Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica)

Oospores of Peronospora manshurica, the causal agent of soybean downy mildew, were stained by a variety of techniques. TTC (tetrazolium chloride) and NBT (nitroblue tetrazolium chloride) primarily stained oospores which were cytologically abnormal and appeared degenerating. Cytological normal oospores were not stained by these compounds presumably because the dyes were excluded from the oospore cytoplasm by the oospore wall or the plasmalemma. Strong autofluorescence of dead/degenerating oospores in the FDA test (fluorescein diacetate) made scoring of the oospore viability by this technique unreliable. Phloxine B was found in a consistent way to stain the degenerating oospores and a small proportion of the oospores which by light microscopic, observations could not be scored cytologically abnormal. Control experiments with live and dead cells of yeast (Saccharomyces cerevisiae) confirm that phloxine B is excluded from live cells and dead cells become stained. The presumed mode of action is that the semipermeability of the plasma membrane of live cells excludes the stain. The phloxine B test described here appears a promising technique for the determination of oospore viability of P. manshurica.     

Passive heat treatment of sweet basil crops suppresses Peronospora belbahrii downy mildew

Sweet basil (Ocimum basilicum) is an annual herb crop grown in polyethylene‐covered structures in Israel. It is Israel's leading herb crop, grown in warm regions of the country. Downy mildew (caused by Peronospora belbahrii) is a severe disease in Israel and in many other crop‐growing regions worldwide. Experiments were carried out to identify potential climate‐management techniques for suppression of this disease on basil in non‐heated greenhouses. Disease severity was evaluated under commercial‐like conditions in three experiments, with 8–10 walk‐in tunnels at each location. Pathogen inoculum was introduced into all walk‐in tunnels. Regression analysis was performed between the disease values and air temperature, relative humidity (RH) and soil temperature. Downy mildew severity was negatively related to high (>25°C) air temperature, RH in the range of 65–85% and high (>21°C) soil temperature. The increase in air temperature did not result in a significant increase in leaf temperature; canopy surface median temperatures only reached 30°C. Symptomless plants from relatively warmer tunnels (peak temperatures of 45–48°C) that were transferred to conditions that promote downy mildew (22 ± 2°C, RH > 95%) became severely diseased, showing sporulation of P. belbahrii, suggesting that infection occurred but at the high temperatures symptom expression/tissue colonisation was suppressed. Pot experiments in which aerial and subterranean plant organs were differentially heated revealed that treating the roots with a high temperature (26–31°C), similar to the soil temperatures in the warmer greenhouses, while maintaining the upper plant parts at ambient temperature (20°C), suppresses canopy downy mildew. The effect lasted for 1–2 weeks after the plants were removed from the heated soil treatments and maintained under optimal conditions for pathogen development. Furthermore, oospores were found in the symptomatic leaves. Oospores are minimally affected by high temperature, and therefore the high temperature presumably did not affect pathogen survival. In conclusion, the effect of high greenhouse temperature on basil downy mildew may not result from a direct negative effect of high temperature on the pathogen but from an indirect high‐temperature effect on the host, rendering it less susceptible to pathogen development.     

Location of Oospores in Buckwheat Seed and Probable Roles of Oospores and Conidia of Peronospora ducometi in the Disease Cycle on Buchwheat

Oospores of Peronospora ducometi, the causal agent of downy mildew of buckwheat (Fagopyrum esculentum), were found in the calyx remnant attached to the seed, on the inside of the seedcoat and in the spermoderm layer between the seedcoat and the endosperm. This constitutes a first report documenting the location of oospores in buckwheat seed. Systemic infection of seedlings occurred from oospore-infested seed. Conidial germination was greater at 14°C than 25°C. Systemic infection also occurred as the result of conidial infection of leaves. It is proposed that primary infection of buckwheat occurs by the germination of seed-borne oospores resulting in systemic invasion of the seedling by the germtubes, and followed by conidial formation on the cotyledons. Secondary infection occurs initially from conidia produced on the cotyledons as a result of the systemic infection from seed and subsequently as the result of repeated infections by conidia produced on leaf lesions as the disease progresses up the plant.     

Phytophthora kernoviae oospore maturity, germination, and infection

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).     

Production of Phytophthora infestans oospores in planta and inoculum potential of in vitro produced oospores under temperate highlands and subtropical plains of India

High moisture content of the host tissue ( 88%) and low ambient r.h. (50-54%) favoured oospore formation under controlled environments. It took 14–16 days for oospores to develop; thereafter the number of oospores increased with time and decreased with moisture content of host tissue. High ambient r.h. (> 80%) did not favour oospore formation under field or controlled conditions. Oospore formation was detected in inoculated plants grown in the field when the ambient r.h. declined to 74% and moisture content of host tissue decreased to 83.7–85.6%. It took 8 days (cv. Kufri Chandramukhi) to 13 days (cv. Kufri Jyoti and Kufri Badshah) for oospores to develop. Cultivars also differed in their response to oospore production, cv. Kufri Chandramukhi being more responsive (4800 oospores g⁻¹ f wt) than cv. Kufri Jyoti and Kufri Badshah (1320 and 390 oospores g⁻¹ f wt respectively). Oospores produced in vitro remained viable when buried in soil in the temperate highlands of Himachal Pradesh and sub-tropical plains of Uttar Pradesh, India for more than 150 days, i.e. beginning of the next crop season. The oospores germinated and initiated late blight infection at the base of the stems after 21–30 days of incubation of the potato plants raised in oospore-infested soil. It took 2 days for newly formed oospores to germinate and this delay time increased to 75–77 days after 180-days burial. It took 15 days for their germination (47%) in soil extract as compared to 50 days in sterilised distilled water.     

