龙舌兰麻种质资源抗斑马纹病鉴定研究

通过分离培养斑马纹病病原菌,人工接种鉴定不同龙舌兰麻种质的抗斑马纹病的特性.结果表明,番麻、东368、墨引6、墨引12、墨引7、墨引5、假7、马盖麻、东109、金边孤叶龙舌和兰墨引4号11份种质为高抗种质,病斑扩展速度和病情严重度可作为龙舌兰麻抗病性快速鉴定技术手段.     

RAPD PCR for Diagnosis of Phytophthora parasitica var. nicotianae Isolates which Cause Black Shank on Tobacco

Phytophthora parasitica var . nicotianae is the fungal pathotype of tobacco black shank (TBS, Disease severity ≥ 2.0). Random amplified polymorphic DNA (RAPD) analysis was used to differentiate isolates which cause TBS from those which do not. Greenhouse assays combined with zoospore inoculation were performed to assess the virulence of the fungal isolates, and the results were compared with the RAPD pattern analysis. The RAPD results exhibited total correlation with the virulence assay results. Amplification patterns generated by RAPD reactions were used to generate a phenogram depicting the genetically distinct nature of the cluster defined by the TBS isolates. This cluster was exclusive and distinct from P. parasitica var . nicotianae isolates which do not cause TBS. Thus, RAPD proved to be a sensitive and highly reliable method for quickly identifying fungal pathotypes which cause TBS.     

Genetic and Pathogenic Variation Among Tobacco Black Shank Strains of Phytophthora parasitica var. nicotianae from the Main Tobacco Growing in China

Pathogenic and genetic variability among seven populations of Phytophthora parasitica var. nicotianae from individual tobacco fields (Yunnan, Shandong, Henan, Heilongjiang, Shanxi, Fujian and Sichuan provinces) were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses; 63 strains were isolated from different fields of seven tobacco growing regions, using tobacco cv. Hongda as a baiting host. Pathogenic variability was evaluated in greenhouse studies using five tobacco cultivars that have different levels of resistance to tobacco black shank; 75 and 73% of the strains were pathogenic on M3 and M4, 29 and 33% on M1 and M2, and 94% were pathogenic on M5, respectively. Disease severity incited by different strains varied significantly on individual tobacco cultivars. The percentage of strains pathogenic on different cultivars varied among locations. Genotypic variation among 63 strains was evaluated by RAPD analysis. Ten primers detected 89 polymorphic bands. Cluster and principal coordinates analysed cluster groups. the minor group contained 26 strains, and major group contained 37 strains. Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.34 within populations to 0.36 among all strains from all populations. Phytophthora parasitica var. nicotianae populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the seven locations.     

In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis

The aim of the present study was to determine the bioactive compounds in four essential oils (EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on eradication of preformed 48?h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.     

Early season root production and zoospore infection of cultivars of flue-cured tobacco that differ in level of partial resistance to Phytophthora parasitica var. nicotianae

Root production of four cultivars of flue-cured tobacco was quantified in the field, greenhouse and phytotron. The cultivars ranged in level of partial resistance to the black shank pathogen, Phytophthora parasitica var. nicotianae, from susceptible to highly resistant. In the field, root-observation plates were installed approximately 10 cm from plants, and in greenhouse and phytotron studies, plants were grown in 4-liter containers with one sloping transparent side for root observation. Root growth was determined weekly for four weeks after transplanting in the field and daily up to 14 days after transplanting in the greenhouse and phytotron. Root tracings were made on acetate sheets placed against the sloping transparent side of the containers or against the transparent observation plates in the field following removal of soil from the outside of the observation plate. Root growth was quantified by retracing the root pattern on the acetate sheets over a digitizing tablet attached to a personal computer. Numbers of roots, root length, and mean and maximum rate of root growth were determined. Cultivars Hicks (susceptible) and K-326 (low level of resistance) had significantly larger root systems than moderately resistant G-28 or highly resistant NC 82. Differences in total root length were due to increased branching that resulted in development of significantly greater numbers of roots in Hicks and K-326. For example, between day 21 and 28, Hicks produced more than three times the number of new roots as NC 82 in the field. The mean rate of root extension observed (2.17 mm hr^–1) was similar in all four cultivars. Infection efficiency on the different cultivars was determined in the field by inoculating roots with zoospores of P. p. nicotianae. Lesions were visible as water soaked areas within 24 hr of inoculation. At 48 hr after inoculation, percentages of inoculations that resulted in lesion formation were 57, 46, 23, and 16% for Hicks, K-326, G-28 and NC 82, respectively. The possible role of rooting intensity as a mechanism of avoidance to P. p. nicotianae in tobacco cultivars is discussed.     

