油菜茎基溃疡病菌的实时荧光PCR检测

 根据油菜茎基溃疡病菌Leptosphaeria maculans与其近似种ITS序列的差异,设计了检测L. maculans的引物Lmb3/R2和探针Probe-M,建立了L. maculans的实时荧光PCR检测方法。试验结果表明,来自加拿大、澳大利亚和乌克兰等国的22株L. maculans菌株都能得到阳性扩增,而供试的30株L. biglobosa菌株和6株其他菌株以及空白对照没有荧光信号的增加。该检测方法的灵敏度达到4 pg菌丝DNA,整个检测过程控制在4 h内,其快速、特异和灵敏的特点可以满足进境油菜籽样品的快速初检以及病菌分离物的快速鉴定。     

Latent infection of winter oilseed rape by Leptosphaeria maculans

Plants of oilseed rape, cultivars Primer and Jet Neuf, were grown in a glasshouse and inoculated at G.S. 2.4–2.7 with pycnidiospores or ascospores of Leptosphaeria maculans. The plants were kept for a further 2–4 weeks at 14°C and then transferred, together with uninoculated plants, to a polythene tunnel in winter. The majority of stems of inoculated plants did not have macroscopic symptoms of L. maculans infection 6 weeks after inoculation. Examination of whole mounts of peripheral tissue and transverse sections of fixed and embedded portions of these stems revealed intercellular septate fungal hyphae, often deep in non-necrotic cortical tissue, in symptomless inoculated plants but not in uninoculated plants. L. maculans was recovered following surface sterilization of adjacent portions of the same stems. When symptomless inoculated plants were transferred to a glasshouse at 18–20°C, cankers soon developed. The significance of these latent mycelial infections to canker development in the field is discussed.     

The occurrence of virulent pathotypes of Leptosphaeria maculans in brassica seed crops in England

Virulent and non-virulent pathotypes of Leptosphaeria maculans were differentiated on the basis of cultural characteristics and virulence to cabbage plants. Surveys of isolates obtained from oilseed rape crops grown in England in 1982 and 1983 showed that virulent pathotypes predominated in some areas whereas in others they were infrequent or absent. Overall 41% of isolates from this crop were of the virulent type. Virulent types usually occurred most frequently in areas with a long history or a high density of oilseed rape. In vegetable and forage brassica seed crops in Essex virulent isolates formed a small proportion of the population, except in one swede crop from which 95% of isolates were virulent. Host specificity was not detected in cross-infection experiments using isolates from different hosts and localities.     

The incidence of Alternaria spp. and Leptosphaeria maculans in commercial brassica seed in the United Kingdom

Tests on samples of oilseed rape seed ( Brassica napus ) sown in the UK between 1981 and 1984 indicated that on average 25% of samples were infected with Alternaria brassicae and 61% with Leptosphaeria maculans , with maximum incidences of infection of 19 and 4.2% respectively. Much infection by Alternaria spp. occurred on vegetable and forage brassica seed produced in the UK between 1979 and 1983. In B. oleracea types A. brassicicola occurred most frequently, affecting 88% of samples and up to 55% of seeds. A. brassicae was detected in 44% of B. oleracea samples and in up to 13% of seeds. Little Alternaria infection occurred in swede or forage rape samples (B. napus ), but A. brassicae affected up to 8–5% of seeds in turnip samples ( B. campestris ). L. maculans occurred in 44% of samples of vegetable and forage brassica seed produced in the UK, with a maximum of 4–6% infected seeds. A. brassicicola was present in 73% of samples of imported B. oleracea seed, affecting up to 25.5% of seeds. A. brassicae was absent from these samples and little L. maculans was detected. Pathogenicity tests on isolates of L. maculans from infected seeds indicated that virulent pathotypes were present in 16 rape seed samples but in only one sample (swede) of vegetable or forage brassica seed. The high incidence of seed infection by these pathogens emphasizes the importance of applying fungicide treatments to all types of brassica seed.     