Use of Oospore Formulations of Pythium oligandrum for Biological Control of Pythium Damping-off in Cress

Oospore preparations of Pythium oligandrum produced by liquid and solid-substrate fermentations were evaluated for biocontrol activity against Pythium damping-off in cress in artificially infested sand and naturally infested soil. Oospore biomass preparations from liquid fermentation of six isolates of P. oligandrum were equally effective in reducing damping-off in sand when tested as seed-coatings, whereas this type of preparation of a single isolate formulated as a kaolin dust, on Perlite and as alginate pellets incorporated into sand gave little or no control. None of the formulations containing oospores produced by solid-substrate fermentation incorporated into sand had any effect. In soil, a formulation containing oospores produced in a barley-Perlite solid-substrate fermentation and all oospore-biomass formulations which were prepared increased seedling survival, but none of these were as effective as a propamocarb HCl drench.     

Survival, Germinability and Infectivity of Oospores of Peronospora viciae f.sp. fabae

A series of experiments was conducted to germinate oospores of Peronospora viciae f.sp. fabae. With rare exceptions, dry-stored oospores did not germinate in water nor did they infect faba bean seedlings in soil. Long-term storage, pre-treatment with KMnO₄ or addition of nutrients to the medium did not induce germination. Survival and infectivity of dry-stored oospores were compared to those of oospores incorporated in a silt loam and a loamy sand soil in the field during 21–22 months. Under dry conditions, the percentage of living oospores did not change as determined by the vital stain tetrazolium bromide. In soil, less than 2% of the oospores had survived after 21 months. Infectivity of oospores was determined by a bioassay 17 and 21 months after oospores had been incorporated in soil. Diseased seedlings were obtained after inoculation of faba bean seeds with oospores extracted from the soil but not with the drystored ones. Soil samples from two field plots naturally infested with oospores 2 and 3 years before the bioassay were infective. Oospores collected with diseased plant material on one of these plots and subsequently stored dry for 3 years were not infective. The results suggested that oospores need a period of natural weathering to become germinable and infective.     

ANNUAL GERMINATION WINFOW IN OOSPORES OF NITELLA FURCATA (CHAROPHYCEAE)1

Oospores of Nitella furcata subsp. megacarpa (Allen emend. Wood) were collected from an oospore bank in the sediments of Lake George, New York. Incubated at constant temperatures, all or nearly all of the oospores germinated when exposed to a brief pulse of red light when the annual window of germinability was open. The window seems related to the annual cycle of sediment temperatures. It is open in spring and closes wit the onset of a secondary dormacncy in the summer. Oospores in storage follow a parallel path if held at 18°C, a summer equivalent temperature; the window remains open indefinitely if the oospores are held at 4°C. Attention is drawn to the similarity if the cyclic window of germinability in seeds of summer annuals and oospores of N. furcata.     

Oospore dimensions and morphology in North American Tolypella (Charophyceae,Charophyta)

Characteristics of the oospores have been used to delimit sections and, in some cases, species in the genus Tolypella A. Braun. To test the utility of oospore characters for identifying North American species of Tolypella, we investigated oospores from field‐collected and herbarium specimens. Oospore dimensions (length, width, and length to width ratio) and morphology (color, ridge number and shape, wall ornamentation, and basal impression number) were measured. Oospore dimensions were statistically analyzed and oospore morphology was studied with light and scanning electron microscopy. Statistical analyses showed significant differences in length, width, and length to width ratios among most Tolypella species and populations but there was considerable overlap, which suggested that species identification based on oospore measurements alone is not wholly reliable. In addition, oospore morphology was not unique for every species.     

Seed treatment with aqueous extract of Viscum album induces resistance to pearl millet downy mildew pathogen

Abstract

Downy mildew (Sclerospora graminicola [Sacc.] Schroet.) is a serious agricultural problem for pearl millet (Pennisetum glaucum [L.] R. Br.) grain production under field conditions. Six medicinally important plant species Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and Viscum album were evaluated for their efficacy against pearl millet downy mildew. Seeds of pearl millet were treated with different concentrations of aqueous extract of the plants to examine their efficacy in controlling downy mildew. Among the plant extracts tested, V. album treatment was found to be more effective in enhancing seed quality parameters and also in inducing resistance against downy mildew disease. Germination and seedling vigor was improved in seeds treated with V. album extracts over control. Seeds treated with 10% concentration of V. album showed maximum protection against downy mildew disease under greenhouse and field conditions. The downy mildew disease protection varied from 44–70% with different concentrations. Leaf extract of V. album did not inhibit sporulation and zoospore release from sporangia of Sclerospora graminicola, indicating that the disease-controlling effect was attributed to induced resistance. Seed treatment with V. album extract increased pearl millet grain yield considerably. In V. album, treated pearl millet seedlings increased activities of peroxidase, and phenylalanine ammonia-lyase enzyme was detected. FTIR analysis of V. album extracts showed the presence of amides and other aromatic compounds which are antimicrobial compounds involved in plant defense.     

