Development of an effective screening method for partial resistance to Alternaria brassicicola (dark leaf spot) in Brassica rapa

In order to develop a method to measure resistance to Alternaria brassicicola (cause of dark leaf spot disease) in Brassica rapa, the effects of inoculum concentration, leaf stage, leaf age and incubation temperature of inoculation on infection were studied under controlled conditions using several B. rapa genotypes. Three inoculation methods (cotyledon, detached leaf and seedling inoculation) were evaluated for this purpose. The detached leaf inoculation test was the most suitable for screening B. rapa genotypes because clear symptoms were observed on the leaves in less than 24 h, and there was a significant positive correlation between the results from the detached leaf inoculation test and the seedling inoculation test, an established method considered to yield reliable results. In addition, it was very easy to screen plants for resistance on a large scale and to maintain standard physical conditions using detached leaves. For successful infection, inoculum concentration should be adjusted to 5 × 10⁴ conidia ml⁻¹, and incubation temperature should be between 20 °C and 25 °C. The 3rd/4th true leaves from 30 day-old plants were optimal for inoculation. In a screening test using 52 cultivars of B. rapa, the detached leaf test effectively discriminated between various levels of partial resistance among cultivars. As a result, we identified two cultivars, viz Saori and Edononatsu, as highly resistant and five cultivars, viz Tokinashi Taisai, Yajima Kabu, Purara, Norin-F₁-Bekana and Tateiwa Kabu, as having borderline resistance.     

Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar- S-methyl (ASM)

The benzothiadiazole compound acibenzolar- S -methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa , agent of witches' broom, and Verticillium dahliae , agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches' broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches' broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.     

Genetics of resistance to cotton leaf curl disease in Gossypium hirsutum

Twenty-two cotton varieties were screened for resistance to cotton leaf curl disease (CLCuD), a disease of viral origin, using three procedures: field evaluation, whitefly transmission assay and graft inoculation. Viral infection of cotton varieties was determined by visual symptom assessment as well as dot-blot and multiplex PCR diagnostic techniques. Crosses were made between the most susceptible variety (S-12) and highly resistant varieties (CP-15/2, LRA-5166 and CIM-443). All F₁ plants of these crosses were resistant, showing dominant expression of the resistance as well as the absence of extrachromosomal inheritance. The F₂ plants of the crosses CP-15/2 × S12, LRA-5166 × S-12 and CIM-443 × S12 exhibited a ratio of 13 resistant (symptomless) to three susceptible (with symptoms). Screening of the F₂ generation for virus infection by multiplex PCR further subdivided the resistant class into those exhibiting a high level of resistance (HR; PCR-negative) and those exhibiting resistance (R; symptomless, yet showing virus replication by PCR analysis). Hence, the final ratio was 3:10:3 (HR:resistant:susceptible). The F₃ progeny of susceptible F₂ plants segregated for resistance, indicating the probable presence of a suppressor gene ( S ). These findings are consistent with three genes being involved in G. hirsutum resistance to CLCuD, two for resistance ( R _1CLCuDhir and R _2CLCuDhir ) and a suppressor of resistance ( S _CLCuDhir ).     

Evaluation of cacao (Theobroma cacao) clones against seven Colombian isolates of Moniliophthora roreri from four pathogen genetic groups

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1·2 × 10⁵ spores mL⁻¹): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri . Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.     

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao (cocoa) varied between isolates of Moniliophthora (formerly Crinipellis ) perniciosa , the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 × Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.     

番茄白粉病苗期抗病性鉴定方法及抗病种质资源筛选

采用不同接种方法、不同接种苗龄、不同接种浓度三因素三水平正交试验设计对番茄白粉病苗期抗性鉴定方法进行筛选。结果表明,番茄苗期接种白粉病最好的方法是喷雾法,接种苗龄为四叶期,接种的最适浓度为106孢子/mL。在此基础上,对东北农业大学番茄研究所提供的54份番茄材料进行白粉病苗期接种鉴定,结果表明,54份材料中,有抗病品种4份、中抗品种16份、感病品种17份、极感病品种17份。     