Bipolar heterothallism in B-group isolates of Leptosphaeria maculans

Pairwise combinations were carried out between the eight isolates originating from each of three asci of the B-group of Leptosphaeria maculans . Some of the pairings produced mature pseudothecia when incubated on wheat straws at 18°C and under a 12-h photoperiod of blue light. Isolates within a given tetrad could be attributed to one of two sexual compatibility groups, on the basis of combinations of isolates leading to fertile crosses. Within each tetrad, mating type segregated in a 1:1 ratio, suggesting the existence of one mating type gene ( MAT1 ) with two alleles MAT 1–1 and MAT 1–2. Under these same conditions, each individual isolate from an A-group ascus originating from Brassica juncea produced pseudothecia with one or other of two compatible A-group testers originating from B. napus . However, attempts to mate these eight A-group single-ascospore isolates with two B-group single-ascospore isolates of opposite mating types remained unsuccessful. This work is the first successful report of in vitro sexual reproduction between B-group isolates, and provides evidence for bipolar heterothallism in B-group isolates of L. maculans . The implications of this mating protocol for genetic studies of B-group isolates and the consequences of this work for the taxonomy of L. maculans are discussed.     

Identification of genomic regions associated with resistance to blackleg (Leptosphaeria maculans) in canola using genome wide association study

Black rot of crucifers is one of the most important diseases of wild rocket (Diplotaxis tenuifolia L. (D.C.)) caused by the seedborne pathogen Xanthomonas campestris pv. campestris. From 2005, it frequently affected this cultivation in the south of Italy, leading to heavy crop losses. In the present work, we aimed to describe the physiological and molecular characteristics of twenty X. campestris pv. campestris strains isolated from plants and seeds. Ten Xanthomonas spp. strains contaminating seeds were identified on the basis of molecular characterization and in vivo pathogenicity on a discriminating host range. Some of seed-borne isolates were ascribed to the species Xanthomonas campestris pv. raphani and X. campestris pv. incanae, indicating the occurrence of non-host pathogenic Xanthomonas on wild rocket seeds. As well as the presence of pathogenic bacteria, even non-pathogenic Xanthomonas spp. strains were detected on the seeds, underlying the importance of identifying them to evaluate the suitability of lots intended for sowing. A phylogeny using 69 Gyrase B (gyrB) sequences retrieved from the literature, was also carried out, highlighting species relatedness. Overall, this study provides a comprehensive framework for Xanthomonas species affecting wild rocket in Southern Italy.

     

Genetic Linkage Maps and Genomic Organization in Leptosphaeria maculans

Leptosphaeria maculans is a haploid outcrossing ascomycete with a genome size of about 34 Megabases (Mb) which is predicted to have between 10,000 and 12,000 genes. The chromosomes of L. maculans are of a size range (0.7–3.5 Mb) and number (15–16) that can be readily resolved by pulsed field gel electrophoresis. Chromosome length polymorphisms are generated by meiosis giving rise to size differences as high as 57%, in the case of the ribosomal DNA-harbouring chromosome whose size varies between 1.8 and 4.2 Mb. Genetic maps are characterised by linkage groups comprising an accumulation of markers based on retrotransposon sequences. This, along with sequencing of pericentromeric regions and stretches of ORF-rich regions, suggest that the genome of L. maculans consists of a mosaic of GC-equilibrated coding regions with no or few transposons, and of stretches of highly degenerated and truncated retrotransposons, encompassing very few genes. Chromosome length polymorphisms are linked with the loss of dispensable regions. We suggest that the degree of length polymorphism for a particular chromosome correlates to the amount of dispensable retrotransposons, and that some gene-rich chromosomes may be less prone to undergo chromosome length polymorphisms than other chromosomes.     