广西岩溶区烟草黑胫病拮抗细菌的筛选鉴定及其抗病机理

从广西岩溶区靖西县优质烟叶生产区分离烟草黑胫病病原及土壤细菌,通过拮抗试验、离体及田间抗病能力测试等方法筛选拮抗细菌;利用形态观察、生理生化测试和16SrDNA序列分析三种方法相结合,对抗病效果良好的菌株进行分类鉴定;并对拮抗菌抗黑胫病机理进行初步研究。结果表明:从8份土壤样品中,共分离出土壤细菌340株,获得抗黑胫病效果良好的拮抗细菌3株,编号为8-23、6-70和13,它们分别属芽孢杆菌属、溶菌杆属和假单胞杆菌属细菌;三个拮抗细菌的抗黑胫病机理是通过胞外分泌一些可溶解病原菌细胞壁的酶或其它化学物质,破坏菌丝的细胞壁和细胞膜等生理结构,使细胞质渗漏、凝集,从而导致病原死亡;其中,菌株8-23和13的抗病活性物质主要为蛋白质类化合物;而菌株6-70除蛋白质外,还有其它一些非蛋白因子起作用。     

Antifungal activity of the dill (Anethum graveolens L.) seed essential oil against strawberry anthracnose under in vitro and in vivo conditions

Strawberry anthracnose, caused by Colletotrichum nymphaeae, is mainly controlled by the application of synthetic chemical fungicides. The present study assessed the antimicrobial activity of essential oils (EOs) from dill (Anethum graveolens L.) seed against C. nymphaeae. The antifungal effects of dill seed EO on C. nymphaeae was initially evaluated in vitro and further extended as in vivo condition. The results indicate that in the contact and volatile assays, dill seed EO significantly inhibited mycelial growth of C. nymphaeae at all concentrations tested. Conidia germination was also significantly inhibited at concentrations of 250 – 1000 ppm. Disease incidence and severity of anthracnose on strawberry fruits were significantly reduced compared with infected control, from concentrations of 500 and 50 ppm, respectively. The results confirm the efficacy of dill seed EO against C. nymphaeae, which may represent an alternative to synthetic chemical fungicides to control strawberry anthracnose pre- and post-harvest.     

Distribution of Phytophthora citrophthora R.E. Sm. and E.H. Sm. and P. parasitica Dastur within citrus orchards in Kerman province,Iran

In this research, distribution of Phytophthora species were determined in Kerman Province (Bam, Shahdad and Arzuiyeh) during 2004–2007. The Phytophthora species were isolated from infected root, crown and soil. Root and crown pieces were washed and cultured on a CMA-PARPH medium. The isolation from infected soil was performed by bating method using citrus leaves. It was identified by morphological and several physiological characteristics. Distribution of species was determined by recording the number of isolates recovered from samples from each city. In this study, from 220 soil samples collected from 52 citrus orchards, 80 isolates of Phytophthora were recovered. Among of all isolates of Phytophthora, P. parasitica and P. citrophthora were the most important species of causal agent of gummosis on Citrus sp. Distribution of P. citrophthora was highest in Arzuiyeh and lowest in Bam and Shahdad cities, while distribution of P. parasitica was highest in Bam and Shahdad cities. The majority of soil samples contained only P. parasitica, but a few of percentage samples containing P. citrophthora alone and both of fungi in cites samples.     

