Retraction of flagella by Phytophthora parasitica var. nicotiana zoospores

Summary Light and electron microscope evidence is presented to show that retraction of flagella may occur in Phytophthora parasitica zoospores during encystment.     

Production of Kestoses (Fructosylsucroses) by Phytophthora parasitica var. nicotianae

The synthesis of kestoses (trisaccharides composed of two fructose units and one glucose unit) by races 0 and 1 of Phytophthora parasitica var. nicotianae is shown. The trisaccharide is found in culture filtrates of isolates grown in liquid media containing 3% sucrose. The utilization of sucrose and trisaccharide formation by the organisms over a 16-day period is described. The kestoses were identified by chemical and enzymatic analysis, and two of three possible isomers were found.     

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.     

Chemical and Physical Factors Influencing Growth of Phytophthora parasitica var. nicotianae on Vegetable Oils

Factors influencing the growth of Phytophthora parasition var. nicotiona on vegetable oil liquid media were studied. This fungus grew poorly on oleic acid, triolein, and tristearin. Cholesterol and α-tocopherol reversed the toxicity of oleic acid. Growth was often stimulated by addition of mineral oil to a glucose medium, and sterols dissolved in mineral oil frequently stimulated grwoth. Shaking produced a 5-fold or more increase in growth on either glucose or cottonseed oil media. Cholesterol (20 μ/ml further increased growth on glucose meidum in shake culture by at least 100%. Growth was usually better at pH 6.7 than at 5.4 or 7.7. Growth in shake culture on a glucose medium supplemented with cholesterol and α-tocopherol approached that occuring on vegetable oil media.     

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.     

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.     

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.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

Cultural and physiological studies of phytophthora parasitica dast. var. macrospora ashby,causing fruit rot of anona squamosa L.

Summary This paper gives an account of some cultural and physiological studies of an isolate ofPhytophthora parasiticaDast. var.macrosporaAshby, the causal agent of fruit rot ofAnona squamosa L. Among the various culture media studied, non-synthetic ones like oat meal agar, corn meal agar, lima-bean agar, carrot extract agar, soya-bean extract agar and steamed rice agar were the best, on which the organism showed marked growth and sporulation. Non-synthetic types were poor in this direction. Regarding the effect of various carbon sources, sucrose, lactose, maltose, raffinose, inulin, dextrin, dulcitol, glycogen, rhamnose and xylose supported growth and sporulation of the organism. Sodium nitrate, ammonium nitrate, magnesium nitrate, potassium nitrate, ammonium lactate and asparagin were the best sources of nitrogen. 6.5 was found to be the best pH for the growth and sporulation of the organism.A portion of Senior Author's M.Sc. Agric. Thesis, University of Poona, India.     

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.     

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.     

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.     

Parasitism of grain legumes by Alectra species (Scrophulariaceae)

In glasshouse pot experiments in the United Kingdom, the host preference of nine seed samples of Alectra vogelii from Eastern, Western and Southern Africa and of two samples of A. picta from Cameroon and Ethiopia, to cultivars of cowpea, groundnut, bambara and mung bean, was assessed. A susceptible cowpea cultivar, Blackeye, and four cultivars of groundnut were attacked by all samples of both parasitic species regardless of whether the host of origin was cowpea, groundnut or bambara. Five “strains” of A. vogelii were distinguished using two criteria: their ability to parasitise bambara and/or mung bean and their ability to parasitise cowpea B301 and bambara TVU 870. The latter proved in an associated experiment to be resistant to collections of the parasite from some locations. Strain 1, including populations from Mali, Nigeria and Cameroon, attacked all groundnuts, cowpea cultivar Blackeye, but not cowpea line B301, mung bean or bambara. Strain 2, from Botswana, differed in attacking B301 and mung bean. Three other strains were identified which attacked susceptible lines of all four legume species. Strain 3 from Kenya failed to attack either cowpea B301 or bambara TVU 870, strain 4 from Malawi attacked cowpea B301, but not bambara TVU 870, while strain 5 from Northern Transvaal, South Africa, attacked bambara TVU 870, but not cowpea B301. Cowpea B359 was resistant to A. vogelii samples from all locations and also to A. picta, which has a similar host preference to strain 1 populations of A. vogelii from West Africa. Two out of 13 groundnut lines tested showed low susceptibility to A. vogelii from Cameroon suggesting there is scope for selecting resistance in this crop also.     

