Resistance of sugar beet to the cyst-nematode Heterodera schachtii Schm.

Lines exhibiting heritable resistance and susceptibility to beet cyst nematode were selected from a heterogeneous cultivated variety of beet but no major gene resistance was detected although many hundreds of lines of wild and cultivated varieties were tested. Recurrent selection improved the resistance of one line, selected originally for yield and non-bolting qualities, when progenies in each generation were compared with susceptible controls and with the original ancestral lines. Resistance was inherited by progenies from selections crossed with susceptible plants, but evidence of some reduction in resistance was detectable where fewer eelworms were available to invade from the soil. There was no evidence of heritable cytoplasmic influence on the resistance. Resistance is probably controlled by a polygene system in sugar beet, although immunity in Beta procumbens and B. webbiana and near-immunity in B. patellaris suggest that in other species of Beta major gene resistance may occur.     

Genetic localization of four genes for nematode (Heterodera schachtii Schm.) resistance in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is highly susceptible to the beet cyst nematode (Heterodera schachtii Schm.). Three resistance genes originating from the wild beets B. procumbens (Hs1 ^pro-1) and B. webbiana (Hs1 ^web-1, Hs2 ^web-7) have been transferred to sugar beet via species hybridization. We describe the genetic localization of the nematode resistance genes in four different sugar beet lines using segregating F₂ populations and RFLP markers from our current sugar beet linkage map. The mapping studies yielded a surprising result. Although the four parental lines carrying the wild beet translocations were not related to each other, the four genes mapped to the same locus in sugar beet independent of the original translocation event. Close linkage (0–4.6 cM) was found with marker loci at one end of linkage group IV. In two populations, RFLP loci showed segregation distortion due to gametic selection. For the first time, the non-randomness of the translocation process promoting gene transfer from the wild beet to the sugar beet is demonstrated. The data suggest that the resistance genes were incorporated into the sugar beet chromosomes by non-allelic homologous recombination. The finding that the different resistance genes are allelic will have major implications on future attempts to breed sugar beet combining the different resistance genes.     

Field performance of chitinase transgenic silver birches (<Emphasis Type="Italic">Betula pendula</Emphasis>): resistance to fungal diseases

A field trial of 15 transgenic birch lines expressing a sugar beet chitinase IV gene and the corresponding controls was established in southern Finland to study the effects of the level of sugar beet chitinase IV expression on birch resistance to fungal diseases. The symptoms caused by natural infections of two fungal pathogens, Pyrenopeziza betulicola (leaf spot disease) and Melampsoridium betulinum (birch rust), were analysed in the field during a period of 3 years. The lines that had shown a high level of sugar beet chitinase IV mRNA accumulation in the greenhouse also showed high sugar beet chitinase IV expression after 3 years in the field. The level of sugar beet chitinase IV expression did not significantly improve the resistance of transgenic birches to leaf spot disease. Instead, some transgenic lines were significantly more susceptible to leaf spot than the controls. The level of sugar beet chitinase IV expression did have an improving effect on most parameters of birch rust; the groups of lines showing high or intermediate transgene expression were more resistant to birch rust than those showing low expression. This result indicates that the tested transformation may provide a tool for increasing the resistance of silver birch to birch rust.     

DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome additions and translocations originating from wild beets of the Procumbentes section

Summary Genes conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) have been transferred to sugar beet (Beta vulgaris L.) from three wild species of the Procumbentes section using monosomic addition and translocation lines, because no meiotic recombination occurs between chromosomes of cultured and wild species. In the course of a project to isolate the nematode resistance genes by strategies of reverse genetics, probes were cloned from DNA of a fragmented B. procumbens chromosome carrying a resistance gene, which had been isolated by pulsed-field gel electrophoresis. One probe (pRK643) hybridized with a short dispersed repetitive DNA element, which was found only in wild beets, and thus may be used as a molecular marker for nematode resistance to progenies of monosomic addition lines segregating resistant and susceptible individuals. Additional probes for the resistance gene region were obtained with a polymerase chain reaction (PCR)-based strategy using repetitive primers to amplify DNA located between repetitive elements. One of these probes established the existence of at least six different chromosomes from wild beet species, each conferring resistance independently of the others. A strict correlation between the length of the wild beet chromatin introduced in fragment addition and translocation lines and the repeat copy number has been used physically to map the region conferring resistance to a chromosome segment of 0.5-3 Mb.     

Inherited resistance of sugar beet to aphid colonization

Results of glasshouse experiments have confirmed that inbred lines of sugar beet differ in each of three types of resistance to Myzus persicae Sulz. and Aphis fabae Scop., namely: resistance to settling, resistance to multiplication, and tolerance. Resistance to multiplication was not invariably associated with resistance to settling, although plants of some lines showed both forms of resistance. Plants that were resistant to settling of alatae were not always resistant to apterae of the same species, and there was not a close relationship between resistance to M. persicae and to A. fabae. The mechanisms involved in resistance to aphids in sugar beet are not understood. Progenies of plants, selected for resistance to aphids from inbred lines, were often more resistant than progenies of unselected plants. Inheritance of each type of resistance is probably polygenic. The potential value of the different kinds of resistance, in reducing direct feeding damage and controlling the spread of virus yellows in the field, is discussed. The ultimate breeding objective is to produce commercial varieties in which appropriate kinds of resistance to aphids are combined with resistance to virus yellows. The use of such varieties would reduce the need to control aphids in the field by applications of chemicals.     

Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis

 Thirty sugar beet (Beta vulgaris) lines conferring complete resistance to the beet cyst nematode (BCN, Heterodera schachtii) originating from interspecific crosses with wild beets of the section Procumbentes (B. procumbens, B. webbiana and B. patellaris) were investigated by morphology and wild beet-specific molecular markers. The beet lines carrying chromosome mutations consisted of monosomic additions (2n=18+1), fragment additions (2n=18+fragment) and translocations (2n=18) from the wild beets. Genome-specific single-copy, satellite and repetitive probes were applied to study the origin, chromosomal assignment and presence of nematode resistance genes. Within the wild beet species at least three different resistance genes located on different chromosomes were distinguished: Hs1 on the homoelogous chromosomes I of each species, Hs2 on the homoelogous chromosomes VII of B. procumbens and B. webbiana and Hs3 on chromosome VIII of B. webbiana. A clear distinction between the three chromosomes was possible by morphological and molecular means. The translocation lines were separated into two different groups: one containing the resistance gene Hs1 from chromosome I and the other carrying a different nematode resistance gene. The molecular data combined with sequence analyses of Hs1 of the three wild beet species revealed a clear distinction between B. procumbens and B. webbiana. The evolutionary and taxonomical relationship of these species supporting the idea of three different species originating from a common ancestor is discussed. Received: 6 April 1998 / Accepted: 22 April 1998     

The control of Alternaria species on leaves of sugar beet infected with yellowing viruses

Leaves of virus-free sugar-beet plants rarely became infected with Alternaria spp. in two field experiments at Cambridge in 1965. Infection with beet yellows virus (BYV) increased susceptibility of plants to Alternaria only slightly but infection with beet mild yellowing virus (BMYV) increased it greatly. There was a close association between the severity of Alternaria symptoms, shown by different breeding lines and varieties of sugar beet, and the losses of sugar yield which they sustained after infection with BYV and BMYV. Many lines and varieties were resistant to Alternaria even when infected with BMYV and their resistance seemed to be inherited as a dominant character. Individual plants of any one line or variety differed greatly in resistance to Alternaria, suggesting that selection should improve the present level of resistance. Spraying the foliage of Alternaria-susceptible varieties with fungicides had little effect on the severity of Alternaria symptoms or on sugar yield. This was probably because the wet summer of 1965 was ideal for the spread of Alternaria and because rain washed the fungicide deposits from the sprayed leaves.     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants’ phenotypic and molecular markers’ segregation was carried out. At a highly infested field site, some 187 F₂ and 66 F₃ individuals, derived from a cross between lines ‘NK-310mm-O’ (highly resistant) and ‘NK-184mm-O’ (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 ‘NK-310mm-O’-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F₂ population (in the year 2002) and 65% in F₃ lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.     

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.     

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.     

Breeding for resistance to infection with yellowing viruses in sugar beet

Differences in resistance to infection with beet yellows virus (BYV) and beet mild yellowing virus (BMYV) have been observed in virus-tolerant sugar-beet breeding material. The results of glasshouse virus-susceptibility tests usually agreed well with those of field experiments in which plants were exposed to artificial, or natural, infestation with viruliferous aphids. Breeding lines and varieties, which showed resistance to BYV when Myzus persicae Sulz, was used as vector, generally showed a similar resistance to this virus when Aphis fabae Scop. was used. Varieties which were resistant to infection with one virus were not necessarily resistant to the other, although some showed resistance to both BYV and BMYV. Preliminary results suggest that resistance to infection may be controlled by recessive genes which occur widely in sugar-beet cultivars. The mechanism of this form of resistance is not understood, but it does not appear to be closely associated with resistance to the aphid vectors of the viruses. The observed differences in resistance to infection demonstrate the possibility of breeding a sugar-beet variety in which two forms of resistance to virus yellows, tolerance and resistance to infection, are combined.     

The Absence of TIR-Type Resistance Gene Analogues in the Sugar Beet (Beta vulgaris L.) Genome

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. These authors (Yanyan Tian, Longjiang Fan) contributed equally to this work.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Intraspecific variation of Myzus persicae on sugar beet (Beta vulgaris)

Differences in inherited resistance among seven sugar-beet stocks had similar effects on Myzus persicae clones representing the range of variation in aphid response to resistant and susceptible sugar beet observed in fifty-eight clones collected between 1969 and 1971. Three sugar-beet stocks were consistently resistant. Statistically significant interactions between beet stocks and aphid clones did not indicate the existence of biotypes with specific abilities to overcome resistance. M. persicae clones differed in their vigour of colonizing sugar beet, irrespective of the differences between beet stocks. The readiness of adult aphids to settle determined the size of aphid population produced and included a component related to the response of the aphid clone to sugar beet as a host, and a component related to the resistance ranking of the beet stock. Breeding sugar beet with resistance to aphids will be simplified, as the results indicate that, at present, differences between aphid biotypes need not be considered a problem.     

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Rhizobium radiobacter conjugation and callus-independent shoot regeneration used to introduce the cercosporin export gene cfp from Cercospora into sugar beet (Beta vulgaris L.)

Leaf spot disease caused by Cercospora is responsible for crop and profitability losses in sugar beet crops in the US and worldwide. The cfp gene that encodes a protein that exports phytotoxic cercosporins from Cercospora was conjugally transferred to sugar beet using Rhizobium radiobacter (Agrobacterium tumefaciens), to improve Cercospora-induced leafspot resistance. Conditions for shoot regeneration were optimized to increase regeneration/transformation efficiencies. Low-light and room-temperature conditions were favorable to sugar beet regeneration without callus when cytokinin had been added to the tissue culture medium. Using this procedure adventitious shoots from leaf pieces were obtained in a simple, one-step regeneration procedure. T7, a cfp-transgenic clone verified by PCR with gene-specific primers, is being propagated for leaf spot disease resistance evaluation.     

The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.