Cloning,structural features,and expression analysis of resistance gene analogs in Tobacco

Using degenerate primers based on the conserved nucleotide binding site (NBS) and protein kinase domain (PKD), 100 resistance gene analogs (RGAs) were isolated from tobacco variety Nicotiana repanda. BLASTx search against the GenBank database revealed that 27 belong to the NBS class and 73 belong to the protein kinase (PK) class. Cluster analysis and multiple sequence alignment of the deduced protein sequences indicate that RGAs of the NBS class can be divided into two groups: toll/interleukin receptor (TIR) and non-TIR types. Both types possess 6 conserved motifs (P-loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, GLPL). Based on their sequence similarity, the tobacco RGAs of the PK class were assigned to 8 subclasses. We examined their expression after infection with either Tobacco mosaic virus (TMV) or the tobacco black shank pathogen (Phytophthora parasitica var. nicotianae). The expression levels of 4 RGAs of the PK class were significantly elevated by TMV and 1 RGA of the PK class and 3 RGAs of the NBS class were up-regulated by P. parasitica var. nicotianae. The expression of two RGAs of the PK class was induced by P. parasitica var. nicotianae. Infection by either TMV or P. parasitica var. nicotianae enhanced the expression of NtRGA2, a RGA of the PK class. The present study shows that RGAs are abundant in the tobacco genome and the identification of tobacco RGAs induced by pathogens should provide valuable information for cloning related resistance genes in tobacco.     

Microassay for biological and chemical control of infection of tobacco byPhytophthora parasitica var.nicotianae

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

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.     

The effect of size and acetylation degree of chitosan derivatives on tobacco plant protection against Phytophthora parasitica nicotianae

Enzymatic degradation of chitosan polymer with Pectinex Ultra SPL was used to obtain derivatives with biological potential as protective agents against Phytophthora parasitica nicotianae (Ppn) in tobacco plants. The 24 h hydrolysate showed the highest Ppn antipathogenic activity and the chitosan native polymer the lowest. The in vitro growth inhibition of several Phytophthora parasitica strains by two chitosans of different DA was compared. While less acetylated chitosan (DA 1%) fully inhibited three P. parasitica strains at the doses 500 and 1000 mg/l the second polymer (DA 36.5%) never completely inhibited such strains. When comparing two polymers of similar molecular weight and different DA, again the highest antipathogenic activity was for the less acetylated polymer. However, degraded chitosan always showed the highest pathogen growth inhibition. Additionally, a bioassay in tobacco seedlings to test plant protection against Ppn by foliar application demonstrated that partially acetylated chitosan and its hydrolysate induced systemic resistance and higher levels of glucanase activity than less acetylated chitosan. Similarly, when treatments were applied as seeds coating before planting, about 46% of plant protection was obtained using chitosan hydrolysate. It was concluded that, while less acetylated and degraded chitosan are better for direct inhibition of pathogen growth, partially acetylated and degraded chitosan are suitable to protect tobacco against P. parasitica by systemic induction of plant resistance.     

A Novel screening method of cellulase-producing bacteria based on <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent     

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.     

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.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Effects of extracts of Alnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions

The amount of root mortality caused by root pathogens such as Phytophthora nicotianae (syn. Phytophthora parasitica) has typically been inferred from the net change in root length density in sequential soil cores. Because such measurements give information only on net changes in root populations, the actual rate of root turnover is often underestimated. We used minirhizotrons to track the fate of a large number of individual fine roots of mature field-grown citrus trees over a 6-month period. This method enabled us to examine the effect of P. nicotianae population levels on fine-root mortality. Seasonal and genotypic variation in patterns of citrus fine root mortality were associated with variation in population levels of P. nicotianae. Fine root lifespans were shorter when populations of P. nicotianae were high. Fine roots of the Phytophthora-susceptible rootstock, rough lemon (Citrus jamibhiri), had shorter median lifespans and supported larger populations of P. nicotianae than the fine roots of the more tolerant rootstock, Volkamer lemon (Citrus volkameriana). Rates of root mortality were either relatively constant for roots of all ages, or increased with age; the latter pattern was most pronounced for Volkamer lemon roots. Differences in the age-dependence of root mortality may, therefore, play a role in genotypic differences in tolerance of Phytophthora root rot by these two rootstocks. H Lambers Section editor     