A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation

Physiological and molecular research on resistance responses of Solanum tuberosum cultivars and partially resistant Solanum species to Phytophthora infestans requires a reliable resistance test that can be used in the laboratory. Laboratory tests performed on detached leaves and intact plants were compared with field tests for similarity of late blight reactions. Detached leaves from field-grown plants were as resistant as detached leaves from climate chamber-grown plants when challenged with P. infestans. However, detached leaves incubated in covered trays at high relative humidity were more susceptible than detached leaves kept in open trays or leaves on intact plants. The incubation conditions of detached leaves in covered trays rather than detachment itself appeared to affect the resistance expression. Detached leaves of some wild Solanum genotypes became partially infected, whereas intact plants were completely resistant when inoculated. Inoculation of leaves on intact plants, however, resulted in lower infection efficiencies. These limitations should be taken into account when choosing the appropriate inoculation method for specific purposes. For resistance screening, laboratory tests proved to be a good alternative for field tests. The ranking of resistance levels for twenty plant genotypes was similar under laboratory and field conditions.     

Comparison of responses to floret and seedling inoculation in wheat- Ustilago tritici and barley- U. nuda combinations

The floret and seedling methods for inoculating wheat and barley with Ustilago tritici and U. nuda, respectively, were optimized and then compared. Floret inoculation gave higher infection levels than did seedling inoculation, especially for wheat, but problems of plant mortality and standardization of tissue age were greater with the former method. Floret inoculation identified wheat and barley varieties exhibiting embryo resistance (class 1 or 2 resistance) or seedling resistance (class 3), but only class 3 wheat and barley varieties (and one class 2 wheat variety) were resistant to seedling inoculation. Using near-isogenic wheat lines expressing different levels of partial resistance to floret inoculation, positive correlations were obtained between percentage infection levels resulting from the two inoculation protocols. In addition, race-specific host-pathogen interactions such as hypersensitivity and partial smutting were expressed following both floret and seedling inoculation, indicating that these interactions operate in the developing plant. The potential value of the seedling inoculation method in fundamental and applied research into the cereal-loose smut interaction is discussed.     

Effect of method of inoculation, inoculum density and seedling age at inoculation on the expression of resistance of cocoa (Theobroma cacao L.) to Verticillium dahliae Kleb.

Soil drench and stem puncture inoculation were compared as methods for selecting cocoa cultivars with resistance to Verticillium dahliae. Disease progress was more rapid and induced symptoms were more severe following stem puncture and, under glasshouse conditions, differences between cultivars were detected 15 days after inoculation. Moreover, using stem puncture, inoculum densities of 10⁴ conidia/ml were sufficient to differentiate resistant and susceptible cultivars, whereas with the soil drench method, inoculum densities of 10⁷ conidia/ml were necessary. Although a substantially higher proportion of plants were affected by stem puncture inoculation, the resistance of cultivar Pound-7 remained effective at high inoculum densities of 10⁸ conidia/ml. With either method, older seedlings were more susceptible to V. dahliae than younger ones. However, with stem puncture, 15-day-old seedlings were sufficiently susceptible for a valid disease assessment. In contrast, with soil inoculation, 60-day-old plants were required. In a nursery trial with 15-day-old seedlings, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to Verticillium dahliae , on the basis of root inoculation, were ranked in the same order when stem punctured. Stem puncture inoculation of young seedlings is cost-effective in terms of time and space, and is therefore recommended for screening of cocoa for wilt resistance, especially in large-scale breeding programmes.     

Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markers

Screening of genotypes of melon ( Cucumis melo ) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth-chamber experiments. Disease incidence varied from 0 to 100% in a genotype-dependent manner. Using four combinations of light (60 and 90  µ E m⁻² s⁻¹) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker-assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence-characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom-2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker-assisted selection.     

Response of strawberry genotypes to inoculation with isolates of Verticillium dahliae differing in host origin

Eight strawberry genotypes known to differ in susceptibility to verticillium wilt were inoculated with eight isolates of Verticillium dahliae originally obtained from six different host crops: strawberry, potato, watermelon, mint, eggplant (aubergine) and cauliflower. Inoculation experiments were conducted in replicated field trials during two successive years. Known susceptible genotypes developed typical symptoms of verticillium wilt in both years. Although isolates manifested a wide range of aggressiveness, differences were significant only on the most susceptible strawberry genotype. Two isolates originating from strawberry were among the most aggressive of the eight tested, whereas the least aggressive isolate was obtained from cauliflower. Six strawberry genotypes that were regarded as resistant to verticillium wilt based on previous tests were also resistant in the present study, regardless of the isolate used. Overall, strawberry genotypes represented the largest source of variation in these experiments, with variance components approximately 10-fold greater than those associated with either isolate or the isolate × genotype interaction. The results suggest it should be possible to develop resistance to verticillium wilt in strawberry that is broadly effective against isolates of diverse host origin.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

