Development Of New Resistant Cultivars Research Articles

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.

Identification and Characterization of NBS Resistance Genes in Akebia trifoliata.

Akebia trifoliata is an important multiuse perennial plant that often suffers attacks from various pathogens due to its long growth cycle, seriously affecting its commercial value. The absence of research on the resistance (R) genes of A. trifoliata has greatly limited progress in the breeding of resistant varieties. Genes encoding proteins containing nucleotide binding sites (NBSs) and C-terminal leucine-rich repeats (LRRs), the largest family of plant resistance (R) genes, are vital for plant disease resistance. A comprehensive genome-wide analysis showed that there were only 73 NBS genes in the A. trifoliata genome, including three main subfamilies (50 coiled coil (CC)-NBS-LRR (CNL), 19 Toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) and four resistance to powdery mildew8 (RPW8)-NBS-LRR (RNL) genes). Additionally, 64 mapped NBS candidates were unevenly distributed on 14 chromosomes, most of which were assigned to the chromosome ends; 41 of these genes were located in clusters, and the remaining 23 genes were singletons. Both the CNLs and TNLs were further divided into four subgroups, and the CNLs had fewer exons than the TNLs. Structurally, all eight previously reported conserved motifs were identified in the NBS domains, and both their order and their amino acid sequences exhibited high conservation. Evolutionarily, tandem and dispersed duplications were shown to be the two main forces responsible for NBS expansion, producing 33 and 29 genes, respectively. A transcriptome analysis of three fruit tissues at four developmental stages showed that NBS genes were generally expressed at low levels, while a few of these genes showed relatively high expression during later development in rind tissues. Overall, this research is the first to identify and characterize A. trifoliata NBS genes and is valuable for both the development of new resistant cultivars and the study of molecular mechanisms of resistance.

Breeding for resistance to tomato bacterial wilt: Identification of potential sources of genetic resistance by field evaluation of 28 tomato cultivars in Daloa, Côte d’Ivoire

Bacterial wilt caused by Ralstonia solanacearum is one of the most damaging tomato diseases in the world. The best strategy for controlling this disease is to use genetically resistant tomato plants. In this study, 28 tomato cultivars were evaluated in the field for their resistance to bacterial wilt in Daloa, one of the most important agricultural regions of Côte d'Ivoire. The experiments were carried out during the dry and rainy season and the experimental design was a Randomized Complete Block Design (RCBD) with three replications. The results showed the presence of wilt disease. The streaming test and bacterial culture on growth medium proved the bacterial origin of the wilt symptoms observed. The results of the staining reactions revealed short, straight, rod and Gram negative bacteria similar to R. solanacearum. The severity of the disease was found to be higher in the dry season than in the rainy season. Of 28 cultivars evaluated, 7 cultivars were resistant and did not show any diseased plants during the two study periods. These cultivars constitute interesting sources of resistance for a breeding program aimed at the development of new resistant cultivars adapted to the region of Daloa. Keywords: Bacterial wilt, Ralstonia solanacearum, resistance, tomato, Solanum lycopersicum, breeding program.

Elucidating genetic variability and population structure in Venturia inaequalis associated with apple scab diseaseusing SSR markers.

Apple scab caused by Venturia inaequalis Cooke (Wint.) is one the important diseases of trade and industrial significance in apple. In present study variability studies in pathogen isolates were studied, which is one of the most important factors for devising management studies of scab disease in apple. Genetic diversity of 30 Venturia inaequalis isolates from 12 districts of two geographical distinct regions of Jammu and Kashmir was calculated based on the allele frequencies of 28 SSR markers and the internal transcribed spacer (ITS) region of the ribosomal DNA. The ITS based characterized sequences were submitted to NCBI GenBank and accession numbers were sanctioned. Dendrogram showed that all the accessions formed 2 main clusters with various degree of sub clustering within the clusters. Analysis based on SSR study reveals that the heterozygosity ranged from 0.0 and 0.5, with an average value of 0.39. The expected heterozygosis or gene diversity (He) ranged from 0.0 to 0.50 with an average of 0.40. The Fst value ranges from 0 to 0.6 with an average of 0.194. Diversity within each population (HS) values ranging from 0.26 to 0.33. Average differentiation among populations (GST) was 0.11 and populations were isolated by significant distance (r 2 = 0.50, P < 0.01). From the AMOVA analysis, 25% of variation was observed among population, 9% among individuals and 66% within individuals observed in the population. Structure analysis grouped isolates into two populations. Principle coordinate analysis explained variation of 36.6% in population 1, 14.30% in population 2 and 13.10% in population 3(Admixture) with 64.07% as overall cumulative percentage of variation. This indicates that extensive short-distance gene flow occurs in Kashmir region that dispersal over longer distances also appears to occur frequently enough to prevent differentiation due to genetic drift. Also it is evident that Jammu and Kashmir most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the Jammu region compared to the mountainous inland Kashmir region. The results of present study would help to understand the genetic diversity of V. inaequalis from Jammu and Kashmir that would lead in the development of more effective management strategies and development of new resistant cultivars through marker-assisted selection.

