Screening for scab resistance by RAPD markers in cultivars of apple (Malus spp.)

Genetic markers are a much faster and more practical alternative to classical methods for the identification of genes for scab resistance present in different apple cultivars. In our study, 28 scab-resistant cultivars, four wild sources of the resistance genes and 10 susceptible cultivars were screened for the presence of the RAPD fragments OPM18/900, OPD20/600 and OPA15/900, which are reported to be linked to the Vf gene. All three marker fragments were successfully amplified with different protocols in Vf-resistant cultivars including ‘M. floribunda 821’. No marker fragments were amplified in susceptible cultivars, three out of four Va-resistant cultivars, three out of four Vm-resistant cultivars, two Vr-resistant cultivars, ‘Antonovka PI 172612’ and ‘M. pumila R 12740-7A’. All three markers were found in the cv. ‘Nova Easygro’, reported to possess the Vr gene, and the cv. ‘Reglindis’, reported to be Va-resistant. M. atrosanguinea of unknown origin showed the presence of OPD20/600 and OPA 15/900 marker bands. The cvs. ‘Nova Easygro’, ‘Reglindis’ and M. atrosanguinea are probably carriers of the VF gene.     

Efficient low-cost DNA extraction and multiplex fluorescent PCR method for marker-assisted selection in breeding

Marker‐assisted selection (MAS) is an increasingly important tool in current breeding efforts for improved crop plants and animal breeds. It enables detection of favourable alleles in early developmental stages and thus may result in substantial cost savings. Until now, however, the high costs of the required chemicals and materials, together with the still very labour‐intensive methods, have been an obstacle to widespread application of MAS. A new multiplex‐polymerase chain reaction (PCR)‐based method has been developed for reliable low‐cost, high‐throughput screening. By its use 3366 apple seedlings were screened with an average hands‐on time from DNA extraction to data ready for analysis of < 4 h per 96 plants, and at a cost below US$ 0.5 per marker per plant. Factors that have a strong effect on segregation ratios such as elevated levels of outcrossing are easily detected, as a significant correlation was observed between deviation from expected segregation ratios in some affected markers and the level of outcrossing in a cross. The new method is suitable for many crop species and, provided that suitable buffers are used for DNA extraction, for animals too.     

梨黑星病菌产毒条件及毒素毒性检测

研究梨黑星病病原菌株在不同培养基、光照、温度、pH值、培养时间和震荡培养方式等条件下产生毒素的情况,并用砀山酥梨种子发芽法、叶片测定法、试管苗根、茎生长测定法检测毒素的毒性.结果表明,适宜梨黑星病菌粗毒素培养的培养基为PDA液体培养基,培养基pH值为6.0,培养温度22℃,在黑暗下110 r·min-1连续震荡培养22 d时产生的毒素毒性最强.采用4种方法都可检测到毒素的活性.     

Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain.     

Microsatellite primers indicate the presence of asexual populations of<Emphasis Type="Italic">Venturia inaequalis</Emphasis> in coastal Israeli apple orchards

This study was initiated to determine whether differences in genotypic diversity among populations ofVenturia inaequalis (Cke.) Wint., as detected using neutral genetic markers, were related to the ecological conditions in which apples are grown in Israel. Since sexual reproduction in this fungal pathogen has an obligate requirement for sustained low winter temperatures, and since these requirements in Israel are met only on the Golan Heights, we were interested in whether lower elevation populations of this pathogen might be comprised of asexual clonal lineages. Unlike temperate apple growing regions, where the primary spring inoculum is ascosporic derived from overwintered pseudothecia, Israeli apple orchards at lower elevations in the Hula Valley and along the coastal plain rarely if ever experience low winter temperatures and pseudothecia have never been recovered. Two orchards were sampled from the Golan Heights (El Rom and Ortal, n=38) and three orchards from the Hula Valley and coastal plain (Sede Eliezer, Ginaton and Be’er Tuvia, n=40). Microsatellite primers were used to analyze population structure and the resulting binary data analyzed by both cluster and parsimony analysis. Populations from the coastal plain were genetically uniform within each of the orchards sampled, whereas populations from the Golan Heights showed levels of genotypic diversity ten times as high. The data support field observations that this pathogen does not reproduce sexually in regions characterized by the absence of low winter temperatures and is instead composed of clonal lineages. This may have bearing on control strategies for the disease in Israel. http://www.phytoparasitica.org posting April 21, 2003.     

