Identification of a 2,6-dichloroisonicotinic-acid-sensitive protein kinase from tobacco by affinity chromatography on benzothiadiazole-sepharose and NIM-metal chelate adsorbent

In the search for the target site of inducers of systemic acquired resistance (SAR), a BTH-binding protein kinase (BBPK) has been identified from tobacco by affinity chromatography on benzothiadiazole-sepharose (CGA 324041-sepharose) and NIM-metal chelate affinity resin. The substrate selectivity of the isolated enzyme (phosphorylation of histone type III-S, I kappa B alpha,IkB alpha S32A/S36A and NIM1) suggested a possible BBPK-mediated regulation of NIM1 in tobacco. The measurement of the effect of different SAR-inducers showed an inhibition of BBPK by 2,6-dichloroisonicotinic acid (INA) and, to a lower extent, by benzothiadiazoles and salicylic acid. Comparison between BBPK cell-free inhibition and in vivo PR-1 induction revealed that BBPK could be the target site of INA.     

Induction of systemic resistance to Sclerotiniasclerotiorum by oxalic acid in oilseed rape

Treatment of the lowermost leaf of flowering plants of oilseed rape cv. Linetta with 20 m m oxalic acid induced systemic disease resistance in the three next youngest leaves. Resistance was not caused by the translocation of fungitoxic concentrations of oxalic acid. Resistance was also expressed in the stem; in oxalic acid-treated plants the vertical spread of stem lesions was halted and the signal travelled both acropetally and basipetally. Expression of resistance in the leaves was detected 6 h after treatment and continued for at least 5 weeks. Twenty-four h after treatment, resistance was detectable in the leaves following the use of as little as 0.5 m m oxalic acid. Local resistance in the oxalic acid-sprayed leaf was detectable only at certain times (12 and 48 but not 24 h after treatment) and at very low (0.25 m m ) or high (≥40 m m ) oxalic acid concentrations. When oxalic acid was applied to a discrete area of the leaf, significant local resistance was expressed in the surrounding leaf tissue, maximum resistance being exhibited by the tissue closest to the site of petiole attachment.     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

Induction of Systemic Acquired Resistance in Pepper Plants by Acibenzolar-S-methyl against Bacterial Spot Disease

The ability of acibenzolar-S-methyl to induce resistance in pepper plants against Xanthomonas campestris pv. vesicatoria was investigated in both growth chamber and open field conditions. Growth chamber experiments showed that acibenzolar-S-methyl (300M) treatment protects pepper plants systemically and locally against X. campestris pv. vesicatoria. Evidence for this was a reduction in the number and diameter of bacterial spots and bacterial growth in planta. Systemic protection was also exerted by the acibenzolar-S-methyl acid derivative, CGA 210007, which may be produced by hydrolysis in the plant. The efficacy of acibenzolar-S-methyl was also found in open field conditions, where both leaves and fruit were protected from the disease. The highest efficacy (about 67%) was obtained by spraying the plants 6–7 times every 8–12 days with a mixture of acibenzolar-S-methyl and copper hydroxide (2.5 + 40ghl^–1 active ingredient). Persistence and translocation data obtained from the growth chamber experiments revealed a persistence of acibenzolar-S-methyl lasting five days after treatment with rapid translocation and negligible levels of acid derivative formation. Since the protection exerted by acibenzolar-S-methyl against bacterial spot disease was observed when the inducer was completely degraded, it would appear to be due to SAR activation.     

Identification of Greenhouse Characteristics which Affect the Incidence of Sclerotinia sclerotiorum (Lib.) de Bary in Pepper Crops in a Mediterranean Climate

The effect of the main construction factors and design features of greenhouses on the incidence of white rot disease, caused by Sclerotinia sclerotiorum (Lib.) de Bary, was studied in peppers grown under plastic-roofed greenhouses typical of Almería (SE Spain). At the height of the pepper-growing season in the province, fifty greenhouses were repeatedly sampled. Incidence of the disease was measured and the different construction features of the greenhouses were evaluated: types of roof cover, colour and age of the plastic cover, dimensions (ground plan, surface area, average height), estimated ventilation capacity and the presence of netting on the sides of the greenhouses (laterals). Statistical analyses were undertaken to see if the choice of various design options for each factor had any significant influence on the incidence of the disease. To achieve this single factor ANOVA was undertaken. The incidence of this disease was significantly affected (p<0.05) by the following greenhouse characteristics: ground plan, surface area, mean height, colour of the plastic cover, estimated ventilation and use of lateral netting.     