Use of morphometric analysis for characterization of tobacco root growth in relation to infection byPhytophthora parasitica var.nicotianae

Development of tobacco root systems was characterized under controlled environmental conditions by use of morphometric root analysis. According to the classification scheme of this system, roots terminating in apical meristems are defined as first-order roots. Elements of second-order roots begin where two first-order roots merge, and so forth. Growth of root systems was similar for susceptible and resistant tobacco cultivars in nonautoclaved and autoclaved soils. During 15 days of growth subsequent to transplanting of 2-week-old plants, relative multiplication and extension rates of first-order and second-order roots were constant. Apparent unit extension rates of first-order and second-order root elements increased through 15 days of root system growth. Classification of tobacco root systems by the morphometric scheme provided a useful means of partitioning susceptibility of tissues to infection byPhytophthora parasitica var.nicotianae. Zoospores applied at the tips of first-order roots were most successful in causing infections; 73.3% of the roots inoculated with 16 zoospores per root tip became infected. Percentages of infections after inoculation of first-order root tissues 2 cm behind root tips or after inoculation of second-order roots were 10 and 4.3%, respectively.Florida Agricultural Experiment Station, Journal Series Paper 8106.     

Aggressiveness and diversity of Phytophthora capsici on vegetable crops in Georgia

Phytophthora blight induced by Phytophthora capsici causes significant yield loss in a number of vegetable crops. It is imperative to understand the diversity and aggressiveness of the pathogen to design more efficient disease management programs. A collection of P. capsici strains isolated from different vegetable crops in Georgia, USA, were characterised in this study. Of the 49 isolates tested, 24 were A1 and 25 were A2 mating type, respectively, with both mating types found in the same fields. Variability of the isolates was assessed in terms of their aggressiveness on six pepper genotypes. The isolates differed in their aggressiveness on different pepper cultivars with 10 pathotypes identified. No correlation between aggressiveness of the isolates and their host origin or geographical location of isolation was observed. Randomly amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic variability among P. capsici populations. RAPD analysis using 15 random primers resulted in 133 reproducible bands and cluster analysis separated the isolates into 5 groups. Analysis of molecular variance showed that there was moderate genetic differentiation associated with host origin and geographical location of the isolates. No correlation was found between RAPD groups and pathotypes or mating types. These results indicate that P. capsici populations infecting vegetable crops in Georgia were genetically diverse, which should be taken into account in developing resistant cultivars or other disease management programmes.     

Behaviour of Phytophthora citrophthora and P. nicotianae var. in soil, and Differences in Their Tolerance to Antimicrobial Components of Selective Media Used for Isolation of Phytophthora spp.

Phytophthora citrophthora was inhibited to a greater extent than P. nicotianac var. parasitica by chloramphenicol, hymexazol, PCNB and pimaricin at concentrations used in selective media for the isolation of Phytophthora spp. Reduced concentrations of the antimicrobial components of the selective media to tolerant levels for P. citrophthora markedly increased the recovery of the two brown rot pathogens from soil. Mycelium of both Phytophthora spp. survived in air-dried soil for at least 5 months while mycelium of most Phytophthora spp. do not survive in dry soil. In moist soil, P. nicotianae var. parasitica produced hyphal swellings, sporangia and chlamydospores. P. citrophthora produced hyphal swellings and sporangia, but no chlamydospores. No oospores were produced, even in pairing cultures on agar plates with isolates obtained from several locations of citrus groves andfruits by both species. Sporania were obtained in both species in citrus groves on mycelium mats, in the rhizosphere, in infected leaves and fruits buried at soil depths of 5–35 cm. Numbers of propagules declined during the incubation period, but conside, rable numbers survived throughout the experimental period (6 months). Although P. nicotianae var. parasitica produced chlamydospores while P. citrophthora did not, numbers of surviving propagules recovered from soil after 6 months were comparable with both species. The brown rot pathogens survived in soil both as colonizers of detached leaves and fruits and as parasites in living root tissues.     

