Mutants of Downy Mildew Resistance in Lactuca Sativa (Lettuce)

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from γ- or fast neutron-irradiated seed. In two separate Dm1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.     

The Possible Involvement of Lipoxygenase in Downy Mildew Resistance in Pearl Millet

The downy mildew disease, incited by Sclerospora graminicola,is a major biotic constraint for pearl millet production inthe semi-arid tropics. Sources of resistance to this diseasehave been identified. However, the mechanism of host resistancestill remains obscure. The enzyme lipoxygenase (LOX) is knownto play a role in disease resistance in many host-pathosystems.In the present study, LOX activity was tested in seeds of differentgenotypes of pearl millet with different susceptibility to downymildew. The LOX assay of the seeds indicated a good correlationbetween enzyme activity and their downy mildew reaction in thefield. Maximum activity was recorded in seeds of highly resistantgenotypes and minimum activity was found in the highly susceptiblegenotypes. Seeds obtained from plants recovered from the downymildew disease had more LOX activity than that of the originalparent seeds. Thus, in seeds, the LOX activity can be used asa biochemical marker for screening different genotypes of pearlmillet for downy mildew. The study, carried out in the susceptiblegenotype of pearl millet seedlings, showed that LOX activitydecreased after inoculating with S. graminicola zoospores whencompared with uninoculated controls. However, a significantincrease in the enzyme activity was observed on the second andthird days after inoculation in resistant seedlings. The possiblerole of LOX in conferring resistance to downy mildew infectionof pearl millet is discussed. Key words: Lipoxygenase, pearl millet, downy mildew     

At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

黄瓜霜霉病抗性相关基因的AFLP标记

运用AFLP技术,采用集群分析法研究与黄瓜霜霉病抗性基因相关的分子标记.结果表明,在F2群体中E25M63-103标记与霜霉病病情指数的相关系数(0.337)和回归分析的F值(20.98)都达到了极显著水平.用E25M63-103标记对国内外的其它27份黄瓜材料进行检测,该标记与霜霉病病情指数的相关系数为0.555,也达到极显著相关水平,进一步证明该标记与控制黄瓜霜霉病感病的相关基因是连锁的.E25M63-103片段长度为103 bp,通过BLAST查询,该片段的同源性较小,表明E25M63-103标记可能是黄瓜基因组特有的一段DNA序列.     

黄瓜霜霉病抗性遗传分析

通过2个抗感杂交组合,采用多世代联合的分离分析方法研究了黄瓜霜霉病抗性的遗传机制.结果显示,2个组合的最适遗传模型分别是2对加性-显性-上位性主基因 加性-显性-上位性多基因模型和2对等加性主基因 加性-显性多基因模型.组合I最优模型的主基因遗传率是56.84%~87.16%,多基因遗传率是0~34.93%;2个主基因的加性效应均为-15.191,加性效应较强,显性效应较弱,它们之间的加性与加性和加性与显性上位性效应较强.组合Ⅱ最优模型的主基因和多基因遗传率分别为48.92%和42.11%;2个主基因的加性效应皆为-13.505,显性效性均为0,它们之间不存在互作效应.结果表明,黄瓜霜霉病抗性,以加性效应为主,主基因遗传力较高,但是微效多基因效应也占相当的比重,所以,在霜霉病抗性育种中,要重视主基因,同时兼顾多基因效应.     

Phytoalexin-Deficient Mutants of Arabidopsis Reveal That Pad4 Encodes a Regulatory Factor and That Four Pad Genes Contribute to Downy Mildew Resistance

We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.     

Comparison of Different Inoculation Techniques to Screen Resistance of Pea Lines to Downy Mildew

Pea lines with partial resistance to downy mildew were tested with different inoculation techniques. Lines with superior resistance to leaf and pod infection in the field also show superior resistance to artificial inoculation of germinating seeds or seedlings at different ages in greenhouse experiments. Increased resistance of older seedlings was found for all genotypes including the susceptible control. Juvenile leaves at the top of the seedlings plants at time of inoculation, developed the strongest symptoms. Selection for partial resistance can probably be carried out in the greenhouse to improve resistance of adult plants in field.     