Sensitivity to iprodione and boscalid of Sclerotinia sclerotiorum isolates collected from rapeseed in China

Baseline sensitivity of Sclerotinia sclerotiorum, causal agent of stem rot of rapeseed, to a dicarboximide fungicide iprodione was determined using 50 isolates (historic population) collected in 2001 from the rapeseed fields without a previous history of dicarboximide usage. The 50% effective concentration (EC₅₀) values to iprodione of these wild-type isolates ranged from 0.163 to 0.734 μg/ml with a mean of 0.428 μg/ml. In 2007 and 2008, 111 isolates (current population) were collected from rapeseed fields with 4–5 years of iprodione application. The EC₅₀ values of these 111 isolates ranged from 0.117 to 0.634 μg/ml. The historic and current populations were not significantly (P > 0.05) different in sensitivity to iprodione. The EC₅₀ values of these 161 isolates to a carboxamide fungicide boscalid ranged from 0.002 to 0.391 μg/ml with a mean of 0.042 μg/ml. In the laboratory, three iprodione-resistant (IR) isolates HA17-IR, SZ31-IR, and SZ45-IR were induced from wild-type isolates HA17, SZ31, and SZ45, respectively. The EC₅₀ values of the IR isolates were 200-fold higher than those of the original wild-type parents. The IR isolates showed an increase in osmotic sensitivity. The IR isolate HA17-IR lost its ability to produce sclerotia, and showed a significantly lower virulence on rapeseed leaves than its parent isolate HA17. In contrast, the IR isolate SZ31-IR had a significantly higher virulence than its wild-type parent SZ31. PCR assays showed that the partial two-component histidine kinase (os-1) gene, which is the putative target gene of iprodione, was deleted in the low virulent IR isolate HA17-IR. DNA sequence analysis showed that each of the other two IR isolates SZ31-IR and SZ45-IR had two point mutations in their partial os-1 genes. These results indicate that the mutations in os-1 gene may be associated with dicarboximide sensitivity, sclerotial development, and virulence in S. sclerotiorum.     

桂枝主要活性物质对新余蜜橘青霉病抗性和防御体系的诱导

为探讨桂枝主要活性物质肉桂醛和肉桂酸对新余蜜橘青霉病的抗性及其机理,以500倍和1 000倍咪鲜胺稀释液为阳性对照,蜜橘损伤接种后,测定了防护效果最佳时肉桂醛、肉桂酸的抑菌浓度,分析了对果实活体防效和防御酶活性的影响。结果表明,25、8 mg/m L肉桂醛以及30、15mg/m L肉桂酸是最佳抑菌浓度;与对照组相比,各浓度药液处理均能显著降低病原菌的致病力,贮藏第6天,25、8 mg/m L肉桂醛处理组的病情指数分别为54.4%和66.4%,30、15 mg/m L肉桂酸处理组分别为49.6%和63.2%;各浓度药液处理均能降低新余蜜橘的MDA和H_2O_2含量,显著提高并维持SOD、PPO等防御酶的活性,以25 mg/m L肉桂醛和30 mg/m L肉桂酸处理效果最佳。研究表明,肉桂醛和肉桂酸能有效增强果实活体防护效果以及诱导果实的抗病性,且抗性的产生可能与果实内相关防御酶活性提高有关。     

Accumulation of phytoalexins in leaves of plane tree (Platanus spp.) expressing susceptibility or resistance toCeratocystis fimbriata f. sp.platani

Inoculation of leaves of resistantPlatanus occidentalis and susceptiblePlatanus acerifolia leaves withCeratocystis fimbriata f. sp.platani, the canker stain disease agent, induced foliar necrosis and biosynthesis of two phytoalexins, scopoletin and umbelliferone. Foliar symptoms keep localized and accumulation of coumarin phytoalexins was rapid for incompatible interactions. Necrosis spread widely and accumulation of these phenolic compounds was much later and lower for compatible interactions. The differential response could be used in a genetic improvement program for resistance against canker stain.     

Induced resistance in tomato plants to the toxin-dependent necrotrophic pathogen Alternaria alternata

A necrotrophic pathogen, the tomato pathotype of Alternaria alternata (Aa) causes Alternaria stem canker on tomato. Its pathogenicity depends on the production of host-specific AAL-toxin. Pre-inoculation with nonpathogenic Aa or pretreatment an elicitor prepared from Aa reduced disease symptoms by the pathogen. Salicylic acid (SA)- and jasmonic acid (JA)-dependent defense responses in tomato are not involved in the resistance to the pathogen induced by nonpathogenic Aa. The results suggest that an alternative and unknown signaling pathway independent of SA- and JA-signaling might modulate the induced resistance by activating the expression of the multiple defense genes.     

