Screening for fire blight resistance in apple (Malus pumila) using excised leaf assays from in vitro and in vivo grown material

The apple cultivars Greensleeves and Novole and somaclones derived from Greensleeves were tested for resistance to fire blight (Erwinia amylovora) using excised leaf bioassays. Cv. Greensleeves was consistently susceptible to fire blight in in vitro, and in glasshouse and excised leaf bioassays whilst cv. Novole was resistant. Greensleeves somaclones, identified as having intermediate levels of fire blight resistance in glasshouse and in vitro screening tests, also showed intermediate levels of resistance in excised leaf bioassays. The length of leaves and the inoculum concentration employed affected the severity of symptoms observed. Excised leaf bioassays were unsuitable as tests for fire blight resistance using field grown material.     

The susceptibility of stems of different potato cultivars to blackleg caused by Erwinia carotovora subsp. atroseptica

The susceptibility of stems of six potato cultivars to Erwinia carotovora subsp. atroseptica was assessed in two years (1981 and 1982) either by direct inoculation in the field or by inoculation of detached stems in the laboratory. These six and a further 22 cultivars were also assessed in three years (1982-84) by inoculating stems of glasshouse-grown plants. Different methods of inoculation and types of inocula were tested. In the field, wooden toothpicks rubbed in bacterial slime were more successful in establishing infection than when dipped in a bacterial suspension, but injection of bacterial suspension with a hypodermic needle was reliable in establishing infection over a range of concentrations. Detached stems were more readily infected and gave more consistent results compared with inoculation in the field. The range of reaction of the six cultivars was similar in both detached stem and glasshouse tests. The early cultivars Pentland Javelin and Ulster Sceptre were most susceptible and of the maincrop cultivars, Maris Piper was intermediate and Desiree and King Edward least susceptible whereas Pentland Crown showed greater resistance in the glasshouse than in the field. Glasshouse tests using hypodermic inoculation indicated a range of susceptibilities; the early cultivars Manna, Maris Bard and Estima were most susceptible and the maincrop Pentland cultivars Crown, Dell, Hawk, Ivory and Squire least susceptible.     

Fiji disease resistance in sugarcane: Relationship to cultivar preference in field populations of the planthopper vector Perkinsiella saccharicida

Populations of the planthopper vector Perkinsiella saccharicida on sugarcane cultivars resistant (cvs QUO and Q87), moderately resistant (cvs Q90 and Q124) and susceptible (cvs NCo310 and Q102) to Fiji disease with known field resistance scores were monitored on the plant (2000–2001) and ratoon (2001–2002) crops. In both crops, the vector population remained very low, reaching its peak in the autumn. The vector population was significantly higher on cultivars susceptible to Fiji disease than on cultivars moderately resistant and resistant to Fiji disease. The number of P. saccharicida adults, nymphs and oviposition sites per plant increased with the increase in the Fiji disease susceptibility. The results suggest that under low vector density, cultivar preference by the planthopper vector mediates Fiji disease resistance in sugarcane. To obtain resistance ratings in the glasshouse that reflect field resistance, glasshouse‐screening trials should be conducted under both low and high vector densities, and the cultivar preference of the planthopper vector recorded along with Fiji disease incidence.     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

Inheritance of resistance to yellow rust (Phragmidium rubi-idaei) in red raspberry

The inheritance of resistance in red raspberry (Rubus idaeus) to yellow rust (Phragmidium rubi-idaei) was studied in a diallel cross among the cultivars Boyne, Meeker, Mailing Jewel, Glen Prosen and Glen Clova. The progenies and clonally propagated parents were exposed initially in a rust-infected plantation where the incidence and severity of infection was assessed at the telial stage. The following spring leaves on new canes of the same plants were inoculated with urediniospores in a glasshouse at 18 ± 2 °C. The latent period and number of uredinia per cm² of abaxial leaf surface were determined 13 and 18 days after inoculation. Cv. Boyne developed only chlorotic flecking on inoculated leaves in the glasshouse and no sporulation was observed. It is postulated that this reaction was determined by a major gene which is designated Yr. Some of the other parents showed incomplete resistance of the slow rusting type, and segregants of Boyne selfed that were susceptible also showed low levels of rust, which suggests that Boyne carries both major- and minor-gene resistance. Diallel analyses of both the field and glasshouse data from the susceptible segregates indicated that the degree of incomplete resistance present was determined predominantly by additive gene action, though small but significant non-additive effects also occurred: cv. Meeker was the most resistant parent and contributed the most resistance to the progenies. The Meeker progenies also segregated for gene H, which determines cane hairiness and is known to be associated with resistance to other diseases. Segregants without hairs (genotypes hh) were found to be the more resistant to rust.     