Effects of Temperature and Wetness Duration on Infection of Oilseed Rape Leaves by Ascospores of Leptosphaeria maculans (Stem Canker)

In controlled environment experiments, ascospores of Leptosphaeria maculans (stem canker) infected oilseed rape (cv. Nickel) leaves and caused phoma leaf spots at temperatures from 8°C to 24°C and leaf wetness durations from 8 h to 72 h. The conditions that produced the greatest numbers of leaf spot lesions were a leaf wetness duration of 48 h at 20°C; numbers of lesions decreased with decreasing leaf wetness duration and increasing or decreasing temperature. At 20°C with 48 h of leaf wetness, it was estimated that one out of four spores infected leaves to cause a lesion whereas with 8 h of leaf wetness only one out of 300 spores caused a lesion. As temperature increased from 8°C to 20°C, the time from inoculation to the appearance of the first lesions (a measure of the incubation period) decreased from 15 to 5 days but leaf wetness duration affected the length of the incubation period only at sub-optimal temperatures. Analyses suggested that, within the optimal ranges, there was little effect of temperature or wetness duration on incubation period expressed as degree-days; the time until appearance of 50% of the lesions was ca. 145 degree-days. A linear regression of % leaves with lesions (P_l) (square-root transformed) on % plants with lesions (P_p) accounted for 93% of the variance: P_l=1.31+0.061P_p. This relationship was also investigated in winter oilseed rape field experiments in unsprayed plots from October to April in 1995/96 (cv. Envol), 1996/97 (cv. Envol), 1997/98 (cvs Bristol and Capitol) and 1998/99 (cvs Apex, Bristol and Capitol) seasons. The linear regression of % leaves with lesions (square-root transformed) on % plants with lesions accounted for 90% of the variance and had a similar slope to the controlled environment relationship: P_l=0.81+0.051P_p. These results were used to examine relationships between the development of phoma leaf spot on plants in winter oilseed rape crops, the incubation period of L. maculans and the occurrence of infection criteria (temperature, rainfall) in the autumns of 1996, 1997 and 1998.     

Quantitative resistance against an isolate of Leptosphaeria maculans (blackleg) in selected Canadian canola cultivars remains effective under increased temperatures

Blackleg disease, caused by Leptosphaeria maculans, is a serious threat to canola production in western Canada. While specific major resistance (R) genes can be effective, they can also be eroded rapidly by a shift in pathogen race composition. Quantitative resistance (QR) has the potential to provide more durable, if less complete, protection. However, the effectiveness of QR may vary widely in the field. It has long been suspected that elevated temperatures may limit the expression of QR. To test this hypothesis, the infection development of blackleg was assessed on three common Canadian canola cultivars (74‐44 BL, PV 530 G and 45H29) showing QR, with and without a heat treatment of 7 h daily exposure to 32 °C for 1 week during rosette to early flowering under controlled environment conditions. The impact of elevated temperature on the susceptibility to blackleg was compared with that of a moderate temperature with a 22 °C daytime high. A susceptible cultivar, Westar, was used as a control. When data from both temperatures were pooled, all three QR cultivars showed lower blackleg severity relative to Westar. Elevated temperatures increased blackleg severity in Westar only (in terms of the stem‐lesion length from the inoculation of the first true‐leaf petiole in trials involving Westar and 74‐44 BL) and in pooled data for disease severity index in trials involving Westar, PV 530 G and 45H29. These findings suggest that the QR traits in 74‐44 BL, PV 530 G and 45H29 are useful for blackleg management in western Canada, especially under warmer growing conditions when plants are at the rosette to early flowering stages.     

Clonal populations of Leptosphaeria maculans contaminating cabbage in Mexico

The race structure and genotypic diversity of four Leptosphaeria maculans populations isolated from Brassica oleracea (broccoli, cauliflower, cabbage, etc.) in central Mexico (Aguascalientes, Guanajuato and Zacatecas states) were analysed. Race structure was characterized by an unusually low diversity at three locations out of four. Fourteen minisatellite markers revealed a high proportion of repeated multilocus genotypes in these populations, combined with a significant linkage disequilibrium and strong clonal fraction (65–87%). The occurrence of the mating‐type idiomorphs always significantly departed from the 1:1 proportion expected in the case of random mating. Each population thus consists of a few (four to nine) multilocus genotypes which are specific to each location. These data strongly support the hypothesis of exclusive, or a high rate of, clonal multiplication. Comparison of cropping practices between B. oleracea and B. napus indicate that the shift in reproductive behaviour of the fungus is chiefly man‐mediated.     

