Components of quantitative resistance to leaf rust in wheat cultivars: diversity,variability and specificity

The aim of this study was to investigate the potential diversity and pathogen‐specificity of sources of quantitative resistance to leaf rust caused by Puccinia triticina in French wheat germplasm. From a set of 86 genotypes displaying a range of quantitative resistance levels during field epidemics, eight wheat genotypes were selected and challenged in a greenhouse with three isolates of the pathogen, belonging to different pathotypes. Five components of resistance were assessed: infection efficiency, for which an original methodology was developed, latent period, lesion size, spore production per lesion, and spore production per unit of sporulating tissue. High diversity and variability for all these components were expressed in the host × pathotype combinations investigated; isolate‐specificity was found for all the components. The host genotypes displayed various resistance profiles, based on both the components affected and the isolate‐specificity of the interaction. Their usefulness as sources of quantitative resistance was assessed: line LD7 probably combines diversified mechanisms of resistance, being highly resistant for all the components, but displaying isolate‐specificity for all the components; cv. Apache did not show isolate specificity for any of the components, which could be related to the durability of its quantitative resistance in the field over more than 11 years.     

Assessment of inoculation methods for screening black alder resistance to Phytophthora ×alni

Identification of resistance to Phytophthora ×alni could provide the basis for a management strategy against alder decline in riparian ecosystems in Europe. This study aimed to test methods to evaluate the resistance of riparian alders to the disease, and to screen alder genotypes for resistance. Phytophthora ×alni isolates were compared for their aggressiveness (lesion length on stem) and sporulation capacity (sporangia). While no difference in lesion lengths was found between isolates, sporangia production was dependent on isolate, highlighting the need for careful selection of isolates used for zoospore inoculation methods. Inoculation tests carried out at different periods of the year revealed a seasonal change in susceptibility to the disease, with the period from June to September being the most efficient for inoculation tests. Stem‐wounded inoculations, carried out on excised shoots, were unreliable for evaluating the level of resistance of alder genotypes to P. ×alni infection, with divergent results between two successive years or between two inoculation periods during the same year. In contrast, a method that mimics the natural conditions of infection, based on flooding of rooted cuttings in artificially infected river water, was found promising. Another method, based on the inoculation of foliated terminal shoots with zoospore suspensions, was found to be repeatable and could be used for high‐throughput analyses. Altogether, the results show a continuous resistance response from highly susceptible to moderately resistant genotypes. This suggests that breeding might be a useful strategy to manage alder decline caused by P. ×alni.     

The reliability of leaf bioassays for predicting disease resistance on fruit: a case study on grapevine resistance to downy and powdery mildew

This study was designed to assess the reliability of grapevine leaf bioassays for predicting disease resistance on fruit in the field. The efficacy of various grapevine quantitative trait loci (QTLs) for conferring resistance to downy and powdery mildew was evaluated in bioassays and in a 2‐year field experiment for downy mildew. The resistance genes studied were inherited from Muscadinia rotundifolia (Rpv1 and Run1) and from American Vitis species through cv. Regent (QTLRgP and QTLRgD). In bioassays, genotypes carrying Run1 blocked powdery mildew development at early stages. Genotypes combining Run1 with QTLRgP displayed no greater level of resistance. For downy mildew, genotypes carrying Rpv1 and/or QTLRgD were more resistant than the susceptible cv. Merlot, and showed a high level of leaf resistance in the field (<10% severity). Disease levels on bunches were much higher than those on leaves, with a high variability between Rpv1 genotypes (1–48%). A Bayesian decision theory framework predicted that an OIV‐452 threshold of 5 in leaf bioassays allowed accurate selection of grapevine genotypes (P = 0·83) with satisfactory disease severity on bunches. Therefore, this study validates that the use of early bioassays on leaves, as currently performed by grapevine breeders, ensures a satisfactory level of resistance to downy mildew of bunches in the field.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Absence of detectable yield penalty associated with insensitivity to Pleosporales necrotrophic effectors in wheat grown in the West Australian wheat belt

Genetic disease resistance is widely assumed, and occasionally proven, to cause host yield or fitness penalties due to inappropriate activation of defence response mechanisms or diversion of resources to surplus preformed defences. The study of resistance gene trade‐offs has so far been restricted to biotrophic pathogens. In some Pleosporales necrotrophic interactions, quantitative resistance is positively associated with insensitivity to effectors. Host lines that differ in sensitivity can easily be identified amongst current cultivars and advanced breeding lines. Large wheat cultivar trials were used to test whether lines sensitive or insensitive to three necrotrophic effectors from Pyrenophora tritici‐repentis and Parastagonospora nodorum differed in yield when subjected to natural disease and stress pressures in the West Australian wheat belt. There was no significant yield penalty associated with insensitivity to the fungal effectors ToxA, SnTox1 and SnTox3. Some yield gains were associated with insensitivity and some of these gains could be attributed to increased disease resistance. It is concluded that insensitivity to these effectors does not render such plants more vulnerable to any relevant biotic or abiotic stress present in these trials. These results suggest that the elimination of sensitivity alleles for necrotrophic effectors is a safe and facile strategy for improving disease resistance whilst maintaining or improving other desirable traits.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