Studies on the sporulation of Phytophthora parasitica Dastur var. piperina Dast. Responsible for Leaf and Foot-rot of Pan,Piper betle. L.

Summary Sporangial formation was found restricted within the range of temperature from 20–31° C. Atmospheric humidity of 100 % did not favour very much in developing sporangia. Free water appeared to be essential in sporangial formation. The range of temperature within which the liberation of zoospores took place was even narrower, 19 to 25° C. Temperature from 21 to 23° C appeared to be best condition for maximum liberation of zoospores. Similarly as with sporangial development atmospheric humidity as high as 100 % had very little effect on the liberation of zoospores unless there was condensation of water. The liberated zoospores in free water were found to germinate only within 20–24° C. At about 22° C the germination rate was found optimum.     

Bleach-induced Germination and Breakage of Dormancy of Seeds of Alectra vogelii

The effects of solutions of sodium hypochlorite and calcium hypochlorite (bleach) on dormancy and germination of Alectra vogelii seeds were investigated. Dry (non-pretreated) and 10-day water-pretreated seeds were exposed to various bleach concentrations, as well as to the host (Vigna unguiculata) root exudate. The 15-month-old Alectra seeds used were partially dormant in that only 40% or less of the 10-day pretreated seeds could be stimulated to germinate by the standard host root exudate. Comparable percentage germination of nonpretreated seeds was achieved with certain concentrations of bleach and the halogens, chlorine and bromine. Bleach and the halogens served not only as germination stimulant for pretreated Alectra seeds but appropriate concentrations induced also high percentage (70–90%) germination indicating a breakage of seed dormancy as well. The activity of the bleach in stimulating high percentage germination could be significantly reduced when the optimally bleach-treated seeds were rinsed daily with water during the germination period. Vigna root exudate, but not bleach, was shown to act as root stimulant for Alectra seedlings. The possible mechanism of the bleach effect on germination and dormancy of Alectra seeds is discussed.     

An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus

The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 × 10⁻¹³ mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium.     

Staurastrum dilatatum var.thomassonii var. nov.

The new varietyStaurastrum dilatatum Ehr. var.thomassonii is described and illustrated.     

Streptococcus faecium var. casselifavus, nov. var

Streptococcus faecium var. casseliflavus is a gram-positive, spherical cell. The cells occur chiefly as pairs within chains and elongate to ogive-shaped cells during growth. Growth is good on 5% bile salts-agar and in broth at 10 C, and in broth adjusted to pH 9.6 or containing 6.5% NaCl, but many strains fail to grow at 45 C. Litmus is reduced rapidly prior to formation of an acid curd. Few strains release ammonia from arginine or serine. The organism is not proteolytic and does not produce H(2)S or acetylmethylcarbinol, reduce nitrate, decarboxylate tyrosine, or produce slime on sucrose-agar. Most strains survive heating to 60 C for 30 min. It produces gray colonies on potassium tellurite agar, reduces 2,3,5-triphenyltetrazolium-HCl to a pink color, and ferments cellobiose, dextrin, maltose, mannose, and sorbitol, thus resembling S. faecalis. Like S. faecium, it produces peroxidase but not catalase on heated blood media, dissimilates malate, and ferments arabinose, melibiose, and salicin, but not melezitose. Like both species, it ferments dextrose, galactose, lactose, mannitol, sucrose, trehalose, and citrate. Properties peculiar to the variant include the high pH limiting initiation and termination of growth; the fermentation of alpha-methyl-d-glucoside, raffinose, and xylose; motility; and growth without blue button formation in ethyl violet broth. The water-soluble, pale lemon-yellow pigment is released into the aqueous phase only after the cell envelope is altered by fat solvents. The bacterium thrives as an epiphyte on plants.