Inheritance of Resistance to Leptosphaeria maculans in Brassica juncea

ABSTRACT The inheritance of resistance to Leptosphaeria maculans, the causal agent of black leg of crucifers, was studied in Brassica juncea. Three resistant accessions (UM3021, UM3043, and UM3323) and one susceptible accession (UM3132) of B. juncea were crossed in a complete diallel. Parents, F(1), and F(2) progenies were evaluated for all crosses using both cotyledon and stem inoculation. Cotyledon reaction was evaluated with two isolates of L. maculans, but stem reaction was evaluated with one isolate. Disease reactions observed for individual plants were the same for both inoculation methods and for both isolates of the pathogen for cotyledon reaction. No segregation was observed for the crosses between resistant accessions (UM3043 x UM3323 and UM3021 x UM3323), but a few susceptible plants were observed in the F(2) progeny of crosses between resistant parents (UM3021 x UM3043). This was probably due to heterozygosity in some parental plants of UM3021. For crosses be tween the susceptible parent and resistant parents, F(1) plants for two crosses were all resistant. For cross UM3132 x UM3021, some susceptible plants occurred, which was also suggestive of heterozygosity in UM3021. Although resistance in F(1) was dominant, for F(2) populations, segregation fit either 13:3, 3:1, or 1:3 ratios, indicating that resistance can be either adominant or recessive trait. F(3) families derived from some susceptible F(2) plants from crosses UM3021 x UM3132 and UM3043 x UM3132 were evaluated using the cotyledon inoculation method only. Segregation of F(2) plants and F(3) families in crosses involving resistant and susceptible parents indicated that the resistance to L. maculans in B. juncea is controlled by two nuclear genes with dominant recessive epistatic gene action.     

The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans

In a series of growth room experiments in which leaves of Brassica napus var. oleifera were inoculated with ascospores or pycnidiospores of Leptosphaeria maculans successful infections progressed through three consecutive phases. Initial establishment in the mesophyll was succeeded by a phase of intercellular exploration, when hyphal proliferation was highly variable and host cell necrosis always ensued, and then by a systemic phase when hyphae were consistently sparse. Host cells associated with the hyphal front were capable of autofluorescence, accumulation of vital stains and plasmolysis, indicating that they were viable and that the pathogen was biotrophic throughout this sequence. During either of the first two phases permanent fungistatic containment, involving the formation of vesicles by disintegration of the hyphae, often occurred. Localization at the first phase was symptomless; at the second it was signified by a lesion with a clearly defined margin.
There was a negative correlation between biotrophic potential and necrotrophic potential of three pathogenic isolates, on both the moderately susceptible cultivar Primor and the resistant cultivar Jet Neuf. As leaves aged, a progressively larger proportion of infections failed to become systemic. With increasing inoculum load, symptomless localization of infection diminished, the phase of necrosis extended, and the probability of irreversible systemic development increased.     

A systemic pathway in the infection of oilseed rape plants by Leptosphaeria maculans

In growth room regimes arranged to simulate field conditions which coincide with natural infection of oilseed rape by Leptosphaeria maculans , leaf inoculation resulted in systemic infection. After colonizing intercellular spaces in the spongy mesophyll of the lamina, the fungus reached a vascular strand and spread down the petiole mainly in xylem vessels or between cells of the xylem parenchyma and cortex, eventually invading and killing cells of the stem cortex and causing the stem canker symptom. The intercellular systemic phase of growth, which was biotrophic and virtually sytnptomless, occurred under a wide range of temperatures.     

