars. Gene editing is an indispensable part of plant breeding to obtain desirable traits in crops. CRISPR (Clustered Regular Interspaced Palindromic Repeats)/Cas9 (CRISPR- associated protein) is a recent breakthrough in gene editing technology. It can be utilized to exploit defensive mechanism in plants against pathogen attack with recognition and degradation of the invading pathogenic genes by bacterial immune system. Advances in plant breeding with integration of CRISPR/Cas9 have facilitated the production of cultivars with heritable resistance to viral and bacterial disease. CRISPR/Cas9 mediated genetically engineered resistance can be inherited to further generation of crops after segregation of Cas9/sgRNA transgene in F1 generation. The segregation of Cas9/sgRNA transgene prevents undesirable genome modification in successive generation and makes use of CRISPR/Cas9 safe in plant breeding. CRISPR/Cas9 proves itself as a fascinating tool to revolutionize plant breeding for the development of various disease resistant cultivars however, effects of CRISPR/Cas9 system on different physiological process of plants still needs to be studied.Int. J. Appl. Sci. Biotechnol. Vol 5(4): 403-409

Genome-Wide Distribution, Organisation and Functional Characterization of Disease Resistance and Defence Response Genes across Rice Species.

The resistance (R) genes and defense response (DR) genes have become very important resources for the development of disease resistant cultivars. In the present investigation, genome-wide identification, expression, phylogenetic and synteny analysis was done for R and DR-genes across three species of rice viz: Oryza sativa ssp indica cv 93-11, Oryza sativa ssp japonica and wild rice species, Oryza brachyantha. We used the in silico approach to identify and map 786 R -genes and 167 DR-genes, 672 R-genes and 142 DR-genes, 251 R-genes and 86 DR-genes in the japonica, indica and O. brachyanth a genomes, respectively. Our analysis showed that 60.5% and 55.6% of the R-genes are tandemly repeated within clusters and distributed over all the rice chromosomes in indica and japonica genomes, respectively. The phylogenetic analysis along with motif distribution shows high degree of conservation of R- and DR-genes in clusters. In silico expression analysis of R-genes and DR-genes showed more than 85% were expressed genes showing corresponding EST matches in the databases. This study gave special emphasis on mechanisms of gene evolution and duplication for R and DR genes across species. Analysis of paralogs across rice species indicated 17% and 4.38% R-genes, 29% and 11.63% DR-genes duplication in indica and Oryza brachyantha, as compared to 20% and 26% duplication of R-genes and DR-genes in japonica respectively. We found that during the course of duplication only 9.5% of R- and DR-genes changed their function and rest of the genes have maintained their identity. Syntenic relationship across three genomes inferred that more orthology is shared between indica and japonica genomes as compared to brachyantha genome. Genome wide identification of R-genes and DR-genes in the rice genome will help in allele mining and functional validation of these genes, and to understand molecular mechanism of disease resistance and their evolution in rice and related species.

Mapping of resistance to spot blotch disease caused by Bipolaris sorokiniana in spring wheat

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat growing regions of the world. The development of disease resistant cultivars is considered as the most effective control strategy for spot blotch. An intervarietal mapping population in the form of recombinant inbred lines (RILs) was developed from a cross 'Yangmai 6' (a Chinese source of resistance) x 'Sonalika' (a spot blotch susceptible cultivar). The 139 single seed descent (SSD) derived F(6), F(7), F(8) lines of 'Yangmai 6' x 'Sonalika' were evaluated for resistance to spot blotch in three blocks in each of the 3 years. Joint and/or single year analysis by composite interval mapping (CIM) and likelihood of odd ratio (LOD) >2.2, identified four quantitative trait loci (QTL) on the chromosomes 2AL, 2BS, 5BL and 6DL. These QTLs were designated as QSb.bhu-2A, QSb.bhu-2B, QSb.bhu-5B and QSb.bhu-6D, respectively. A total of 63.10% of phenotypic variation was explained by these QTLs based on the mean over years. Two QTLs on chromosomes 2B and 5B with major effects were consistent over 3 years. All QTL alleles for resistance were derived from the resistant parent 'Yangmai 6'.

