Cellulysin induces downy mildew disease resistance in pearl millet driven through defense response

The responses to cellulysin as an immune inducer in pearl millet that confers downy mildew resistance mediated through lipoxygenase (LOX), a jasmonate-dependent enzyme involved in defence signalling, are discussed in this paper. The susceptible pearl millet cultivar 7042S was treated with cellulysin at 10, 15, 20, 30 and 50 μg/ml concentrations. All tested concentrations showed enhanced seed germination and seedling vigour when compared with the untreated control. Maximum seed germination of 92 % and seedling vigour was obtained following 20 μg/ml cellulysin treatment. Significant (P?<?0.05) downy mildew disease protection of 67 % and 71 % was observed when cellulysin was used at 20 μg/ml under greenhouse and field conditions, respectively. Further studies showed that the resistance induced by cellulysin treatment in pearl millet plant was systemic, required a minimum of 4 days to achieve maximum resistance development after pathogen inoculation seedling inoculation (five-day-old), and was sustained throughout the plant’s life. Plants raised from cellulysin-treated seeds and challenge inoculated at tillering (25-day-old) and inflorescence (45-day-old) showed persistence in resistance till the end of the crop period. A notable increase in LOX activity was observed in all the tested concentrations of cellulysin in plants inoculated with the pathogen at 24 h, compared to the control. However, a maximum 6-fold increase in LOX activity was noticed using a cellulysin concentration of 20 μg/ml 48 hours post inoculation. In contrast, glucanase (GLU) activity was high in control inoculated seedlings, but was low in cellulysin treated samples at all time intervals. The optimal cellulysin treatment (20 μg/ml) provided enhanced vegetative and reproductive parameters that resulted in higher yield compared to the untreated control.     

Spore cell wall components ofAspergillus niger elicit downy mildew disease resistance in pearl millet

Elicitors derived from the cell wall of fungi are shown to be active in eliciting resistance in plants against a wide range of pathogens. In the present study carbohydrate components from the autoclaved spore cell wall ofAspergillus niger were prepared as aqueous suspensions and tested for defense response in pearl millet (Pennisetum glaucum (L.) R.Br.) against the oomycetous downy mildew pathogenSclerospora graminicola (Sacc.) Schroet. The aqueous suspension derived from the spore cell wall ofA. niger was used as a seed soak treatment at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg ml⁻¹ for time intervals of 3, 6, 9 and 12 h. The concentration of 0.5 mg ml⁻¹ for a 6 h soaking period offered 94% seed germination and seedling vigor index increased to 1526. The seed germination and the seedling vigor were significantly higher than the untreated check. Spore cell wall suspension as seed treatment at a concentration of 0.5 mg ml⁻¹ required a 3-day time interval to provide 67% protection against downy mildew. Histological and biochemical studies were conducted to elucidate the mechanism of defense response in treated seedlings uponS. graminicola infection. Resistance host response was detected in the form of lignin and callose deposition in the epidermal cell wall of pearl millet seedlings, which is the site ofS. graminicola infection. A time course study showed rapid and localized deposition of lignin and callose in epidermal cell wall of carbohydrate components-treated pearl millet seedling coleoptiles. Increased levels of the defense-related enzyme peroxidase were detected in the treated seedlings. Peroxidase activity in elicitor-treated samples reached a peak at 8 h post-infection, which was 45% more than in their respective uninoculated control. Characterization of peroxidase isoforms by isoelectric focusing revealed 16 different isoforms, of which pI 6.8, 7.2 and 8.7 increased in elicitor-treated samples uponS. graminicola infection. http://www.phytoparasitica.org posting Nov. 14, 2005.     