The Rpv3-3 haplotype and stilbenoid induction mediate downy mildew resistance in a grapevine interspecific population

The cultivated Eurasian grapevine (Vitis vinifera L.) is highly susceptible to downy mildew (DM) – caused by the biotrophic oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni – the major disease of temperate-humid climates among various pathogen threats. DM control relies mainly on the massive use of fungicides leading to environmental pollution, development of resistance and residual toxicity. The exploitation of DM-resistant wild genetic resources for the development of new resistant cultivars represents a promising alternative. Taking advantage of a segregating population derived from ‘Merzling’ (M, a mid-resistant hybrid) and ‘Teroldego’ (T, a susceptible landrace), recent studies have highlighted the importance of stilbenoids among phenolic compounds in conferring resistance to this oomycete. In order to elucidate the genetic bases of DM resistance and polyphenol biosynthesis upon P. viticola inoculation, 136 M×T F1 individuals were characterized by an integrative approach combining genetic, phenotypic and gene-expression data. An improved M×T linkage map was obtained by scoring 192 microsatellite markers. The progeny was further screened for degree of resistance and production of 42 phenolic compounds (including 18 different stilbenoids). Quantitative trait locus (QTL) mapping showed that DM resistance is associated with a specific haplotype at the Rpv3 locus – herein named Rpv3-3, derived from the French hybrid ‘Seyval’ – and identified 46 novel metabolic (m)QTLs linked to 30 polyphenol-related parameters. A list of the 76 most relevant candidate genes was generated by specifically exploring the genomic regions underlying the mQTLs associated with the stilbenoids induced by the infection. Finally, expression analysis of 13 genes in Rpv3-3+/- genotypes, displaying divergent DM resistance and stilbenoid accumulation, revealed significant candidates for the genetic control of stilbenoid biosynthesis and oligomerization. These findings emphasize that DM resistance can be mediated by the major Rpv3-3 locus and stilbenoid induction.

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

Soybean genotype analysis for cyst nematode resistance

Some species of soybean cultivars have the characteristic of resistance or alteration in survival skills, and perpetuation of nematodes (female index - FI), whose process is activated when the nematode parasitism starts and the development of new resistant cultivars is primordial to the preservation of plant productivity. The objective of this study was to identify the reaction of 101 soybean genotypes and two standards, one susceptible (M-SOY 8866) and the other resistant (BRSGO Raissa) of the Germplasm Laboratory of Federal University of Uberlândia (LAGER/UFU) improvement program, after inoculation of Heterodera glycines race 3 eggs. The evaluations 35 days after inoculation consisted of the fresh and dry weight of roots, root volume, the number of females (NF), and females index (FI). Genotypes 46 (F5: 6 BRSGO Caiaponia x 100 IAC) and 11 (Engopa 316 RR - Sel. 4) performed moderate resistance; the genotype 61 (F5: 6 Caiaponia x Potenza) had moderate susceptibility; and the rest of the genotypes presented susceptible. Between the genotypes, NF ranged from 9.5 to 159.7 and FI ranged from 17 to 214.8. The coefficient of genotypic determination (H2) was 70.28 (high) for NF, indicating genetic variation in the genotypes tested. Estimates of genotypic (rg), phenotypic (rf), and environmental (ra) correlation coefficients had lower magnitude values (<0.70). Analysis of the simple Pearson correlation was significant and positive between the variables dry weight (g) and root volume (cm3) but was not related to the resistance to the cyst nematode in the assay.