Resistance to Rhynchosporium secalis in the winter barley cultivar Vulcan

The six-rowed winter barley cultivar Vulcan was found to be highly resistant to Rhynchosporium secalis in the field, although this resistance was not apparent in routine screening tests of barley genotypes carried out at the seedling stage. More precise seedling tests, using graded inoculum levels to derive dose-response curves, showed that cv. Vulcan had an ED₅₀ which was 4.8 times that of the susceptible, two-rowed cv. Maris Otter, and 4.1 times that of cv. Maris Puma. The inheritance of resistance was investigated at the adult plant stage in a cross with cv. Maris Otter. Although a clear-cut segregation of the F₃ progeny into resistant, segregating and susceptible phenotypes was not observed, the genetic variance of the F₃ family means was compatible with the hypothesis of monogenic control of resistance. Resistance was inherited independently of the factor for six-rowed head type.     

Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of black

The R10 and R11 late blight differentials of Black (tetraploid clones 3681ad1 and 5008ab6) were crossed with the susceptible potato (Solanum tuberosum) cultivar Maris Piper and the progeny were assessed for blight resistance in a whole plant glasshouse test using race 1,2,3,4,6,7 of Phytophthora infestans. The disease scores for the R10 population displayed a continuous distribution whereas the progeny in the R11 population could be categorised as resistant or susceptible. A bulk segregant analysis using amplified fragment length polymorphism assays was done on the ten most resistant and ten most susceptible progeny in each population and two closely linked markers were found to be associated with resistance. R11 mapped to 8.5 cM from marker PAG/MAAG_172.3 and R10 mapped as a quantitative trait locus in which marker PAC/MATC_264.1 explained 56.9% of the variation in disease scores. The results were consistent with R10 and R11 being allelic versions of genes at the R3 locus on chromosome 11. The implications are discussed for mapping R-genes which fail to give complete immunity to a pathogen.     

Differences in the tolerance of potato cultivars to potato cyst nematodes (Globodera rostochiensis and G. pallida) in field trials with and without nematicides

In field trials Cara, Brio, Maris Piper and Pentland Javelin were consistently more tolerant of damage by Globodera rostochiensis yielding more than Corsair, Pentland Dell, Maris Anchor and Maris Peer, in untreated, heavily infested soil and giving the smallest increases to nematicide treatment. No yield or growth differences were found between plants in untreated and nematicide treated plots at a nematode-free site. The most tolerant cultivars all had a gene (H₁) for resistance to G. rostochiensis derived from Solanum tuberosum ssp. andigena and in soil infested with G. pallida the tolerance of at least one resistant cultivar (Maris Piper) appeared to be lessened. However, some resistant cultivars were comparatively intolerant, even to G. rostochiensis. Early cultivars were generally less tolerant than late maturing cultivars but there were exceptions. Amongst cultivars with resistance derived from Solanum vernei the early cultivar Guardian was more tolerant than the main crop cultivar Corsair. The effect on the yield of several cultivars of a range of densities of G. rostochiensis, produced either by applying different rates of a nematicide or by cropping in the previous year, was examined at two sites. The results indicated that the slope of the regression for yield in relation to nematode density was less for tolerant than intolerant cultivars. At sites infested with G. rostochiensis Maris Piper was found to be consistently more tolerant than Pentland Crown.     

Restricted multiplication of potato leafroll virus in resistant potato genotypes

Plants of a range of potato genotypes differing in rating for field resistance to potato leafroll virus (PLRV) were inoculated with the virus by grafting or by aphids (Myzus persicae). Plants of all genotypes tested became infected by each inoculation method and PLRV was detected by ELISA in the upper leaves of all genotypes within 26 days after grafting. Most genotypes with high resistance ratings developed only mild primary and secondary symptoms whereas those with low resistance ratings developed more pronounced symptoms. However, one genotype (G7461(4)) with a high resistance rating was very severely affected. The concentrations attained by PLRV in genotypes with high resistance ratings were only 1–10% of those in genotypes with low resistance ratings. These differences in virus concentration were found in young leaves of plants with primary or secondary infection, whether inoculated by grafting or by aphids and whether grown in the glasshouse or the field. In older leaves, differences in virus concentration between genotypes were at least as pronounced as those in younger leaves. In contrast, PLRV concentration in vascular tissue at the heel end of tubers of plants with primary infection was similar for all the genotypes tested. Although low PLRV concentration was consistently associated with high resistance rating it is not the only form of resistance to PLRV occurring in potato.     

