肉桂醛对增强辣椒疫霉病抗性的作用机制研究

该实验以辣椒疫霉菌(Phytophthora capsici)致病性菌株NJ01和‘苏椒5号’辣椒幼苗为研究对象,通过肉桂醛对辣椒疫霉菌的体外抑菌作用、室内侵染效果以及对辣椒幼苗防卫反应的调控作用,揭示肉桂醛对增强辣椒疫霉病抗性的作用机制。结果表明:(1)肉桂醛对实验所用致病性菌株NJ01的抑制中浓度(EC50)值为0.81mmol/L;肉桂醛处理可导致NJ01菌丝严重皱缩、畸形、断裂;PI染色显示NJ01菌丝出现明显的细胞死亡现象。(2)单独NJ01菌株接种的辣椒植株表现出明显病症(茎基部变黑褐色、干枯萎缩,植株倒伏,叶片脱落,生物量下降);而肉桂醛处理2h后接种NJ01菌株的辣椒植株长势良好,无明显病症,鲜重和叶绿素含量显著上升至对照水平。(3)与单独接种NJ01菌株处理相比,肉桂醛处理2h后接种NJ01菌株的辣椒植株体内抗氧化酶(CAT、SOD、POD)活性显著上升,抗氧化物质(GSH和ASA)含量显著增加。研究认为,肉桂醛可能通过抑制辣椒疫霉菌的生长及其对辣椒植株的侵染能力,同时调控辣椒植株防卫反应,进而提高辣椒对疫病抗性。     

The Raf-like kinase Raf36 negatively regulates plant resistance against the oomycete pathogen Phytophthora parasitica by targeting MKK2

Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T-DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf-like mitogen-activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus-induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36-conferred resistance to P. parasitica. Taken together, we identified a Raf-like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae

PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism. The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.     

Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.     

The Influence of Phytophthora citricola on Rhizosphere Soil Solution Chemistry and the Nutritional Status of European Beech Seedlings

In a rhizotron experiment the influence of Phytophthora citricola on root development, rhizosphere chemistry and nutritional status of beech seedlings was studied. After infection, the fine root system was seriously damaged and 6 weeks after inoculation some seedlings died. Plants infected with Phytophthora citricola had less steep concentration gradients of K⁺, Ca²⁺, Mg²⁺ and NO in the rhizosphere soil solution as compared with uninfected seedlings, which indicates reduced activity in nutrient uptake. It is concluded that plants try to compensate their deficit in fine root biomass and nutrient acquisition by retranslocation of nutrients.     

Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997