油菜菌核病抗性鉴定方法的比较及抗源的筛选

采用已报道的七种油菜菌核病抗性鉴定方法,在同一条件下对两个感病和两个抗病品种进行鉴定比较。结果表明:草酸浸根(叶)法用于白菜型油菜鉴定效果不好;不同生育期的油菜对菌核病抗性有差异,应该以花期鉴定为主,苗期鉴定为辅;在花期以麦粒叶腋接种、琼脂块叶腋接种和牙签茎杆接种,接种后发病程度依次为牙签>麦粒>琼脂块;三种花期接种方法与大田接种鉴定的结果呈显著正相关(r>0.8000),这三种方法均能有效反映抗、感品种的差异,其中花期牙签茎杆接种法与大田接种鉴定法相关性达极显著水平(r=0.9140)。根据抗性鉴定方法比较的结果,选用花期牙签茎杆接种法,以中油821为对照,对四川农业大学油菜研究中心G1自交系的44份油菜育种材料进行鉴定、筛选,得到12份高抗材料,其中有5份材料的抗性极显著高于对照。     

The infection process of Moniliophthora perniciosa in cacao

The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.     

番茄灰叶斑病原菌的鉴定及抗性种质资源的筛选

<正>番茄灰叶斑病是一种世界性病害,近年来,该病在北京、台湾、海南、山东和浙江等地均有发生,极大地限制了我国番茄生产的竞争力~([1])。该病害主要由Stemphylium spp.不同的4个种引起~([2])。本研究采用形态观察法、ITS和gpd序列分析法对从番茄病株分离得到的病原菌进行鉴定,并对番茄灰叶斑病抗性鉴定方法进行研究,以期为番茄灰叶斑病的抗病育种研究及其利用提供理论依据。     

Slow wilting components in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>(L.) Millsp.)

Partial resistance to Fusarium wilt was characterized based on root inoculation on seven pigeonpea genotypes with a virulent isolate of Fusarium udum. Resistance to wilt seems to be mainly due to localization of the pathogen in roots and vascular systems in the stem. Three components of resistance (wilting rate or incubation period, weighted wilt index and number of colonized plants) were determined and compared to those of susceptible genotypes Bahar and TTB7. There were significant differences between the genotypes for the resistance components. The mechanisms of resistance in the genotypes appeared to be different, with genotype ICP8863, having a longer incubation period, minimum wilt index and minimum pathogen colonization as compared to other resistant genotypes (ICP9174, ICP87119 and ICP8858). Wilting rate or incubation period and number of colonized plant were significantly correlated with resistance in adult plants in the field (AUDPC). Wilt index was useful in discriminating between genotypes that had a similar incubation period and number of colonized plants. The partial character of resistance is probably based on quantitative differences in localization capacity among the genotypes. A quantitative relationship between components, incubation period and number of colonized plants and the AUDPC, if verified for a large number of genotypes, may be used to obtain an index of resistance that may predict resistance levels in the field.     

A high-throughput glasshouse bioassay to detect crown rot resistance in wheat germplasm

A high-throughput and reliable seedling bioassay to screen wheat germplasm for crown rot resistance was developed. Single wheat seedlings were grown in square seedling punnets in a glasshouse and inoculated with a monoconidial Fusarium pseudograminearum isolate 10 days after emergence. The punnets were laid horizontally on their side and a 10- µ L inoculum droplet placed on the stem base. Seedlings were incubated at near-saturated relative humidity, and crown rot severity was assessed 35 days after inoculation. Studies on the duration of incubation period, inoculum concentration and temperature were carried out to optimize these parameters. Seedling growth at 25/15(±5)°C in a glasshouse and 48-h incubation at near-saturated RH in darkness gave the best results. When crown rot resistance rankings of 16 Australian cultivars from the bioassay were compared with their field performance, Spearman's rank correlation coefficient was highly significant. This indicated that the seedling bioassay mimicked field resistance to crown rot in adult plants. A bootstrap resampling analysis showed little or no improvement in the coefficient of variation with an increasing number of replications, indicating a high level of precision and reproducibility. By detecting small but consistent differences in crown rot severity, the bioassay proved effective in large-scale screening for partial resistance: already over 1400 wheat genotypes have been screened. The high degree of precision makes this an invaluable tool in the understanding of pathogen aggressiveness, host specialization and parasitic fitness.     