超敏蛋白对烟草黄瓜花叶病毒的诱导抗性及生长、品质的影响

为明确超敏蛋白对烟草黄瓜花叶病毒(Cucumber mosaic virus,CMV)的诱导抗性及其生长、品质的影响,采用半叶枯斑法和田间试验研究了10%超敏蛋白对枯斑三生烟和普通烟的诱导抗性及促生作用,并分析了对烤烟经济性状和内在质量的影响。结果表明,10%超敏蛋白在活体外对CMV无钝化作用,但预防、治疗效果显著,接种前48 h喷施预防效果达75.70%,接种后24 h喷施治疗效果达65.45%,均显著高于对照;10%超敏蛋白处理后烟草的株高、茎围、最大叶面积均高于其它处理,单位产量比20%吗啉胍·乙铜可湿性粉剂处理增产211.8 kg/hm2,增值3 519.2元/hm2;经10%超敏蛋白处理后的烤烟化学成分均处于优质烟的适宜质量分数范围内。表明超敏蛋白不仅有利于提高烟草抗性和促进烟株生长,且可以明显提高烟叶经济效益和烟叶内在质量。     

Inhibitors of N-linked glycosylation induce systemic acquired resistance in cucumber

Localized treatment of cucumber (Cucumis sativus L. cv. Wisonsin) cotyledons with inhibitors of N-glycosylation such as tunicamycin or amphomycin resulted in systemic acquired resistance in the first leaf to the fungal pathogen Colletotrichum lagenarium. Resistance was maximal as early as 2 days after application and best results were observed when the inhibitor was used at 100 μ . The same treatment also induced salicylic acid accumulation as well as the expression of chitinase and a PR1-like protein. The systemic effect is not caused by the transport of tunicamycin, since tunicamycin was not detected in the leaves. Within 2 h after application tunicamycin inhibited N-glycosylation, but not protein synthesis as indicated by labelling experiments. The amount of large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased after tunicamycin treatment and after pathogen inoculation and the expression of BiP, a protein localized in the endoplasmic reticulum was enhanced. The activation of defense reactions seems to be dependent and sensitive to N-linked glycosylation.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

内生细菌EBS05对烟草诱导抗性的信号转导途径研究

 樟树内生枯草芽胞杆菌EBS05是一株对多种植物病原菌具有较强拮抗活性,并能诱导烟草系统抗性的生防菌株。本文以缺失Surfactin A合成相关基因的突变菌株EBS05^T为材料,研究了内生细菌EBS05对烟草诱导系统抗性的激发子及其信号转导途径。结果表明,菌株EBS05产生的Surfactin A是诱导烟草对TMV系统抗性的有效激发子;Surfactin A诱导处理后,SA信号转导途径下游的关键调节基因NPR1首先被激活,并持续超量表达,进而触发PR1b和PR1a基因持续超量表达,表明Surfactin A诱导烟草对TMV的系统抗性是通过激活SA信号转导途径实现的。同时,Surfactin A诱导处理后24~72 h,JA/ET信号转导途径调节基因PDF1.2被激活,且超量表达,表明在Surfactin A诱导烟草对TMV系统抗性的信号转导过程中,可能存在SA信号途径和JA/ET信号途径的交叉协同作用。     

Biological control of Rhizoctonia solani on potatoes by antagonists. 4. Inoculation of seed tubers with Verticillium biguttatum and other antagonists in field experiments