Molecular Marker Analysis of Soybean Plant Introductions with Resistance toPhytophthora sojae

ABSTRACT Molecular analysis of sources of resistance to plant pathogens should expedite and confirm novel gene discovery and consequently the development of disease resistant cultivars. Recently, soybean plant introductions (PIs) were identified that contain putative novel Rps genes for resistance to Phytophthora sojae. The number of resistance genes that confer resistance to P. sojae isolates OH17 (1b,1d,2,3a,3b,3c,4,5,6,7) and OH25 (1a,1b,1c,1k,7) was then determined in several of the PIs. The objective of this study was to determine if the Rps genes present in these PIs were associated with eight described Rps loci that have been mapped on soybean molecular linkage groups F, G, J, and N. Nine F(2:3) soybean populations were genotyped with simple sequence repeat (SSR) markers linked to previously mapped Rps loci. The nine PI populations all had SSR markers associated (P < 0.01) with resistance to P. sojae isolate OH17 in the Rps1 region. Rps1c is a likely candidate in eight PIs but novel genes may also be possible, while novel genes may confer resistance in one PI to P. sojae isolate OHI7. Two or more Rps genes, including some that are potentially novel, confer resistance to P. sojae isolate OH25 in eight of the populations. However, based on the response to these two isolates, virulence already exists for at least some of the novel genes identified in this study.

Transcriptional profiling of chickpea genes differentially regulated by salicylic acid, methyl jasmonate and aminocyclopropane carboxylic acid to reveal pathways of defence-related gene regulation

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes and grasspea (Lathyrus sativus L.) expressed sequence tags, chickpea responses to treatments with the defence signalling compounds salicylic acid (SA), methyl jasmonate (MeJA) and aminocyclopropane carboxylic acid (ACC) were studied in three chickpea genotypes with ranging levels of resistance to ascochyta blight [Ascochyta rabiei (Pass.) L.]. The experimental system minimised environmental effects and was conducted in reference design, where samples from untreated controls acted as references against post-treatment samples. Microarray observations were also validated by quantitative reverse transcription-polymerase chain reaction. The time-course expression patterns of 715 experimental microarray features resulted in differential expression of 425 transcripts. The A. rabiei resistant chickpea genotypes showed a more substantial range of defence-related gene induction by all treatments, indicating that they may possess stronger abilities to resist pathogens. Further, the involvement of SA, MeJA and ACC signalling was identified for the regulation of some important A. rabiei responsive transcripts, as well as cross-talk between these pathways. In the current study we also found evidence to suggest the involvement of A. rabiei-specific signalling mechanisms for the induction of several transcripts that were previously implicated in A. rabiei resistance. This study characterised the regulatory mechanisms of many chickpea transcripts that may be important in defence against various pathogens, as well as other cellular functions. These results provide novel insights to the molecular control of chickpea cellular processes, which may assist the understanding of chickpea defence mechanisms and allow enhanced development of disease resistant cultivars.

Identification and mapping of AFLP markers linked to peanut (Arachis hypogaea L.) resistance to the aphid vector of groundnut rosette disease.

Groundnut rosette disease is the most destructive viral disease of peanut in Africa and can cause serious yield losses under favourable conditions. The development of disease-resistant cultivars is the most effective control strategy. Resistance to the aphid vector, Aphis craccivora, was identified in the breeding line ICG 12991 and is controlled by a single recessive gene. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) analysis were employed to identify DNA markers linked to aphid resistance and for the development of a partial genetic linkage map. A F(2:3) population was developed from a cross using the aphid-resistant parent ICG 12991. Genotyping was carried out in the F2 generation and phenotyping in the F3 generation. Results were used to assign individual F2 lines as homozygous-resistant, homozygous-susceptible or segregating. A total of 308 AFLP (20 EcoRI+3/MseI+3, 144 MluI+3/MseI+3 and 144 PstI+3/MseI+3) primer combinations were used to identify markers associated with aphid resistance in the F(2:3) population. Twenty putative markers were identified, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. A single recessive gene was mapped on linkage group 1, 3.9 cM from a marker originating from the susceptible parent, that explained 76.1% of the phenotypic variation for aphid resistance. This study represents the first report on the identification of molecular markers closely linked to aphid resistance to groundnut rosette disease and the construction of the first partial genetic linkage map for cultivated peanut.

Mapping of quantitative trait loci for field resistance to Fusarium head blight in an European winter wheat.

Fusarium head blight (FHB) caused by Fusarium culmorum is an economically important disease of wheat that may cause serious yield and quality losses under favorable climate conditions. The development of disease-resistant cultivars is the most effective control strategy. Worldwide, there is heavy reliance on the resistance pool originating from Asian wheats, but excellent field resistance has also been observed among European winter wheats. The objective of this study was to map and characterize quantitative traits loci (QTL) of resistance to FHB among European winter wheats. A population of 194 recombinant inbred lines (RILs) was genotyped from a cross between two winter wheats Renan (resistant)/Récital (susceptible) with microsatellites, AFLP and RFLP markers. RILs were assessed under field conditions For 3 years in one location. Nine QTLs were detected, and together they explained 30-45% of the variance, depending on the year. Three of the QTLs were stable over the 3 years. One stable QTL, QFhs.inra.2b, was mapped to chromosome 2B and two QTLs QFhs.inra.5a2 and QFhs.inra5a3, to chromosome 5A; each of these QTLs explained 6.9-18.6% of the variance. Other QTLs were identified on chromosome 2A, 3A, 3B, 5D, and 6D, but these had a smaller effect on FHB resistance. One of the two QTLs on chromosome 5A was linked to gene B1 controlling the presence of awns. Overlapping QTLs for FHB resistance were those for plant height or/and flowering time. Our results confirm that wheat chromosomes 2A, 3A, 3B, and 5A carry FHB resistance genes, and new resistance factors were identified on chromosome arms 2BS and 5AL. Markers flanking these QTLs should be useful tools for combining the resistance to FHB of Asian and European wheats to increase the resistance level of cultivars.