Fusarium wilt of cotton: Management strategies

Cotton is a globally important crop used for both its natural fiber and seed. Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. vasinfectum, is a major disease of cotton capable of causing significant economic loss. The fungus persists in soil as chlamydospores and in association with the roots of susceptible, resistant and non-cotton hosts as well as in seed. Management of Fusarium wilt is difficult and most successfully achieved through the use of resistant cultivars and pathogen-free cotton seed. Once inoculum has been introduced into the field, strategies such as soil solarization and fumigation are applied to manage inoculum levels. While experimentally successful, these techniques are of limited use in a commercial setting. Management of Fusarium wilt will continue to rely upon the development of new resistant cultivars. Additionally, understanding the mechanism of seed colonization and developing high throughput seed treatment and testing protocols will allow the industry to effectively avoid the introduction of inoculum.

QTL for resistance to root lesion nematode (Pratylenchus thornei) from a synthetic hexaploid wheat source.

A whole genome average interval mapping approach identified eight QTL associated with P. thornei resistance in a DH population from a cross between the synthetic-derived wheat Sokoll and cultivar Krichauff. Pratylenchus thornei are migratory nematodes that feed and reproduce within the wheat root cortex, causing cell death (lesions) resulting in severe yield reductions globally. Genotypic selection using molecular markers closely linked to Pratylenchus resistance genes will accelerate the development of new resistant cultivars by reducing the need for laborious and expensive resistance phenotyping. A doubled haploid wheat population (150 lines) from a cross between the synthetic-derived cultivar Sokoll (P. thornei resistant) and cultivar Krichauff (P. thornei moderately susceptible) was used to identify quantitative trait loci (QTL) associated with P. thornei resistance. The resistance identified in the glasshouse was validated in a field trial. A genetic map was constructed using Diversity Array Technology and the QTL regions identified were further targeted with simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers. Six significant and two suggestive P. thornei resistance QTL were detected using a whole genome average interval mapping approach. Three QTL were identified on chromosome 2B, two on chromosome 6D, and a single QTL on each of chromosomes 2A, 2D and 5D. The QTL on chromosomes 2BS and 6DS mapped to locations previously identified to be associated with Pratylenchus resistance. Together, the QTL on 2B (QRlnt.sk-2B.1-2B.3) and 6D (QRlnt.sk-6D.1 and 6D.2) explained 30 and 48% of the genotypic variation, respectively. Flanking PCR-based markers based on SSRs and SNPs were developed for the major QTL on 2B and 6D and provide a cost-effective high-throughput tool for marker-assisted breeding of wheat with improved P. thornei resistance.

Genetic analysis and identification of DNA markers linked to a novel Phytophthora sojae resistance gene in the Japanese soybean cultivar Waseshiroge

The Glycine max (L.) Merr. cultivar Waseshiroge is highly resistant to several races of Phytophthora sojae in Japan. In order to determine which Rps gene might be present in Waseshiroge, 15 differential cultivars were challenged with 12 P. sojae isolates. None had a reaction pattern identical to that of Waseshiroge, indicating that Waseshiroge may contain a novel Rps gene. In order to characterize the inheritance of Waseshiroge resistance to P. sojae isolates, 98 F2 progeny and 94 F7:8 lines were produced from crosses between the susceptible cultivar Tanbakuro and Waseshiroge. Chi-square tests indicated that segregation fit a 3:1 ratio for resistance and susceptibility in two F2 sub-populations of 42 and 56 seedlings. This and a 46.27:1.46:46.27 (or 63:2:63) ratio for resistance: segregation: susceptibility among the 94 F7:8 lines indicated that resistance was controlled by a single dominant gene. DNA analyses were carried out on Tanbakuro, Waseshiroge and the 94 F7:8 lines, and a linkage map was constructed with 17 SSR markers and nine new primer pairs that amplify marker loci linked to Rps1 on soybean chromosome 3 (linkage group N). The closest markers, Satt009 and T000304487l, map to locations 0.9 and 1.6 cM on each side of the estimated position of the Rps gene, respectively. The results showed that the Rps gene in Waseshiroge is either allelic to Rps1, or resides at a tightly linked locus in a gene cluster. A three-way-contingency table analysis indicated that marker-assisted selection with the two flanking markers could be used in the development of new resistant cultivars.