Overexpression of an Oil Radish Superoxide Dismutase Gene in Broccoli Confers Resistance to Downy Mildew

Superoxide dismutases (SOD) play important roles in plant disease resistance. In this study, a manganese superoxide dismutase gene, designated RsrSOD, was isolated from oil radish (Raphanus sativus var. raphanistroides). RsrSOD was 696?bp in length predicted to encode 231 amino acids. The deduced amino acid sequence contained four putative Mn-binding sites. Under the control of the CaMV35S promoter, RsrSOD was introduced into broccoli (Brassica oleracea var. italica) via Agrobacterium tumefaciens-mediated transformation. Six transgenic lines were obtained out of 35 independent shoots. Both gene expression and enzyme activity of SOD increased significantly in transgenic lines when challenged with Hyaloperonospora parasitica. Three lines, L19, L23, and L25, exhibited the highest resistance against downy mildew with disease symptoms restricted completely. These highly resistant lines would serve as good broccoli breeding materials.     

Lipid Peroxidation and Antioxidant Activities Involved in Resistance Response against Downy Mildew in Opium Poppy

The aim of this study was to observe the lipid peroxidation (LP) of cell membranes and antioxidant systems in response to inoculation of Peronospora arborescens causing downy mildew (DM) in opium poppy. Contents of the LP product, malondialdehyde (MDA) and antioxidant glutathione (GSH) were determined in leaves of two opium poppy genotypes, Pps‐1 (highly resistant to DM) and Jawahar‐16 (highly susceptible to DM) at different time intervals after inoculation (12 h, 24 h, 48 h and 72 h). The provided GSH content corresponded to that of total non‐protein sulfhydryl groups. In leaves of Jawahar‐16, a significant decrease in concentration of GSH and a persistent increase in concentration of MDA were recorded after inoculation in comparison to leaves of control plants. The continuous decrease in GSH content contributed to damage of cell membranes leading to disease development in Jawahar‐16. On the other hand in a resistant genotype (Pps‐1), initially at 12 h after inoculation (hai) the level of GSH was found to be high, but a transient and highly significant decrease in content of GSH and increase in content of MDA was observed at 24 hai in comparison to control plants of same genotype and also in comparison to inoculated plants of susceptible genotype (Jawahar‐16). These results indicate that generation of GSH and MDA is negatively correlated during the infection process as found in the case of DM‐resistant genotype Pps‐1 at 24hai, which also suggests an increased need by the host plant for oxidative stress, required for hypersensitive response mediated defense mechanism.     

草酸诱导黄瓜幼苗对霜霉病的抗性与H2O2的关系

以长春密刺黄瓜幼苗为材料,对经草酸处理或霜霉菌接种后黄瓜叶片的过氧化物酶(POD)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及H2O2含量的变化进行了研究.结果表明:草酸处理或霜霉菌接种均可诱导黄瓜幼苗叶片H2O2含量显著增加,且草酸预处理后接种的叶片比相应对照叶片能更快地积累H2O2;草酸处理后叶片SOD和POD活性均升高,而CAT活性却受到一定程度的抑制.研究发现,H2O2参与了幼苗对霜霉病的抗性诱导;叶片H2O2含量的增加与其SOD、POD活性升高、CAT活性下降有关;通过调节黄瓜叶片H2O2的含量来调控有关黄瓜霜霉病抗性的防御基因表达是草酸诱导抗性的机制之一.     