A green fluorescent protein-based screening method for identification of resistance in anthurium to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae

Resistance of cultivars of Anthurium andraeanum to systemic infection by Xanthomonas axonopodis pv. dieffenbachiae , the causal organism of bacterial blight disease of anthurium, was investigated using a bioengineered bacterial strain containing p519ngfp plasmid. Successful infection establishment in anthurium was found to be cultivar and inoculum density dependent, but independent of plant age. Injection of cut petioles (stage-2 leaf) with 100  µ L inoculum (10⁹ CFU mL⁻¹) resulted in 100% infection establishment in susceptible cultivars on a repeatable basis, and differentiated between various levels of observed field resistance. Time to death (weeks) and proportion of dead plants best differentiated between levels of resistance and cultivars were placed in four groups based on these criteria. The susceptible group (32 cultivars) rapidly declined within 6–12 weeks of inoculation (WAI) and resulted in 100% plant death; the moderately resistant group (10 cultivars) declined within 12 WAI, but resulted in less than 100% plant death; the resistant category had less than 100% plant death with a slow decline taking over 20 weeks; and the highly resistant category (15 cultivars) showed 0% infection. The correlation coefficient between green fluorescent protein (GFP)-fluorescence and eventual death of plants was 0·90, indicating that the final death of individual plants can be reasonably well predicted based on GFP-fluorescence data at 5 WAI. Hence GFP data at 5 WAI can be used for early detection of latently infected plants and may assist screening for resistance in segregating populations of anthurium.     

Production, survival and infectivity of oospores of Phytophthora infestans

The formation of oospores of Phytophthora infestans was studied in tomato and potato crops and volunteer plants under field conditions, and in laboratory tests with leaf discs of potato cultivars differing in their level of race-nonspecific resistance. Oospores were readily detected in blight-affected tomato leaflets and fruits, and in leaflets of field crops and volunteer potato plants. Oospores extracted from blighted potato leaflets yielded 13 oospore-derived progeny. Oospores were also produced following inoculation of leaf discs of eight potato cultivars expressing different levels of race-nonspecific resistance with a mixture of sporangia of A1 and A2 isolates. The highest numbers of oospores were produced in cvs Bintje (susceptible) and Pimpernel (resistant), and the lowest in Nicola (intermediate resistance). The relationship between lesions per leaflet and oospore incidence, affected by varying A1 : A2 ratios, was explored using a simple mathematical model, and validated by comparing actual oospore production in leaflets with multiple lesions of the race-nonspecific-resistant potato clone Lan 22-21 with the predictions generated by the model. Survival of oospores was investigated after their incorporation in either a sandy or a light clay soil in buried clay pots exposed to the local weather conditions. Over 6 years these soils were regularly assessed for their infection potential using floating leaflets in a spore-baiting bioassay. Sandy and clay soils contaminated with oospores remained infectious for 48 and 34 months, respectively, when flooded. Infections of floating potato leaflets occurred within 84–92 h and ceased after 11 days. Soil samples remained infective if dried and re-flooded on two, but not more, occasions.     

Evaluation of carrot resistance to alternaria leaf blight in controlled environments

The objective of this study was to find a technique for plant resistance screening to alternaria leaf blight (ALB), caused by the fungus Alternaria dauci , in controlled environments. Glasshouse and laboratory screening methods were compared using three cultivars and F₂ genotypes segregating for ALB resistance evaluated against self-pollinated F₃ field-grown plants. Plant disease was assessed through a disease index obtained from the size and number of symptoms on carrot leaves. The results indicated the value of glasshouse evaluation and the inadequacy of detached leaf and hypocotyl assays for carrot screening for ALB resistance. Spearman's rank correlation, applied to results obtained with both F₂ plants and their progeny, indicated that the optimal evaluation stage for ALB resistance in carrot is 20 days after inoculation. This test was powerful enough to be used as a prescreening test in breeding programmes.