梨黑星病菌被哈茨木霉感染后抗氧化系统的变化

测定被哈茨木霉感染后梨黑星病菌抗氧化系统变化的结果表明,被哈茨木霉感染后梨黑星病菌过氧化氢酶、过氧化物酶和超氧化物歧化酶的活性逐渐下降;丙二醛含量和超氧阴离子产生速率上升;还原型谷胱甘肽和抗坏血酸含量降低。初步明确,梨黑星病菌被哈茨木霉感染后,活性氧清除系统等被破坏,膜脂过氧化作用加强,是最终导致菌体受害的原因之一。     

长江中下游梨黑星病前期发病程度预测及防治

长江中下游梨黑星病病菌多于病芽鳞片内越冬,从芽萌动至鳞片脱落是初侵染的主要时期。至5月中旬(前期)的病叶率与上年7月的降雨量呈极显著的正相关(r=0.9765,n=16),从而建立直线回归方程(?)=-3.575 0.0586X,以此方程依据7月的降雨量(X)预测次年前期的病叶率((?))。因此,花序露出期喷洒铲除剂和花期雨后(尤其流行年份)及花谢80％喷洒保护剂是控制前期发病与全年为害的关键。     

Effects of cultivar mixtures on scab control in apple orchards

The effects of two mixtures of resistant and susceptible apple cultivars on the development of scab caused by Venturia inequalis were observed in an experimental orchard over four years, initially for two years without fungicides against scab, and subsequently for two years with a moderate fungicide schedule. The row-by-row and within-row mixtures included a susceptible cultivar and a resistant cultivar in equal proportions. Without fungicides, the results showed a significant reduction of disease incidence over both years (7·3 to 21·3%), and severity in the second year (35·4%) in the within-row mixtures, compared to the monoculture of the susceptible cultivar. The best results were obtained when the within-row mixture was associated with moderate fungicide treatments; in this case the reduction in disease incidence reached 75·1% on leaves and 69·7% on fruits during the growth phase. The characteristics of the Venturia inaequalis / Malus  ×  domestica pathosystem and the results obtained in this experiment suggest a moderate but not negligible ability of cultivar mixtures for reducing epidemics of the disease.     

Sensitivity to strobilurin fungicides of Italian Venturia inaequalis populations with different origin and scab control

BACKGROUND: Venturia inaequalis (Cooke) Winter with reduced sensitivity to strobilurins has been reported in several countries, including Italy. This study aimed to characterise the sensitivity to strobilurins of three different types of V. inaequalis population: (a) wild types; (b) from commercial orchards satisfactorily managed with strobilurins; (c) from an experimental orchard with control failures by trifloxystrobin and kresoxim‐methyl. In vitro sensitivity tests included antigerminative activity on population conidia and mycelial growth inhibition on monoconidial isolates. Cleaved amplified polymorphic sequence (CAPS) analysis was used for the detection of G143A substitution. RESULTS: Wild‐type populations showed EC₅₀ values lower than 0.031 mg L^?1, while those of orchards with good performance by strobilurins presented EC₅₀ values never higher than 0.063 mg L^?1. Samples with scab control failures showed a strongly reduced population sensitivity. Similar differences were confirmed in monoconidial isolates. The G143A substitution was always detected in low‐sensitivity populations, only sometimes in well‐controlled populations and generally not in wild types. CONCLUSIONS: In vitro sensitivity assays were able to discriminate the three population types with different scab management, while the qualitative PCR analysis (CAPS) was only partially reliable. High sensitivity differences among V. inaequalis populations with good and poor field control by strobilurins were observed. Copyright © 2011 Society of Chemical Industry