Resistance to Xanthomonas perforans race T4 causing bacterial spot in tomato breeding lines

Tomato (Solanum lycopersicum) is the second most important vegetable crop in the world. Bacterial spot (BS) of tomato, caused by four species of Xanthomonas: X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri, results in severe loss in yield and quality due to defoliation and formation of lesions on fruits, respectively. Currently management practices do not offer effective control under conditions of high disease pressure. Thus, developing BS resistance is a critical priority for tomato growers in order to minimize crop losses. Sixty‐three advanced tomato breeding lines, heirlooms and wild tomato lines with diverse genetic backgrounds were screened under greenhouse and field conditions for BS resistance using X. perforans race T4, which was found to be a prevalent race in North Carolina. Race T4 isolate 9 was used to inoculate the plants by spraying, and disease severity was measured using the Horsfall–Barratt scale. Tomato lines 74L‐1W(2008), NC2CELBR, 081‐12‐1X‐gsms, NC22L‐1 (2008) and 52LB‐1 showed resistance to BS in the field and/or greenhouse trials. These lines were derived from S. pimpinellifolium L3707. Screening L3707 followed by development of a mapping population and mapping resistance genes might be useful for breeding resistance against BS in future breeding programmes.     

Variability and selection of verticillium wilt resistant genotypes in cultivated olive and in the Olea genus

Developing verticillium wilt resistant genotypes is currently a major objective in olive breeding. In this study, 6017 genotypes derived from 48 crosses obtained by open pollination and crosses between olive cultivars, wild olive genotypes and other Olea species and Olea europaea subspecies were individually evaluated for verticillium wilt resistance. More than 800 genotypes were identified as resistant to the disease based on the absence of symptoms. High genetic variability and wide segregation in resistance were observed. The inheritance of resistance was studied, and the best parents and crosses to breed resistant genotypes were identified. According to the results, verticillium wilt resistance in olive appears to be a quantitative trait. The results obtained by comparing the level of resistance between different crosses as well as by estimating heritability suggest that it is possible to breed for verticillium wilt resistance in olive.     

Detection of quantitative resistance loci associated with resistance to rice false smut (Ustilaginoidea virens) using introgression lines

False smut, caused by Ustilaginoidea virens, is a rice disease of increasing importance worldwide, with no source of high‐level resistance in the existing rice germplasm. To facilitate breeding varieties with good levels of field resistance to false smut, quantitative resistance loci (QRL) were identified using 213 introgression lines (ILs) from a cross between Teqing (recipient) and Lemont (donor) evaluated using natural infection at two hotspots of false smut in northeast China. Ten QRL affecting percentages of diseased hills, diseased panicles and diseased spikelets were detected and mapped to rice chromosomes 2, 3, 4, 6, 8, 10, 11 and 12. The Lemont alleles at all QRL increased false smut resistance. Four QRL (qFSR‐6‐7, qFSR‐10‐5, qFSR‐10‐2 and qFSR‐11‐2) had relatively larger and consistent effects across the two testing sites. Promising resistant ILs were identified, most of which had multiple QRL, suggesting that pyramiding multiple QRL by marker‐assisted selection would be an effective strategy for improving rice resistance to false smut. The identified QRL and their linked DNA markers will facilitate this breeding effort in the future.     

Simulation study of the competitive ability of erect, semi-erect and prostrate cowpea (Vigna unguiculata) genotypes

Ecophysiological simulation models provide a quantitative method to predict the effects of management practices, plant characteristics and environmental factors on crop and weed growth and competition. The INTERCOM interplant competition model was parameterised, calibrated by monoculture data for three cowpea (Vigna unguiculata) genotypes that differed in growth habit, common sunflower (Helianthus annuus) and common purslane (Portulaca oleracea), and used to simulate competition of cowpea cover crops with sunflower or purslane. The simulation results were compared with observations from field competition experiments in 2003 and 2004. INTERCOM more accurately simulated actual field data for the competition of cowpea genotypes and sunflower than companion field experiments for the competition of cowpea and purslane. The validated simulation model of cowpea and sunflower at two densities was used to study the effects of cowpea growth habit on final biomass production of cowpea and sunflower. The model suggested that erect growth habit was more competitive than semi‐erect and prostrate growth habit, when cowpea genotypes were grown with sunflower. Cowpea leaf area distribution was important to higher cowpea biomass production, while cowpea height growth was important to reduce sunflower biomass. Our simulation approach is suggested as a method for crop breeders to gauge the likely success of selection for competitive crops before undertaking expensive long‐term breeding experiments.     