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of Verticillium dahliae in the lettuce cultivar La Brillante.

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F(1) and F(2) families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R(2) = 0.49-0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.

POTENTIAL IMPACT OF A NEW HIGHLY VIRULENT RACE OF FUSARIUM OXYSPORUM F. SP. NIVEUM IN WATERMELON IN THE USA

Fusarium wilt of watermelon was first reported in the United States (U.S.) in 1894. Although variation in virulence within the pathogen population, Fusarium oxysporum f. sp. niveum, had been observed, differentiation of isolates into races did not occur for 70 years. Currently, three races (0, 1, and 2) of F. oxysporum f. sp. niveum have been described. Races 0 and 1 were first described in 1963 and race 2 was described in 1973. Although race 1 is the predominant race, race 2 appears to be spreading in the U.S. and its presence has been confirmed in eight states. Losses from Fusarium wilt were managed for many years through the use of cultivars that were resistant to race 1. However, the prevalence and severity of Fusarium wilt in watermelon has been increasing in the eastern U.S. in the past decade. This increase has coincided with a large increase in production of triploid watermelon cultivars. Most triploid cultivars evaluated in 2002 and 2005 in a field with a mixture of races 1 and 2 were rated susceptible to Fusarium wilt. In 2009 cultivars which were reported to be resistant to F. oxysporum f. sp. niveum race 1 were evaluated and many also exhibited severe wilt. During surveys of the watermelon production region in Maryland and Delaware from 2000 to 2007, some isolates of F. oxysporum f. sp. niveum were found that appeared to be more virulent than any of the previously described races. Two isolates of F. oxysporum f. sp. niveum collected in Maryland in 2001 along with reference isolates of races 0, 1, and 2 were compared for virulence, host range and vegetative compatibility. Race identification was made on eight differential watermelon genotypes. Both Maryland isolates were highly virulent, causing 65-100% wilt on all differentials, two of which were PI-296341-FR (FL) and PI-296341-FR (OK) considered highly resistant to race 2. Both isolates were non-pathogenic on muskmelon, cucumber, pumpkin and squash. Two additional isolates collected in Maryland in 2007 also were tested. These studies demonstrated that the four Maryland isolates were more virulent than race 2 and are proposed as a new race 3. Although most fields surveyed had a predominant population of race 1, the presence of F. oxysporum f. sp. niveum race 2 and the discovery of race 3 highlight the need for the development of new resistant cultivars.

Relative susceptibility of banana cultivars to Xanthomonas campestris pv. musacearum

The banana Xanthomonas wilt (BXW) is the most devastating disease of banana in Great Lakes region of East and Central Africa. The pathogen kills plants quickly and spreads rapidly over a large area in a short time making the disease one of the most dreaded in banana. The disease affects almost all varieties of commonly grown banana cultivars. Some knowledge of the relative susceptibility of banana cultivars would be extremely useful and could be a basis for management strategies for BXW. Ten banana cultivars were evaluated for their relative susceptibility to Xanthomonas campestris pv. musacearum. All the ten cultivars were tested by injecting bacterial inoculum in pseudostem of in vitro plantlets as well as potted plants. The various banana cultivars showed significant differences in susceptibility to Xanthomonas campestris pv. musacearum. The beer banana cultivar ‘Pisang Awak’ and dessert cultivars ‘Dwarf Cavendish’, ‘Giant Cavendish’, and ‘FHIA-17’ were found to be highly susceptible, where as East African Highland banana cultivar ‘Nakitembe’ was found to be least susceptible. The other cultivars ‘Mpologoma’, ‘Mbwazirume’, ‘Sukali Ndiizi’ and ‘FHIA-25’, were found to be susceptible. Diploid parent ‘Musa balbisiana’ was found to be resistant. This study clearly highlights the need for development of new resistant cultivars for BXW disease control, as all the commercial cultivars are susceptible.