Comparative efficacy of strobilurin fungicides against downy mildew disease of pearl millet

Three commercial formulations of strobilurins, viz., azoxystrobin, kresoxim-methyl, and trifloxystrobin were evaluated for their efficacy against pearl millet downy mildew disease caused by Sclerospora graminicola. In vitro studies revealed inhibition of S. graminicola sporulation, zoospore release, and zoospore motility at 0.1-2 μg ml⁻¹ of all the three fungicides. The fungicides were evaluated for phytotoxic effects on seed quality parameters and for their effectiveness against downy mildew disease by treating pearl millet by: (1) seed dressing, (2) seed dressing followed by foliar spray, and (3) also by foliar spray alone. The highest non-phytotoxic concentrations of 5, 10, and 10 μg ml⁻¹ for azoxystrobin, trifloxystrobin, and kresoxim-methyl, respectively, were selected for further studies. Under greenhouse conditions, these fungicides showed varying degrees of protection against downy mildew disease. Among the three fungicides, azoxystrobin proved to be the best by offering disease protection of 66%. Further, seed treatment along with foliar application of these fungicides to diseased plants showed enhanced protection against the disease to 93, 82, and 62% in treatments of azoxystrobin, kresoxim-methyl and trifloxystrobin respectively. Foliar spray alone provided significant increase in disease protection levels of 91, 79, and 59% in treatments of azoxystrobin, kresoxim-methyl, and trifloxystrobin, respectively. Disease curative activity of azoxystrobin was higher compared to trifloxystrobin and kresoxim-methyl. Tested fungicides showed weaker translaminar activity, as the disease inhibition was marginal when applied on adaxial leaf surface. Partial systemic activity of azoxystrobin was evident by root uptake, while trifloxystrobin and kresoxim-methyl showed lack of systemic action in pearl millet. A trend in protection against downy mildew disease similar to greenhouse results was evident in the field trials. Grain yield was significantly increased in all strobilurin fungicide treatments over control and maximum increase in yield of 1673 kg ha⁻¹ was observed in combination treatments of seed treatment and foliar spray with azoxystrobin.     

Chitosan enhances disease resistance in pearl millet against downy mildew caused by Sclerospora graminicola and defence-related enzyme activation

BACKGROUND: The present study investigated the effect of chitosan seed priming on the induction of disease resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroet. RESULTS: Pearl millet seeds were primed with chitosan at different concentrations: 0.5, 1.5, 2.5 and 3 g kg^?1 seed. Of the different concentrations, 2.5 g kg^?1 was found to be optimum, with enhanced seed germination of 99% and seedling vigour of 1782, whereas the untreated control recorded values of 87% and 1465 respectively. At optimum concentration, chitosan did not inhibit sporulation and release of zoospores from sporangia. Furthermore, pearl millet seedlings raised after seed treatment with chitosan showed an increased level of the defence‐related enzymes chitosanase and peroxidase as compared with the untreated pearl millet seedlings on downy mildew pathogen inoculation. The effect of chitosan in reducing downy mildew incidence was evaluated in both greenhouse and field conditions, in which respectively 79.08 and 75.8% disease protection was obtained. CONCLUSION: Chitosan was effective in protecting pearl millet plants against downy mildew under both greenhouse and field conditions by inducing resistance against the pathogen. Thus, chitosan formulation can be recommended for seed treatment in the management of downy mildew disease. Copyright © 2008 Society of Chemical Industry     

Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon

The synthetic 1-oxo-indanoyl-l-isoleucine methyl ester (Ind-Ile-Me) represents a highly active mimic of octadecanoic phytohormones, which are involved in plant defenses against pathogens and pests. Seed treatments and foliar spray with Ind-Ile-Me were tested for induced resistance against downy mildew disease caused by the phytopathogenic oomycete Sclerospora graminicola in pearl millet (Pennisetum glaucum) under greenhouse and field conditions. Under greenhouse conditions, a 50% protection level was achieved after seed treatment. Seed treatment in combination with foliar spray resulted in 60% protection. The induction of resistance was correlated with the enhanced activities of defense-related proteins such as phenylalanine-ammonia-lyase, peroxidase, and enhanced level of hydroxyproline-rich glycoproteins. Under field conditions, a maximum protection of 62% was recorded upon seed treatment along with foliar spray. Hence, it infers that Ind-Ile-Me can be used as a valuable protection compound at least in downy mildew disease management.     

Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease

Nitric oxide (NO) donors Nitroso-R-Salt, 2-Nitroso-1-Naphthol and Sodium Nitro Prusside (SNP) were evaluated for their effectiveness in protecting pearl millet [(Pennisetum glaucum L.) R. Br.] plants against downy mildew disease caused by Sclerospora graminicola [(Sacc). Schroet]. Optimization experiments with NO donors showed no adverse effect either on the host or pathogen. Aqueous SNP seed treatment with or without polyethylene glycol (PEG) priming was the most effective in inducing the host resistance against downy mildew both under greenhouse and field conditions. Potassium Ferrocyanide, a structural analog of NO donor lacking NO moiety failed to protect the pearl millet plants from downy mildew indicating a role for NO in induced host resistance. Spatio-temporal studies corroborated that the protection offered by NO donor treatment was systemic in nature and a minimum of 3-day time gap between the inducer treatment and subsequent pathogen inoculation was necessary for maximum resistance development. Disease protection ability of NO donors was also validated as durable in nature. Conversely, prior-treatment with NO scavenger 2-4-carboxyphenyl-4,4,5,5 tetrazoline-1-oxyl-3-oxide potassium salt (C-PTIO) rendered the pearl millet plants relatively susceptible for pathogen infection. Expression of primary defense responses like hypersensitive response, lignin deposition and defense related enzyme phenylalanine ammonialyase −EC 4.3.1.5 (PAL) were enhanced by NO donor treatments.     

Nitric oxide is involved in chitosan‐induced systemic resistance in pearl millet against downy mildew disease

BACKGROUND: The nature and durability of resistance offered by chitosan and the involvement of nitric oxide (NO) in chitosan‐induced defence reactions in pearl millet against downy mildew disease were investigated. RESULTS: It had previously been reported that chitosan seed priming protected pearl millet plants against downy mildew disease. Further elucidation of the mechanism of resistance showed that chitosan seed priming protects the plants systemically. A minimum 4 day time gap is required between the chitosan treatment and pathogen inoculation for maximum resistance development, and it was found to be durable. Chitosan seed priming elevated NO accumulation in pearl millet seedlings, beginning from 2 h post‐inoculation, and it was found to be involved in the activation of early defence reactions such as hypersensitive reaction, callose deposition and PR‐1 protein expression. Pretreatment with NO scavenger C‐PTIO and nitric oxide synthase (NOS) inhibitor L‐NAME before pathogen inoculation reduced the disease‐protecting ability of chitosan, and defence reactions were also downregulated, which indicated a possible role for NO in chitosan‐induced resistance. CONCLUSION: Protection offered by chitosan against pearl millet downy mildew disease is systemic in nature and durable. Chitosan‐induced resistance is activated via NO signalling, as defence reactions induced by chitosan were downregulated under NO deficient conditions. Copyright © 2009 Society of Chemical Industry     

Activity of cyazofamid against Sclerospora graminicola, a downy mildew disease of pearl millet