Genotype-isolate interaction for resistance to black stem in sunflower (Helianthus annuus)

Two experiments were undertaken to determine the partial resistance of sunflower genotypes to seven isolates of Phoma macdonaldii . In the first experiment, 28 genotypes, including recombinant inbred lines and their parents, M6 mutant lines developed by gamma irradiation, and some genotypes from different geographical origins, were used. The experiment consisted of a split-plot design with three replications, each with 12 seedlings per genotype per isolate, in controlled conditions. Seven days after inoculation, plantlets were scored on a 1–9 scale for percentage necrotic area. Highly significant differences were observed among genotypes, isolates and their interactions. The presence of a differential interaction between genotypes and P. macdonaldii isolates was confirmed in a second experiment using 12 genotypes representing large variability for partial resistance to P. macdonaldii isolates, as identified in the first experiment. Inbred lines B454/03, ENSAT-B5 and LC1064C were the most susceptible sunflower genotypes, whereas two American lines SDR19 and SDR18 presented high partial resistance to all P. macdonaldii isolates studied. The least and most aggressive isolates were MA6 and MP3, respectively. Isolates interacted differentially with sunflower genotypes. This study identified two genotypes (AS613 and PAC2) presenting specific resistance to isolate MP8. The results also showed a wide range of isolate-nonspecific partial resistances among the lines tested. The information presented here could assist sunflower breeders to choose parents of crosses for breeding of durable resistance to phoma black stem disease.     

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP‐tagged strain XF3.9.C_(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post‐inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.     

Field response of near‐isogenic brown midrib sorghum lines to fusarium stalk rot,and response of wildtype lines to controlled water deficit

To increase digestibility for ruminant livestock and for lignocellulosic ethanol conversion efficiency in sorghum (Sorghum bicolor), brown midrib (bmr) lines carrying bmr6 or bmr12 and the double mutant (bmr6 bmr12) in two backgrounds (RTx430 and Wheatland) were developed, resulting in lines with significantly reduced lignin, as compared with the near‐isogenic wildtype. Under greenhouse conditions, these lines had previously demonstrated no increased susceptibility, and some lines were more resistant to the highly virulent stalk rot pathogen, Fusarium thapsinum, compared to the wildtype. Fusarium stalk rot of sorghum is a destructive disease that under high temperatures or drought conditions may lead to lodging. To determine if greenhouse observations could be extended to field environments, bmr and near‐isogenic wildtype lines were inoculated with F. thapsinum at field locations, Mead (irrigated) and Havelock (dryland) in Nebraska, USA. Analysis of mean lesion lengths showed those of most bmr lines were statistically similar to the wildtype. Across both genetic backgrounds, bmr6 and bmr6 bmr12 double mutant plants grown at Mead had significantly smaller mean lesion lengths than the corresponding wildtype (P ≤ 0.05). To assess responses of the two genetic backgrounds to controlled (greenhouse) water stress, wildtype RTx430 and Wheatland plants were inoculated with F. thapsinum under well‐watered and water stress conditions. Mean lesion lengths resulting on water deficit plants were significantly larger than those on well‐watered plants (P = 0.01). These results indicate that this bioassay can be used to screen sorghum lines in the greenhouse for increased resistance or tolerance to both drought and fusarium stalk rot.     

Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt disease

Bacterial wilt is a serious problem affecting many important food crops. Recent studies have indicated that treatment with biotic or abiotic stress factors may increase the resistance of plants to bacterial infection. This study investigated the effects of magnesium oxide nanoparticles (MgO NP) on disease resistance in tomato plants against Ralstonia solanacearum, as well as its antibacterial activity. The roots of tomato seedlings were inoculated with R. solanacearum and then immediately treated with MgO NP; the treated plants showed very little inhibition of bacterial wilt. In contrast, when roots were drenched with a MgO NP suspension prior to inoculation with the pathogen, the incidence of disease was significantly reduced. Rapid generation of reactive oxygen species such as O₂^?˙ radicals was observed in tomato roots treated with MgO NP. Further O₂^?˙ was rapidly generated when tomato plant extracts or polyphenols were added to the MgO NP suspension, suggesting that the generation of O₂^?˙ in tomato roots might be due to a reaction between MgO NP and polyphenols present in the roots. Salicylic acid‐inducible PR1, jasmonic acid‐inducible LoxA, ethylene‐inducible Osm, and systemic resistance‐related GluA were up‐regulated in both the roots and hypocotyls of tomato plants after treatment of the plant roots with MgO NP. Histochemical analyses showed that β‐1,3‐glucanase and tyloses accumulated in the xylem and apoplast of pith tissues of the hypocotyls after MgO NP treatment. These results indicate that MgO NP induces systemic resistance in tomato plants against R. solanacearum.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).     