Inheritance of Phytophthora root rot resistance in red raspberry determined by generation means and molecular linkage analysis

Classical and molecular methodologies were used to determine the inheritance of Phytophthora root rot (PRR) resistance in red raspberry. The varieties 'Latham' and 'Titan,' resistant and susceptible, respectively, were used to create F(1), F(2), B(1), B(2), and S(1) populations for analysis. Generational means analysis was used to calculate the components of genetic variation and estimates of narrow and broad sense heritability for the plant disease index and the incidence of petiole lesions. The plant disease index showed additive genetic variation with additional significant interactions, but the incidence of petiole lesions was non-additive. A dominant, two-gene model was shown to be the best fit for the observed segregation ratios when classification for resistance was based on a combination of all criteria measured. Molecular linkage maps were generated from the segregating B(2) population. Linkage maps of both parents were constructed from amplified fragment length polymorphism (AFLP), Random amplified polymorphic DNA (RAPD), and uncharacterized resistant gene analog polymorphism (RGAP) markers with seven linkage groups each totaling 440 and 370 cM of genetic distance, respectively. An analysis of the distributional extremes of the B(2) population identified several RAPD markers clustered on two linkage groups associated with PRR resistance. QTL analysis identified two similar genomic regions on each map that explained significant percentages of phenotypic variation observed for the disease assessment criteria. Genetic mapping supports the dominant two-gene model developed from generational means analysis. The results reconcile conflicting reports on inheritance of PRR resistance, provide a basis for further investigation of durable resistance to Phytophthora caused diseases, and indicates that recurrent selection is the appropriate approach for the development of new resistant cultivars.

Allelic relationships for genes that confer resistance to angular leaf spot in common bean

Angular leaf spot is one of the major diseases of the common bean. The extensive genetic variability of this pathogen requires the constant development of new resistant cultivars. Different sources of resistance have been identified and characterized. For the State of Minas Gerais, Brazil, four main resistance sources were found: Mexico 54, AND 277, MAR 2 and Cornell 49-242. Independent characterization of these genotypes demonstrates that resistance in all four sources is dominant and monogenic. However, there are no studies on the relationship and independence of these genes. In the present work, allelism tests were carried out to understand the relationship among the resistance genes present in these four resistance sources. The data revealed a much higher complexity in the resistance inheritance of these genes than previously reported. It was demonstrated that Cornell 49-242 possesses a dominant gene (Phg-3); Mexico 54 possesses three genes, denominated Phg-2, Phg-5 and Phg-6. In MAR 2, two genes were found, one independent designated Phg-4 and the other, an allelic form of Phg-5, denominated of Phg-52. Allelic forms were also found in AND 277, Phg-22, Phg-32 and Phg-42. These results have special importance for breeding programs aiming to pyramid resistance genes.

Inheritance of Resistance to Orobanche cernua Loefl. in Six Sunflower Lines

Sunflower (Helianthus annuus L.) is severely affected by broomrape (Orobanche cernua Loefl.) in the main crop areas of the Old World. The appearance of new virulent broomrape populations has prompted the search for new sources of resistance. The objectives of this study were to elucidate the inheritance of sources of resistance from different origins and to determine allelic relationships between the resistance genes. Six resistant sunflower lines (one of them with resistance gene Or 5 ), two susceptible lines, the F 1 crosses between resistant and susceptible lines and resistant and resistant lines, and the F 2 and BC 1 generations were evaluated for broomrape resistance with the widespread highly virulent population SE 194. Genetic ratios from segregating generations indicated that resistance to O. cernua in these lines was conferred by a single dominant gene. None of the crosses between resistant lines produced susceptible F 2 or BC 1 plants. However, the reaction of the resistant lines to broomrape populations from different areas and years showed differences to new highly virulent populations. Only two lines were resistant to all populations, indicating that resistance in these lines was conferred by additional dominant alleles at the Or locus or by a cluster of very tightly linked non-allelic genes. The resistance found in the two cultivated lines against the new populations, which overcome the Or 5 resistance gene, is an important finding and will aid the development of new resistant cultivars since the current resistant hybrids are based on this gene. Results from this study can also be used to establish differential lines against the new broomrape populations.