The efficacy of cyazofamid was tested against pearl millet downy mildew disease caused by Sclerospora graminicola Schroet. Significant inhibition of sporangial sporulation, zoospore release and motility was observed at 0.3 mg mL(-1), and this concentration also provided good fungicidal activity under in vitro conditions. Under glasshouse conditions, none of the concentrations tested, either 0.01-2 mg mL(-1) as seed treatment or 1-10 mg mL(-1) by foliar application, was found to be phytotoxic. The effect of cyazofamid was tested by seed treatment alone, seed treatment followed by foliar application and foliar application alone. Seed treatment with cyazofamid offered only 19.7% disease control, but seed treatment followed by a single foliar application to diseased plants provided good control over disease, seed treatment with two foliar applications was significantly superior and foliar application alone showed a high level of activity, with 10 mg mL(-1) giving 97.9% disease control. Lack of systemic activity of cyazofamid was evident, root treatment giving disease levels on a par with the untreated control. The fungicide exhibited strong curative activity, but only moderate translaminar activity, with only marginal (34.8%) disease control after treatment of the adaxial leaf surface at 10 mg mL(-1). Loss of cyazofamid activity over time was very low, indicating stable residual and rainfastness activity. These results indicate that cyazofamid has a high potential to be an effective fungicide for the control of downy mildew disease of pearl millet.     

Mapping of easy to screen SSR markers for selection of RFLP markers-bracketed downy mildew resistance QTLs in pearl millet

There is a growing necessity to replace chemical agents with ecofriendly materials, arising from the impact on the environment and/or human health, which calls for the design of new broad-spectrum fungicides. In this work, chitosan oligomers (COs), propolis (Ps) and silver nanoparticles (AgNPs) mixtures in solution were assessed to control the growth of different phytopathogenic fungi and oomycetes in vitro. Binary solutions of COs-Ps and COs-AgNPs evinced the highest antifungal effect against Fusarium circinatum and Diplodia pinea fungi, respectively, with a ca. 80% reduction in their mycelial growth. The COs solution by itself also proved to be greatly effective against Gremmeniella abietina, Cryphonectria parasitica and Heterobasidion annosum fungi, causing a reduction of 78%, 86% and 93% in their growth rate, respectively. Likewise, COs also attained a 100% growth inhibition on the oomycete Phytophthora cambivora. On the other hand, Ps inhibited totally the growth of Phytophthora ×alni and Phytophthora plurivora. The application of AgNPs reduced the mycelial growth of F. circinatum and D. pinea. However, the AgNPs in some binary and ternary mixtures had a counter-productive effect on the anti-fungal/oomycete activity. In spite of the fact that the anti-fungal/oomycete activity of the different treatments showed a dependence on the particular type of microorganism, these solutions based on natural compounds can be deemed as a promising tool for control of tree diseases.     

Serodiagnosis of pearl millet resistance to downy mildew by quantitating cell wall P/HRGP using polyclonal antiserum Pab-P/HRGP

Proline/hydroxyproline-rich glycoprotein (P/HRGP) level in pearl millet genotypes resistant to downy mildew increase after inoculation with the oomycete pathogen Sclerospora graminicola. Using purified P/HRGPs from pearl millet cell walls, polyclonal antibodies (Pab-P/HRGP) were raised in rabbit. Based on this antiserum, an enzyme immunoassay was developed that displays a linearity detection range from 0.01 to 10 μg P/HRGP. Western blot analysis, confirming the induction of three marker P/HRGPs in the infected resistant genotype, and immunocytochemical studies on P/HRGP localization either in epidermal peelings or in suspension-cultured cells demonstrated the specificity of the antiserum. Besides its characterization, Pab-P/HRGP was employed to screen various genotypes of pearl millet for fast, sensitive and specific detection of induced P/HRGPs upon infections. The results presented are discussed with presumed importance to downy mildew disease and the use of this new antiserum in pearl millet screening for disease resistance.     

不同葡萄品种对霜霉病的抗性

葡萄霜霉病是葡萄生产上重要的病害之一,通过对辽宁省不同葡萄品种进行室内离体叶片接种和田间自然发病情况调查,以期为葡萄抗性品种的选育和葡萄霜霉病的防治提供科学依据。结果表明,在供试品种中,没有对霜霉病完全免疫的品种,室内离体叶片接种和田间调查结果基本一致,不同品种间霜霉病的抗性存在差异。供试的65个品种中,室内离体叶片接种评价高抗品种3个,抗病品种23个,感病品种24个,高感品种15个;田间自然发病调查评价高抗品种1个,抗病品种19个,感病品种35个,高感品种10个。欧美杂交品种(系)相对欧亚杂交品种(系)较抗病。     