Evaluation of strawberry (Fragaria L.) genetic resources for resistance to Botrytis cinerea

The grey mould disease caused by Botrytis cinerea leads to substantial economic losses in strawberry production all over the world. Control of the disease requires an extensive amount of fungicide that is applied in varying complexes because the pathogen easily develops resistance against the active compounds. Planting of resistant cultivars seems to be a promising alternative for fruit growers, but there are currently no cultivars available combining resistance to B. cinerea with attractive horticultural traits. Breeding of new cultivars requires the effective identification of resistant strawberry genotypes; therefore the current study was aimed at the evaluation of strawberry genetic resources under controlled conditions by establishing an artificial inoculation assay. The method presented in this study is an artificial inoculation of ripe fruits with a defined spore suspension under laboratory conditions. The results show that this assay is fast and simple and leads to reproducible results that correlate with field observations. Over 3 years a total of 107 strawberry genotypes of the German National Fruit Genebank at the JKI in Dresden‐Pillnitz were evaluated. Five partly resistant genotypes, cultivars Diana, Joerica and Kimberly, and Fragaria virginiana ‘Wildmare Creek’ and F. vesca subsp. bracteata, were identified with mean disease levels of <20% at 6 days post‐inoculation. The obtained results are discussed with regard to future breeding activities.     

Measuring lesion attributes and analysing their spatial patterns at the leaf scale using digital image analysis

Digital image analysis was used to quantify size, shape and relative positions of individual plant disease lesions to determine their spatial distribution pattern at the leaf scale. Rice brown spot was used as a necrotrophic pathogen causing numerous discrete lesions. A 50‐leaf subsample was selected from an existing data set of 350 images of leaves taken from the field, and analysed for disease severity using image analysis. Further measurements included size, shape and the relative positions of lesions for all leaves with severity > 8% (n = 25) and an additional 25‐leaf sample with severity <8%. A total of 3964 necrotic and/or halo areas were selected using a manually defined threshold in the computer program Assess . There were significant and positive associations (Pearson's r > 0.81; P < 0.001) between the size‐related measurements (lesion area, longest and shortest axis). Coalesced areas, formed by interconnection of lesions and associated haloes, and a high number of small lesions were found with an increase in severity, suggesting a secondary cycle and autoinfection process. Results from quadrat‐based (Poisson distribution and Spatial Analysis by Distance IndicEs) and distance‐based (point‐process Poisson) spatial methods were in good agreement and, together with a Taylor power law model, suggested a shift from random to predominantly aggregated patterns of lesions at severities approaching 10%. This framework, which is applicable to other foliar diseases, proved useful in providing quantitative knowledge of epidemic processes at the leaf scale. Finally, these results may be useful in improving simulation models and disease assessment methods.     

A novel screening method for rice allelopathic potential: the inhibitory‐circle method

Screening crop accessions for allelopathic activity is of paramount importance for crop allelopathy research. Previous bioassays often did not use a mixed culture of donor and target plants, did not use soil and were not conducted under natural conditions. In this study, we designed an inhibitory‐circle method in which a rice accession (donor plant) and Echinochloa crus‐galli (target plant) were cultured together in paddy soil under natural conditions. First, we determined that the highest allelopathic activity of allelopathic rice accession PI312777 was at the 5‐leaf stage, and the suitable distance of rice seedlings and E. crus‐galli was 12 cm apart. This method was then validated by a field test. A further 40 rice accessions were evaluated for allelopathic activity to E. crus‐galli using this method. Two rice accessions, PI312777 and Taichung Native 1, had highly allelopathic activity to E. crus‐galli (inhibitory rate > 50%), while another accession, Lemont, had non‐allelopathic activity. These experimental results were in accordance with previous studies using direct field experiments. The inhibitory‐circle method integrated three necessary conditions, that is donor and target plants grown together, with soil as the medium and under natural conditions for reliable results. The ‘inhibitory‐circle method’, which combined donor and target plants, soil medium and field conditions, can give reliable results in one step, compared with laboratory screening methods. Also, the ‘inhibitory‐circle method’ gave results in 30‐35 days, thereby substantially reducing the requirements for time, labour and cost.