油菜茎基溃疡病菌DPO-PCR检测方法的建立

根据油菜茎基溃疡病菌ITS基因序列,设计特异性DPO引物,建立检测油菜茎基溃疡病菌的DPO-PCR检测方法,并对其特异性、灵敏性进行评价。结果显示与常规PCR检测方法相比,DPO-PCR反应对退火温度不敏感,具有更强的特异性。利用该方法对10批油菜籽样品进行检测,共检出5批阳性样品,检测结果与常规PCR一致,为油菜茎基溃疡病菌的检测提供了新方法。     

油菜茎基溃疡病菌LAMP-LFD检测方法的建立

本文基于环介导等温扩增技术与横向流动试纸条相结合的方法,建立了一种应用于油菜茎基溃疡病菌(Leptosphaeria maculans)的LAMP-LFD快速检测方法。以油菜茎基溃疡病菌的ITS基因序列为靶序列,设计出一套用于LAMP-LFD检测的引物和探针,优化了反应体系与反应条件(63℃,35 min)。结果表明:只有油菜茎基溃疡病菌出现阳性条带,其他参照菌株和阴性对照均未出现阳性条带,说明LAMP-LFD检测特异性强;灵敏度检测表明,对油菜茎基溃疡病菌的检测极限可低至114 fg/μL,灵敏度比传统PCR高10倍;该方法可从进境船载油菜籽样品中成功检测出油菜茎基溃疡病菌,检测结果与传统的鉴定方法一致。LAMP-LFD检测方法能够快速检测油菜茎基溃疡病菌,具有简便、灵敏、特异性高,不依赖特殊检测设备等优点,极具推广前景。     

Effect of defoliation by livestock on stem canker caused by Leptosphaeria maculans in Brassica napus

Brassica napus (canola, oilseed rape), an important break crop for cereals across the Australian wheat belt, is being rapidly adopted as a dual‐purpose (forage and grain) crop in mixed farming systems. Stem canker caused by the fungus Leptosphaeria maculans is the most important disease of B. napus in Australia. The primary source of inoculum is airborne ascospores released during autumn/winter which coincides with the grazing of dual‐purpose crops. Field experiments were defoliated by sheep to determine the effect of grazing on blackleg stem canker severity at plant maturity in B. napus cultivars differing in their resistance level and grazed at different times. One cultivar was sown on different dates to investigate the impact of grazing at the same time, but at different growth stages. Defoliation by mowing was compared to defoliation by livestock. Similar amounts of dry matter remained after defoliation by machinery (0·66 t ha^?1) or livestock (0·52 t ha^?1). However, stem canker severity was higher in the grazed (40% of crown cross‐section diseased) compared with the mown (25%) treatment, which was higher than the ungrazed control (9%). Stem canker severity generally increased with grazing, but the increase was eliminated or reduced in cultivars with good resistance. Grazing during vegetative plant growth minimized the increase in stem canker severity compared with grazing during reproductive growth. Currently, cultivars with good L. maculans resistance are recommended in high disease situations. To avoid excessive yield loss in dual‐purpose B. napus crops due to L. maculans it is recommended that such cultivars are grown even in low‐moderate disease situations.     

Rep-PCR Based Genomic Fingerprinting of Isolates of Leptosphaeria maculans from Poland

Leptosphaeria maculans, the ascomycete fungus which causes blackleg disease of oilseed rape, has been considered for a long time as a single species divided into aggressive and non-aggressive pathogenicity groups which differ in their economic importance. However, the development of accurate biochemical and molecular characterisation methods has demonstrated that the world-wide L. maculans population actually comprises at least two species. The aim of this research was to assess the ability of rep (repetitive element based)-PCR genomic fingerprinting methods, initially developed for bacterial identification, to characterise a collection of 90 isolates of L. maculans from Poland, in comparison with reference isolates from the IBCN (International Blackleg of Crucifers Network) collection. REP (repetitive extragenic palindromic)-, ERIC (enterobacterial repetitive intergenic consensus)-, and BOX primers for rep-PCR genomic fingerprinting, and primers derived from LMR1, a L. maculans specific repeated element, were tested. Rep-PCR and LMR1-based analyses were able to discriminate the different components of the species complex and to evaluate the genetic diversity within each member of the complex. These analyses suggested that Polish populations of L. maculans mainly belong to the non-aggressive species, rather than the aggressive species which is prevalent in Western Europe, Canada and Australia.