Factors affecting the detection of potato leafroll virus in potato foliage by enzyme-linked immunosorbent assay

Using antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme-linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10^-1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme. The detection end-point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10^-2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus-free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested. PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field-grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse-grown plants. In field-grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.     

Screening methods for salinity tolerance: a case study with tetraploid wheat

Fast and effective glasshouse screening techniques that could identify genetic variation in salinity tolerance were tested. The objective was to produce screening techniques for selecting salt-tolerant progeny in breeding programs in which genes for salinity tolerance have been introduced by either conventional breeding or genetic engineering. A set of previously unexplored tetraploid wheat genotypes, from five subspecies of Triticum turgidum, were used in a case study for developing and validating glasshouse screening techniques for selecting for physiologically based traits that confer salinity tolerance. Salinity tolerance was defined as genotypic differences in biomass production in saline versus non-saline conditions over prolonged periods, of 3–4 weeks. Short-term experiments (1 week) measuring either biomass or leaf elongation rates revealed large decreases in growth rate due to the osmotic effect of the salt, but little genotypic differences, although there were genotypic differences in long-term experiments. Specific traits were assessed. Na⁺ exclusion correlated well with salinity tolerance in the durum subspecies, and K⁺/Na⁺ discrimination correlated to a lesser degree. Both traits were environmentally robust, being independent of root temperature and factors that might influence transpiration rates such as light level. In the other four T. turgidum subspecies there was no correlation between salinity tolerance and Na⁺ accumulation or K⁺/Na⁺ discrimination, so other traits were examined. The trait of tolerance of high internal Na⁺ was assessed indirectly, by measuring chlorophyll retention. Five landraces were selected as maintaining green healthy leaves despite high levels of Na⁺ accumulation. Factors affecting field performance of genotypes selected by trait-based techniques are discussed.     

Glandular hairs are a possible means of limiting aphid damage to the potato crop

Glandular hairs on leaves and stems stuck naturally-occurring aphids to field grown plants of Solamim polyadenium, S. berthaultii, and first and second generation hybrid S. tuberosum cv. Pentland Crown ×berthaultii, but none became stuck on Pentland Crown. S. polyadenium had most glandular hairs, stuck most aphids and had fewest free aphids. In the glasshouse, the percentage of stuck aphids was positively correlated with the abundance of glandular hairs, especially on the leaves, so counting the hairs may be a simple preliminary method of screening for aphid-trapping ability.     

Identification of resistance to Peanut bud necrosis virus (PBNV) in wild Arachis germplasm

Eighty three wild Arachis germplasm accessions, belonging to 24 species of five sections and one natural hybrid derivative of a cross between the cultivated and a wild Arachis species, were evaluated along with a susceptible groundnut cultivar for resistance to Peanut bud necrosis virus (PBNV) in a replicated field trial at ICRISAT, Patancheru, India. Thirty days after sowing, the percentage of infected plants were recorded for all the accessions and subsequently young leaflets from all these accessions were tested for the presence of the virus by enzyme linked immunosorbent assay (ELISA). One accession each of A. benensis and A. cardenasii, and two accessions of A. villosa, in the section Arachis, two accessions of A. appressipila in the section Procumbentes, and one accession of A. triseminata under section Triseminatae were not infected by PBNV. These seven field‐resistant accessions were tested under glasshouse conditions for virus resistance by mechanical sap inoculations. One accession of A. cardenasii and two accessions of A. villosa did not show systemic infection. Similarly, in another glasshouse test, where 13 A. cardenasii accessions of section Arachis were evaluated, two accessions did not show systemic infection. In all these resistant accessions, the inoculated leaves showed infection, but the systemic leaves did not show the presence of virus in spite of repeated mechanical sap inoculations. So, the resistance in these accessions appears to be due to a block in systemic movement of the virus. To our knowledge this is the first report on the identification of resistance to PBNV in wild Arachis species. Since both A. cardenasii and A. villosa are the progenitors of cultivated groundnut and can be hybridised with the latter, the resistant accessions are being utilised in conventional breeding programmes to transfer PBNV resistance to widely adapted groundnut cultivars.     