Effect of fungicidal essential oils against Botrytis cinerea and Rhizopus stolonifer rot fungus in vitro conditions

The development of natural crop protection products as alternatives to the use of synthetic fungicides is currently popular. The aim of this study is to evaluate the antifungal effects of several essential oils against the fungal pathogens, Botrytis cinerea and Rhizopus stolonifer, under in vitro condition. Four essential oils (fennel, black caraway, peppermint and thyme) were each tested at five concentrations (0, 200, 400, 600 or 800 μl l^?1). In vitro results showed that the essential oil of black caraway and fennel had the highest fungicidal effect against B. cinerea and R. stolonifer, respectively. The growth of B. cinerea was completely inhibited by the essential oil of black caraway at 400 μl l^?1. Fennel oil perfectly inhibited growth of R. stolonifer fungus colonies at concentration higher than 600 μl L^?1 in potato dextrose agar medium. Percentage of spores germination was the lowest in medium of Fennel and black caraway essential oils, and was the highest in Thyme ones. These results show that plant essential oils can have a strong effect on reducing post-harvest decay. These plant essential oils could provide an alternative to synthetic chemicals to control post-harvest phytopathogenic fungi on fruit.     

Antioxidant,antimicrobial activities and comparative analysis of the composition of essential oils of leaf,stem, flower and aerial part of Nepeta hindostana

Nepeta hindostana (B.Heyne ex Roth) Haines is belonging to lamiaceae family and used as a component of herbal ayurvedic formulation Abana which is useful for the treatment of Hyperlipidemia, Dyslipidemia and Hypercholesterolemia. In the present study, the essential oil from aerial parts (flower, leaves, stem and whole aerial) was collected and the major constituents of essential oils were characterized by GC-FID and GC/MS and further evaluated for their antioxidant and antimicrobial efficacy. The major components of the essential oil were sesquiterpene hydrocarbons (77.2, 80.5, 62.5, 77.8%), oxygenated sesquiterpenes (10.5, 9.2, 20.6, 9.2%) and oxygenated monoterpenes (5.3, 4.2, 2.5, 3.6%) in leaves, stem, flowers and aerial part, respectively. The major compounds in essential oils were identified as β-sesquiphellandrene, cadina-1,4-diene, α-cadinene, (E)-caryophyllene, α-humulene and β-bisabolene. At 100 μg/mL concentration, leaves essential oil showed strong 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical scavenging activity with the IC₅₀ 2.8 μg/mL and 34.0% by β-carotene bleaching assay. Furthermore, the antibacterial activity was tested against four Gram-negative and five Gram-positive pathogenic strains. The essential oil from flower showed potent activity (37.5 μg/ml) against S. aureus, S. mutans but was less active against Gram-negative bacterial strains. In anti-MRSA activity, leaves and flowers exhibited strong activity against S. aureus (SA-2071) and S. aureus (SA-4627) with lowest IC₅₀ value of 50–100 μg/mL. Overall, N. hinodostana (L.) essential oil represented a potential reservoir of molecules having potent antioxidant and antimicrobial potential.     

Achillea ligustica: composition and antimicrobial activity of essential oils from the leaves, flowers and some pure constituents

The composition of the essential oils obtained from the leaves and the flowers of Achillea ligustica (Asteraceae) growing in Sicily has been studied. The main constituents of the leaves were 4-terpineol (19.3%), carvone (8.9%), γ-terpinene (7.2%) and β-phellandrene (6.8%). 4-terpineol (12.0%), carvone (10.0%), and β-phellandrene (5.4%), along with linalool (20.4%) and cedrol (4.3%) were detected in the flower’s oil. Furthermore, the antimicrobial activity of the essential oils and of some of the main constituents were assayed on bacteria and fungi. In memory of Prof. Ivano Morelli (1940–2005)     

Screening of potential antimicrobial compounds against Xanthomonas campestris from 100 essential oils of aromatic plants used in India: an ecofriendly approach

Plants are constantly exposed to and threatened by a variety of pathogenic microorganisms present in their environments. Xanthomonas campestris is a major plant pathogen in the world. It is known to cause significant losses in many crop plants due to leaf spot and leaf blight. In the present study, bacteria isolated from infected Citrus limon fruit and identified by 16S rDNA sequencing was X. campestris. In vitro antimicrobial properties of 100 essential oils were evaluated against X. campestris. The chemical composition of the most active essential oils was investigated by gas chromatography–mass spectral (GC–MS) analysis. The potential of these essential oils as ecofriendly and economical biocontrol in agriculture is discussed.     

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil

Aims:  Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213.
Methods and Results:  The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa , membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus .
Conclusions:  Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed.
Significance and Impact of the Study:  Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.     