不同品种葡萄抗霜霉病特性与叶片POD、PPO活性关系的研究

在霜霉病盛发期,对8804、梅尔诺、品丽珠3个葡萄品种(系)叶片中的PPO和POD活性变化进行了测定.结果显示,8804的PPO和POD活性较大,并保持相当长时间的高活性值,而梅尔诺、品丽珠叶片中PPO和POD活性较小;8804的PPO酶活性变化范围高于其它2个品种,但POD酶活性变化范围却低于后者.葡萄叶片中PPO和POD活性与葡萄霜霉病抗性之间存在一定的相关性,且不同抗感品种间PPO和POD酶活性存在极显著差异.研究结果表明,8804较梅尔诺、品丽珠对霜霉病具有较强的抗性.     

Phenylalanine Ammonia Lyase Activity in Pearl Millet Seedlings and its Relation to Downy Mildew Disease Resistance

Phenylalanine ammonia lyase (PAL) activity was studied in differentgenotypes of pearl millet with varying degrees of susceptibilityto downy mildew disease, after inoculating with Pathotype 1of Sclerospora graminicola. In resistant genotypes, the enzymeactivity significantly increased 24 h after fungal inoculationwhile in the susceptible genotypes, the activity decreased.The increase or decrease in enzyme activity was well-correlatedwith the degree of host resistance to the pathogen. A time-courseof change in activity of PAL after inoculation showed a considerabledifference between resistant and susceptible genotypes. Studieson the activity of PAL in different parts of pearl millet seedlingsrevealed that in the resistant genotype, enzyme activity significantlyincreased at 24 h post-inoculation only in the shoot portion,whereas in mesocotyl and root the activity decreased. In susceptibleseedlings, enzyme activity decreased at 24 h post-inoculationin shoot, mesocotyl and root. The activity of PAL was also foundto be pathotype-specific. Histochemical tests for lignin werepositive in infected cells in the resistant genotypes. The roleof PAL in imparting resistance to pearl millet against downymildew disease is discussed. Key words: Sclerospora graminicola, resistance screening, enzyme activity     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Histopathological Studies of Resistance of Sunflower (Helianthus annuus L.) to Downy Mildew (Plasmopara halstedii)

Histopathological studies of the infection of sunflower seedlings by downy mildew ( Plasmopara halstedii ) have shown that penetration of roots and the lower part of the hypocotyl occurs for both compatible combinations (suseptibility) and incompatible combinations (resistance). After penetrating susceptible genotypes, the parasite develops intercellular hyphae and intracellular haustoria, leading to systemic invasion. In contrast, in resistant plants, as soon as colonization develops, hypersensitive-like reactions occur in the parenchyma, with the appearance of necrotic zones surrounded by dividing cells. Growth of the parasite is strongly inhibited and most hyphae are blocked before they reach the cotyledonary node.     

Red Light Increases Suppression of Downy Mildew in Basil by Chemical and Organic Products

Basil is an economically important herb in the United States and in the world. Recent epidemics of basil downy mildew, caused by Peronospora belbahrii, have significantly affected basil production in the United States. ProPhyt (potassium phosphite), Actigard (acibenzolar‐S‐methyl) and Organocide (sesame oil) were evaluated in the greenhouse in the presence or absence of red light for their effects on the severity of downy mildew and sporangial production by P. belbahrii. Red light at intensity of 12 μmol photons/m²/s significantly (P < 0.05) reduced severity of downy mildew in basil. ProPhyt‐treated basil plants had the lowest disease severity irrespective of red light exposure. Basil plants treated with Actigard and Organocide under red light had significantly lower disease severity compared to plants under dark conditions with the same fungicide treatments 14 and 13 days after inoculation (DAI) in experiments 1 and 2, respectively. Red light significantly reduced AUDPC in the treatments of Actigard and Organocide in both experiments. Basil plants treated with Actigard and Organocide under red light had significantly reduced number of P. belbahrii sporangia than those under dark conditions receiving the same fungicide treatments. This is the first report demonstrating red light in combination with Actigard and Organocide for improved management of downy mildew in greenhouse‐grown basil.