Pathogen‐inducible Ta‐Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects

Plant diseases are a serious threat to crop production. The informed use of naturally occurring disease resistance in plant breeding can greatly contribute to sustainably reduce yield losses caused by plant pathogens. The Ta‐Lr34res gene encodes an ABC transporter protein and confers partial, durable, and broad spectrum resistance against several fungal pathogens in wheat. Transgenic barley lines expressing Ta‐Lr34res showed enhanced resistance against powdery mildew and leaf rust of barley. While Ta‐Lr34res is only active at adult stage in wheat, Ta‐Lr34res was found to be highly expressed already at the seedling stage in transgenic barley resulting in severe negative effects on growth. Here, we expressed Ta‐Lr34res under the control of the pathogen‐inducible Hv‐Ger4c promoter in barley. Sixteen independent barley transformants showed strong resistance against leaf rust and powdery mildew. Infection assays and growth parameter measurements were performed under standard glasshouse and near‐field conditions using a convertible glasshouse. Two Hv‐Ger4c::Ta‐Lr34res transgenic events were analysed in detail. Plants of one transformation event had similar grain production compared to wild‐type under glasshouse and near‐field conditions. Our results showed that negative effects caused by constitutive high expression of Ta‐Lr34res driven by the endogenous wheat promoter in barley can be eliminated by inducible expression without compromising disease resistance. These data demonstrate that Ta‐Lr34res is agronomically useful in barley. We conclude that the generation of a large number of transformants in different barley cultivars followed by early field testing will allow identifying barley lines suitable for breeding.     

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.     

Rooted leaf cuttings as an aid in screening for resistance to Plasmodiophora brassicae

Rooted leaf cuttings were used to screen a range of genotypes and cultivars from Brassica spp. and Raphanus sativus for resistance to Plasmodiophora brassicae. The optimum conditions for obtaining equivalent disease incidence and disease severity on leaf cuttings and on seedlings were investigated. In the method used leaves were cut near the base of the petiole before the lamina was fully expanded. They were kept in a mist propagator for 6 to 18 days and then transferred to a clubroot test bench and individually inoculated with a suspension of resting spores. There was an optimum period for each cultivar between cutting and inoculation which gave the maximum incidence of infection: c. 15 days for Doon Major (swede); 10–15 days for Civasto (stubble turnip) and Giant Rape (forage rape); c. 22 days for Maris Kestrel (marrow stem kale) and c. 10 days for Slobolt (fodder radish). Gall size on rooted leaf cuttings gave a quantitative measure of disease severity equivalent to that on seedlings in tests using the European Clubroot Differential host set, the five above-mentioned cultivars and four swede cultivars. There was little callus production except on some B. campestris hosts and on cuttings with shoot tissue; microscopic examination was used to confirm the presence or absence of infection in galls. Hormone rooting powder did not increase the rate of root production in two cultivars of forage rape. Root development was less extensive in B. campestris than in B. oleracea or B. napus. Apparently normal shoots developed on 96% of leaf cuttings (inoculated and uninoculated) in which a fragment of axillary bud tissue was included. The results are discussed in relation to screening for resistance in whole plants and in tissue culture.     

Generation and screening of recombinant inbred lines of rice for yellow stemborer resistance

Based on the results of studies on varietal screening, antixenosis (egg laying preference) and antibiosis (larval survival and adult emergence), rice varieties W1263 and CO43 were selected as resistant and susceptible parents, respectively, for yellow stemborer (YSB) infestation. A mapping population was developed using above parents following single seed descent method. Screening for YSB reaction in F1 and F2, generations under field and glasshouse conditions for both dead hearts and white ears, established the polygenic nature of inheritance for YSB resistance. Field screening for YSB resistance at F9 generation revealed the difference in the reactions among recombinant inbred lines (RILs) between vegetative and reproductive stages. The experiments under field and glasshouse screening of RILs for dead hearts showed significant positive association. However, the reaction was more towards susceptibility in glasshouse screening due to no choice test. Scoring of 250 RILs (F8) for various morphological traits showed wide range of variation indicating the suitability for QTL mapping.     

Disease spread of non-specialised fungal pathogens from inoculated point sources in intraspecific mixed stands of cereal cultivars

Spread of Septoria nodorum from inoculated point sources was examined in pure stands and mixtures of two spring wheat cultivars Kolibri and Maris Butler. Gradients in disease were observed soon after inoculation; the presence of the more resistant cultivar (Maris Butler) in the mixtures retarded the outward spread of disease compared with the susceptible pure stand (Kolibri). Regression analysis suggested that gradients in incidence were influenced by nearness to the source whereas gradients in severity were not. Spread of disease was also examined in pure stands and mixtures for the host/pathogen combinations, winter wheat (cvs Maris Huntsman and Maris Ranger)/S. nodorum and winter barley (cvs Maris Otter and Hoppel)/Rhynchosporium secalis. In contrast to the spring wheat experiment, no gradients were observed; explanations for the uniform distribution of disease were (a) extensive spread prior to the period of assessment, (b) no physical barrier to dispersal due to the juvenile growth stage of the crop and (c) exhaustion of the point-source.