Resistência de linhagens de feijoeiro a quatro raças de Uromyces appendiculatus isoladas em Minas Gerais, Brasil

A identificação de fontes de resistência é uma etapa básica em programas de melhoramento que visam o desenvolvimento de cultivares resistentes a doenças. Neste trabalho, foram inoculadas, sob condições controladas, oito linhagens de feijoeiro-comum (Phaseolus vulgaris) desenvolvidas pelo Departamento de Agricultura dos Estados Unidos, com quatro raças conhecidas de Uromyces appendiculatus coletadas no estado de Minas Gerais, Brasil. A cultivar Ouro Negro foi utilizada como padrão de resistência e US Pinto 111 (suscetível universal) como padrão de suscetibilidade. Foi avaliada a freqüência de infecção (FI) e estimado o tamanho médio das pústulas (TMP). As linhagens BelMiDak-RMR-12 e BelMiDak-RR-3 destacaram-se como as mais resistentes, apresentando, para todas as raças, apenas pústulas (com diâmetro &lt; 300mim) na face abaxial da folha. As linhagens BelDakMi-RMR-10 e BelNeb RR-2 apresentaram baixa FI média e foram resistentes a quatro e três raças, respectivamente. A linhagem BelDak-RR-2, apesar de sua resistência a todas as raças, apresentou uma FI média relativamente alta. As linhagens BelMiNeb-RMR-3 e BelDakMi RR-7 também foram resistentes às quatro raças testadas, porém apresentaram alta FI média e algumas pústulas com diâmetro maior que 300 mim. A linhagem BelDakMi-RMR-11 foi resistente às raças 45, 46 e 49 e moderadamente resistente à raça 52. A cultivar Ouro Negro foi resistente às quatro raças, com um nível de resistência igual às melhores linhagens norte americanas.

Disease Pressure on Soybean in Illinois

Development of disease resistant cultivars is a primary emphasis of many soybean (Glycine max) breeding programs. By understanding the magnitude and frequency of economic loss caused by soybean pathogens, breeding programs can establish breeding priorities. Therefore, 13 yr of disease monitoring data comprising 89 trials and 52 cultivars were analyzed to determine disease pressure on soybeans in Illinois. Of the 12 diseases observed, brown spot (Septoria glycines) was the predominant foliar disease, while brown stem rot (Phialophora gregata) was the most frequent soilborne disease

Use of Detached Leaves to Evaluate Tobacco Haploids and Doubled Haploids for Resistance to Tobacco Mosaic Virus,Meloidogyne incognita,andPseudomonas syringaepv.tabaci

Rufty, R. C., Wernsman, E. A., and Gooding, G. V., Jr. 1987. Use of detached leaves to evaluate tobacco haploids and doubled haploids for resistance to tobacco mosaic virus, Meloidogyne incognita, and Pseudomonas syringae pv. tabaci. Phytopathology 77:60-62. Development of disease-resistant cultivars in self-pollinated crops like virus Y. Detached leaves were maintained by immersing their petioles in tobacco (Nicotiana tabacum) can be greatly accelerated by evaluating water until symptoms appeared. Symptoms in detached leaves were similar populations of haploid plants derived from F1 hybrids resistant to various to those in intact plants, and disease reactions corresponded with wholediseases. A limitation of haploid breeding is the need to assess reactions to plant determinations. The technique can also be used with doubled haploid multiple pathogens on single plants. To avoid confounding systemic or or diploid populations segregating for disease resistance. The original lethal effects from inoculations with multiple pathogens, detached leaves intact plants with identified resistance may be evaluated for other traits, were inoculated separately with tobacco mosaic virus and Pseudomonas and susceptible genotypes may be discarded. Field evaluations for syringae pv. tabaci. Resistance to Meloidogyne incognita was identified by agronomic characteristics can then be performed on a population fixed for the associated reactions of detached leaves to the MsN R strain of potato disease resistance genes.