Interactions between the soilborne root pathogenPhytophthora nicotianae var.parasitica and the arbuscular mycorrhizal fungusGlomus mosseae in tomato plants

In order to study the influence of Arbuscular Mycorrhiza (AM) on the development of root rot infection, tomato plants were raised with or withoutGlomus mosseae and/orPhytophthora nicotianae var.parasitica in a sand culture system. All plants were fed with a nutrient solution containing one of two phosphorus (P) levels, 32µM (I P) or 96µM (II P), to test the consequence of enhanced P nutrition by the AM fungus on disease dynamics. Mycorrhizal plants had a similar development to that of control plants. Treatment withPhytophthora nicotianae var.parasitica resulted in a visible reduction in plant weight and in a widespread root necrosis in plants without mycorrhiza. The presence of the AM fungus decreased both weight reduction and root necrosis. The percentage reduction of adventitious root necrosis and of necrotic root apices ranged between 63 and 89% The enhancement of P nutrition increased plant development, but did not appreciably decrease disease spread. In our system, mycorrhiza increased plant resistance toP. nicotianae var.parasitica infection. Although a contribution of P nutrition by mycorrhiza cannot be excluded, other mechanisms appear to play a crucial role.     

Antifungal activity of essential oils and their combinations in in vitro and in vivo conditions

Natural additives are in demand for the control of microbial growth in foods. Several natural compounds including essential oils (EOs) are being explored for food uses. In the present investigation, the antifungal activity of cinnamaldehyde, eugenol, peppermint and clove EOs and their combinations was evaluated against 12 species of Aspergillus, Fusarium, Penicillium and Rhizopus in in vitro and tomato fruit system (in-vivo). The EOs were able to inhibit complete growth of tested fungi at or below 0.6% level and 80?μL of EOs (except peppermint oil) in in vitro condition and tomato system, respectively. The fractional inhibitory studies showed either additive or indifferent effect by combining eugenol and peppermint, and indifferent or antagonist effect by combining the cinnamaldehyde and clove in both in vitro and in vivo studies. The findings may be useful for application of these EOs in foods, but their effects on organoleptic quality of foods need to be investigated.     

九种植物精油对腌肉及咸鱼害虫大头金蝇的杀卵作用

为开发腌肉和咸鱼害蝇的安全性杀卵剂,采用改进的浸卵法,进行了9种植物精油对大头金蝇的杀卵活性测定.结果表明:肉桂、丁香、八角茴香3种植物精油较为有效,它们的杀卵毒力(LC50)依次为0.428、1.605和2.489mg.ml-1,以肉桂油的毒力最强.通过GC/MC成分分析,从肉桂油检出22个成分,主成分桂皮醛占92.33%.合成桂皮醛(含量97.33%)的毒力为0.281mg.ml-1,略高于肉桂油,表明桂皮醛是肉桂油的主要杀卵活性成分,可替代肉桂油作为害蝇杀卵剂利用.     

植物精油对大菱鲆弧菌的体外和体内抗菌活性

【目的】研究天然植物精油对大菱鲆弧菌的体外和体内抗菌活性。【方法】采用纸片扩散法和微量肉汤稀释法对14种植物精油或其组分的体外抑菌活性进行检测;通过细菌形态透射电镜观察、胞内乳酸脱氢酶及核酸释放研究山苍子精油对大菱鲆弧菌的膜损伤作用;采用大菱鲆人工攻毒感染实验研究山苍子精油的体内抗菌作用。【结果】14种植物精油或其组分对大菱鲆弧菌具有不同程度的抑制效果,其中肉桂醛的抗菌活性最强,最低抑菌浓度为0.25μL/m L;百里香酚、丁香酚、柠檬醛和山苍子的抗菌活性次之,最低抑菌浓度为0.5μL/m L;山苍子精油可破坏大菱鲆弧菌的细胞膜,并导致胞内蛋白酶和核酸外泄;经200μL/L山苍子精油浸浴后,大菱鲆攻毒后死亡率由对照组50%降至0。【结论】富含芳香醛、芳香酚和柠檬醛的植物精油对大菱鲆弧菌具有良好抗菌活性,有望替代抗生素用于大菱鲆弧菌病的防治。