Identification and classification of celery cultivars with RAPD markers

Summary Twenty-one celery (Apium graveolens L. var. dulce) cultivars, one celeriac (var. rapaceum) and one annual smallage (var. secalinum) cultivar were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with 28 arbitrary 10-mer primers. Among a total of 309 bands observed, 29 (9.3%) were polymorphic in the 23 cultivars screened, but only 19 (6.1%) markers were polymorphic within the 21 type dulce cultivars. These markers were sufficient to distinguish each of the cultivars used. The average marker difference was 6.4 between two celery cultivars, 16.7 between celery and annual smallage, 14.7 between celery and celeriac, and 12.0 between annual smallage and celeriac. The celery cultivars surveyed were classified into three groups based on the marker differences. The relationship among the dulce-type cultivars concluded from this research is basically consistent with the known lineage of the cultivars and the previous study using stem protein and isozyme markers. RAPD technology provides a new alternative for cultivar identification and classification in celery.     

Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae

An elicitor preparation obtained from Phytophthora parasitica var. nicotianae, a pathogen of tobacco, induced an accumulation of proteinase inhibitors and a stimulation of ethylene synthesis in a tobacco (Nicotiana tabacum) cell suspension culture. About 30 micrograms per milliliter of elicitor were necessary for maximal induction of proteinase inhibitor accumulation, and the response was detectable after 12 hours of incubation with elicitor. Accumulation of proteinase inhibitors required de novo protein synthesis, since cycloheximide completely inhibited its elicitation, and actinomycin D inhibited it partially. One of the inhibitors was purified by a procedure that included heating, (NH₄)₂SO₄ precipitation, ion-exchange chromatography, and affinity chromatography. The purified inhibitor was shown to be a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 10,500. It inhibited trypsin but not chymotrypsin.     

Partial molecular characterization of some kiwi fruit cultivars by RAPD markers

Molecular variability among seven cultivars of A. deliciosa var. deliciosa was investigated through RAPD markers. Thirty four decamer primers were screened generating polymorphic patterns of amplified DNA for these cultivars. Twenty one selected primers gave clear and reporducible patterns. A total of 430 bands were produced and 29.37% of them were polymorphic. The patterns distinguished between the cultivars and their analysis established an approach to classification within A. deliciosa var. deliciosa based on RAPD markers. The dendrogram clearly differentiated male from female cultivars. While abbot and allison female cultivars were closely related, bruno and abbot female cultivars showed maximum dissimilarity.     

Detection of the patulin-producing potential of some <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> strains isolated from the air samples of Jeddah City,Saudi Arabia,using the RAPD-PCR technique

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was used to detect the patulin-producing potential among seven different strains of Paecilomyces variotii isolated from air samples collected in Jeddah City, Saudi Arabia. Using five different primers, the strains showed some similarities and distinct RAPD patterns. A correlation between isolation source and clustering was noted in the constructed dendrogram. Patulin-producing strains showed identical RAPD patterns. Primer M13 produced a distinct fragment with toxin-producing strains.     

烤烟品种资源的RAPD分析

从156个随机引物中筛选出30个引物,对来自国内外的31个烤烟品种进行了遗传多样性和亲缘关系的RAPD分析。在检测的246个位点中,127个位点为多态位点（52％）。聚类分析表明,不同烤烟品种之间存在明显差异,31个品种基本上可分为4大类,品种最多的第一大类（19个）主要由来自美国的Orinoco烤烟选育而成,反映了我国现在推广的烤烟品种遗传基因比较狭窄。研究结果表明,RAPD技术可用于烤烟品种的鉴别和纯度测定。研究为烤烟杂种育种中亲本的选配提供了一定的理论依据。