葡萄霜霉病抗病性鉴定方法及品种抗病性测定

本文通过对葡萄霜霉病抗病性鉴定方法进行优化,建立了一种更为快捷、方便、可靠的鉴定方法,并对32个葡萄品种对霜霉病抗病性进行了鉴定,为葡萄抗病品种的选育和应用提供依据。结果显示:以田间混合的霜霉病菌为接种体采用叶盘法鉴定葡萄品种的抗病性更加快捷、方便。供试的32个葡萄品种对葡萄霜霉病的抗性存在显著差异。其中免疫品种有‘康拜尔早生';高抗的有‘阳光玫瑰'、‘美乐'、‘Ms27-31'等5个品种;中抗的有‘贝达'、‘小芒森'、‘2E-16-2'等6个品种;低抗的有‘瑞都红玉'、‘早黑宝'、‘摩尔多瓦'等10个品种;感病的有‘里扎马特'、‘玫瑰香'、‘香妃'等10个品种。     

Infection of the pearl millet (Pennisetum americanum) inflorescence by the downy mildew fungus (Sclerospora graminicola)

Different developmental stages of the inflorescence of pearl millet (Pennisetum americanum) were inoculated with zoospores of the downy mildew fungus (Sclerospora graminicola). Individual florets within a panicle were infected, with resultant malformation of any floral organs that were not fully differentiated at the time of infection. 'Green-ear' symptoms resulting from hyperplasia and hypertrophy of the host tissues were accompanied by both sexual and asexual sporulation of the fungus on the malformed plant parts. No grain set occurred in affected florets, indicating that secondary inoculum was able to cause yield reductions even at late stages in the host development. Infection of differentiated stigmas led to rapid dissolution and necrosis of tissue and prevented colonization by the pathogen. This failure suggests that seeds are unlikely to be infected internally.     

Seed priming with plant gum biopolymers enhances efficacy of metalaxyl 35 SD against pearl millet downy mildew

‘Priming’ the plant and seed induces a physiological state in which plants are able to activate defense responses. Plant-based exudates are excellent gum biopolymers which contain plant growth-regulating hormones with priming potential without any side effects. In this study, gum exudates of Acacia arabica, Moringa oleifera, Carica papaya and Azadirachta indica were evaluated for synergistic effects of seed priming with exuded gum biopolymer combined with metalaxyl (Apron 35 SD) on pearl millet seed quality, growth parameters, and resistance to Sclerospora graminicola. Seeds of 7042S were primed with gum biopolymers and metalaxyl 35 SD and evaluated under laboratory and greenhouse conditions. Seed germination and vigor were synergistically enhanced using gum biopolymers solution (1:2 w/v) with 3 g kg⁻¹ metalaxyl 35 SD. A. arabica and A. indica gum biopolymers alone or with 3 g kg⁻¹ of metalaxyl 35 SD resulted in seed germination of >91%. Seed priming with 6 g kg⁻¹ of metalaxyl 35 SD gave 89% seed germination and was not significantly different from control. A similar trend in vigor was observed among treatments. Seed priming with gum biopolymers alone provided varied disease protection levels when compared with the control. A. arabica or A. indica gum with 3 g kg⁻¹ of metalaxyl 35 SD was the superior treatment, offering significant 86% disease reduction while exhibiting a growth-promoting effect. Synergistic use of gum biopolymers and metalaxyl 35 SD by seed priming is highly effective in growth promotion and management of pearl millet downy mildew disease.     