Inoculation of seed potatoes with the mycoparasiteVerticillium biguttatum, isolate M73 (combined withGliocladium roseum in 1981, either alone or mixed with isolate M180 plus antibiotics-producing isolates ofAzotobacter chroococcum in 1982) repeatedly proved successful in reducingRhizoctonia solani on stolons and stems. In field experiments, this ultimately led to a reduced formation of sclerotia on new tubers, particularly in neutral sandy loam and clay loam soils. In 1981 inoculation with antagonists led, when compared with no inoculation, to average reductions of 22 and 42% for the harvest from clean, and 15 and 26% for the harvest from infected seed tubers grown on slightly acid sandy soils and on neutral loam soils, respectively. The harvest from clean, inoculated seed tubers had the lowest sclerotium index. In 1982 inoculation of seed tubers planted in slightly acid sandy soils gave reductions of the sclerotium index of up to 22%. In the neutral marine loam soils considerable reductions were often achieved, viz., in slightly infected loams 51–68% and in rather heavily infected ones 4–43%. Chemical disinfection of seed tubers proved effective only in loam soils that were slightly infested withR. solani. In both years inoculation of seed tubers with antagonists led to significantly lower sclerotium indices of the harvest (p=0.1% in 1981; p=5% in 1982). V. biguttatum was present more frequently and in greater densities on stems and stolons of plants from inoculated than from non-inoculated seed tubers. The latter were colonized by wildV. biguttatum strains from the soil, apparently less effective antagonists.Early in the season, the soil temperature was too low for growth ofV. biguttatum. Nevertheless, inoculation of tubers that were planted early resulted in a considerable cotrol ofR. solani.Samenvatting Het beënten van poters met de opRhizoctonia solani parasiterende schimmelVerticillium biguttatum isolaat M73 in combinatie metGliocladium roseum (1981) of metV. biguttatum M73 alleen of in combinatie met isolaat M180 plus antibiotische isolaten van de bacterieAzotobacter chroococcum (1982), bleek effectief in het terugdringen of het onderdrukken vanR. solani op stengels en stolonen en het verminderen van de aantasting.Beënting van het pootgoed leidde tot een vermindering van de sclerotium (lakschurft)-vorming op de nieuwe knollen, vooral in klei-en zavelgronden. In 1981 leidde beënting van poters tot reductie in de sclerotiumvorming van gemiddeld 22 en 42% voor de oogst uit schoon en 15 en 26% voor de oogst uit besmet pootgoed geteeld op respectievelijk zandgrond en klei- en zavelgrond.In 1982 leidde beënten van de poters uitgeplant in licht zure zandgrond tot een gemiddelde reductie van de sclerotiumindex van de oogst van 22%. In zwaar besmette zandgrond trad evenwel geen reductie op; de infectiedruk was hier te groot. In de neutrale zavel- en kleigronden, vaak ook in de zwaarder besmette percelen werden aanzienlijke reducties bereikt, in de licht besmette gemiddeld 51–68% en in de zwaarder besmette 4–43%. Ontsmetten van pootgoed bleek alleen effectief in percelen die licht metR. solani waren besmet.In beide jaren bleek beënten van pootgoed met antagonisten te resulteren in een significant lagere sclerotiumindex van de oogst (p=0,1% in 1981; p=5% in 1982). V. biguttatum was veel vaker en meer aanwezig op de ondergrondse stengeldelen en stolonen van planten uit beënt pootgoed dan op die van niet beënte poters. De laatsten werden gekoloniseerd door wilde stammen vanV. biguttatum uit de grond, die vaak minder effectieve antagonisten waren. Beënting van vroeg gepote knollen — als de temperatuur nog te laag is voor de groei vanV. biguttatum — leverde toch gunstige resultaten op.     

Ribonucleases in the Seedlings of Pearl Millet and their Involvement in Resistance Against Downy Mildew Disease

Tissue homogenates of pearl millet seedlings (cultivars HB 3, 843 B, ICMP 451 and IP 18292), with varying degree of resistance to downy mildew disease were tested for ribonuclease (RNase) enzyme activity and the profile of major RNase isozymes by substrate based gel assay. Polyacrylamide gel electrophoresis (PAGE) of the four pearl millet homogenates revealed 15–20 isozymes, varying in size from 6.5 to 121.0kDa. Most of the RNases were highly active between pH 6 and 8 with maximum activity at pH 7. Tissue specific expression of RNase was observed with more activity in the root, i.e., 38.84, 59.61, 39.90 and 49.23 units in HB 3, 843 B, ICMP 451 and IP 18292, respectively than in shoot 11.54, 9.95, 9.46 and 9.49 units in HB 3, 843 B, ICMP 451 and IP 18292, respectively. Effect of metal ions on the RNase profile indicates Zn⁺⁺ at 2, 20 and 200M concentrations to be inhibitory. Ca⁺⁺ and Mg⁺⁺ at 1mM concentration enhanced the enzyme activity while at 10mM inhibition of enzyme activity was observed. Inoculation with the downy mildew pathogen Sclerospora graminicola reduced RNase activity by 4–13% in compatible interactions while in incompatible combinations, the enzyme activity increased by 10–27%. The significance of RNase in pearl millet-downy mildew interaction and its involvement of in systemic acquired resistance of pearl millet against the downy mildew pathogen are discussed.     

Suppression of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> by antifungal substances produced by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in MCD (MCD_cm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCD_cm. Sclerotia of Ss soaked in the filtrates of MCD_cm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCD_cm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCD_cm. Treatment of leaves of oilseed rape with cultural filtrates of MCD_cm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.