BTH诱导黄瓜对霜霉病的抗性

 由黄瓜霜霉病菌(Pseudoperonospora cubensis)引起的病害是黄瓜生产上的主要病害,利用化学药剂是当前我国菜农的主要防病措施。而频繁使用化学杀菌剂,不仅易使病菌产生抗药性,且影响环境安全和人体健康。     

Influence of dosage,storage time and temperature on efficacy of metalaxyl-treated seed for the control of pearl millet downy mildew

Metalaxyl (Apron 35WS) as a seed treatment has been used extensively to control downy mildew (caused by Sclerospora graminicola) in pearl millet in India. However, the extent of disease control has varied across cultivars, years and locations. We investigated the effects of fungicide dosage, storage time and storage temperature of metalaxyl-treated seed on disease incidence in four pearl millet lines having varying levels of resistance. A linear relationship was found between fungicide dosage (0.5, 1.5 and 2 g a.i. kg⁻¹ seed) and reduction in disease incidence up to 40 days after emergence in all the lines. The normal fungicide dose (2 g a.i. kg⁻¹ seed) protected the crop for up to 20, 40 and 50 days after emergence in highly susceptible (7042S), moderately susceptible (4042R), and moderately resistant (ICMP 451) lines, respectively. However, the quarter and half the normal dosage of fungicide provided protection only up to 20 days after emergence in 7042R and 40 days after emergence in ICMP 451. Storage duration of metalaxyl-treated seed (2 g a.i. kg⁻¹) up to 9 months at 25 ± 2°C did not affect fungicide efficacy. Storage temperatures (5, 25 and 40°C) and duration (30, 60 and 90 days) of metalaxyl-treated seed (2 g a.i. kg⁻¹) showed differential effects in two pearl millet lines 7042S and 843B with downy mildew incidence being significantly lower in 7042S than in 843B. Metalaxyl-treated seed of 7042S and 843B stored at 40°C for different durations showed phytotoxic effects and it was more pronounced in 843B stored for 60 and 90 days where seed germination was inhibited in pot soil.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

葡萄品种对霜霉病抗性鉴定的生化指标研究

 通过盆栽接种和田间自然发病鉴定,从22个供试葡萄品种中鉴定筛选到1个高抗霜霉病的品种超藤和8个中抗品种饭刚黑、信浓乐、弗雷无核、黑蜜、优选皮奥萘、高妻、京秀、峰后。相关性分析表明,22个供试葡萄品种接种前和接种后2d,PPO、PAL活性和CAT比活性与霜霉病病情指数呈极显著负相关,SOD活性与霜霉病病情指数相关不显著。PPO、PAL活性和CAT比活性与葡萄品种对霜霉病的抗性呈极显著正相关,可利用这3种酶的活性作为葡萄品种霜霉病抗性鉴定的辅助评价指标。     

Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew

Levels of resistance to Plasmopara viticola, from susceptible to highly resistant, of different grapevine cultivars were observed in vineyards and confirmed by the symptoms developed after inoculations. On the abaxial surface of infected leaves, P. viticola developed abundant sporangiophores on susceptible cultivars (Chasselas, Gamay, Gamaret and Pinot Noir), whereas on less susceptible cultivars (Seyval Blanc and Johanniter) the parasite produced few sporangiophores and some necrotic spots at the site of infection. On resistant cultivars (Bronner, Solaris, IRAC 2091), P. viticola induced a hypersensitive response and only necrotic spots were visible and the disease ceased to develop. Stilbenes were analyzed 4, 7, 24, 48 h post-infection (hpi) on small leaf samples cut from the site of infection. Large differences were observed between the cultivars at 24 and 48 hpi. Susceptible grapevines produced resveratrol and its glycoside, piceide. In contrast, resistant plants produced high concentration of ε- and δ-viniferin. Resveratrol and piceide have little or no toxicity activity against P. viticola, whereas δ-viniferin is highly toxic and can be considered an important marker for resistance of grapevine to downy mildew. The importance of oxidative dimerization of resveratrol in comparison to the extent of its glycosylation in defense reaction of grapevines against P. viticola is discussed.