Reproductive isolation in Chara aspera populations

Possible reproductive isolation between freshwater and brackish water populations of the dioecious charophyte Chara aspera was studied by means of cross-fertilization experiments and AFLP (Amplified Fragment Length Polymorphism). Three Swedish freshwater populations and three (German and Swedish) Baltic Sea populations of C. aspera were sampled. Cross-fertilization experiments were performed in a full combination setup of all populations and with two different salinities (0 and 10 PSU). Both freshwater and brackish water females formed about 70% more gametangia at 0 than at 10 PSU. Male individuals collected from freshwater had higher fertility than brackish water males at both salinities. 57% of all gametangia of females from freshwater developed into oospores compared to only 8% of gametangia of brackish water females. 42% of all oospores were fertilized in crosses between habitats (freshwater–brackish water) compared to 36% in crosses within habitats, the difference was not significant.Oospore and bulbil germination was investigated using propagules from freshwater and brackish water populations and incubation salinities of 0, 5, 10 and 20 PSU. None of the oospores collected from brackish water germinated. Germination of oospores and bulbils from freshwater was higher at 0 and 5 PSU than at higher salinities. Only around 40% of bulbils from brackish water germinated at 20 PSU compared to around 70% at the other three salinities. Germination of all bulbils was delayed at 20 PSU compared to other salinities.Genetic similarities (Jaccard indices of AFLP data) were higher within than between populations, but comparisons within habitat (freshwater–freshwater and brackish water–brackish water) were not different from comparisons between habitats.Our results did not identify any reproductive isolation between freshwater and brackish water populations, but indicate low gene flow between the two habitats. Oospore and bulbil germination success were highest at salinities corresponding to the conditions of their original habitat, suggesting genetic adaptation to their environmental conditions and indicating that propagules transported from freshwater to brackish water or vice versa will hardly develop into fertile plants. Additionally, brackish water plants perform poorer in all aspects of sexual reproduction than freshwater plants. Possibly, successful dispersal of oospores is not subjected to high selective pressure within the Baltic Sea where new sites easily can be colonized by means of vegetative reproduction. We assume that these adaptations will favour speciation within C. aspera and support the idea of the geologically young Baltic Sea as a “cradle of plant evolution”.     

Detection of Seed-Borne Inoculum of Peronosclerospora sorghi in Sorghum

An alkali maceration technique has been developed to detect internally seed-borne inoculum of Peronosclerospora sorghi in sorghum seeds. Optimum period for maceration was found to be 36 h. Oospores in the glumes and mycelia in the pericarp and endosperm were clearly demonstrated. Mycelium was found in the pericarp of 40 % of the seeds tested and in the endosperm of 5 % of seeds. The possibility of using this technique to detect seed-borne downy mildew infection has been emphasized.     

The Infection Court of Faba Bean Seedlings for Oospores of Peronospora viciae f.sp. fabae in Soil

The infection court of Faba bean seedlings for oospores of Peronospora viciae f.sp. fabae in soil was determined. Soil naturally infested with oospores was placed as 3-cm thick layers at four different depths relative to Faba bean seeds. Seedlings with downy mildew were obtained only from seeds sown in the middle of a 3-cm layer of oosporeinfested soil. No infection was obtained from oosporeinfested soil placed more than 1.5 cm above or below seeds. Histological observations showed that the hypocotyl and first part of the main root were the most probable sites of infection.     

STORAGE AND GERMINATION OF CHARA OOSPORES

Germination tests were conducted on 39 collections of Chara oospores stored under 4 different conditions for periods of approximately 4 yr. In general, storage of dried oospores at low temperatures (3 C) provided the most satisfactory means for long-term preservation of viable disseminules. Oospore germination zuas higher in light than in darkness. Effects of temperature and substrate upon germination were explored briefly.     

Some factors affecting germination of Peronospora farinosa conidia in water

The germination of conidia of Peronospora farinosa f. sp. betae, collected from sugar beet and suspended in deionized water, was inhibited by dilution with 10% solutions of glycerol, glucose or sucrose and with sap from sugar-beet leaves. Germination was stimulated by diluting with deionized water but not with tap water or biological saline. Substances that diffused from excised buds of sugar-beet plants into deionized water also stimulated germination of conidia but diffusates from leaves did not. This may partly explain why buds are more susceptible to downy mildew than leaves in sugar beet. Germination of conidia was apparently stimulated more by diffusates from buds of seedlings than by those from buds of older plants; this may help to explain why sugar-beet seedlings are more susceptible to downy mildew than older plants. Diffusates from plants of four sugar-beet stocks, that differed from each other in susceptibility to downy mildew, had very similar effects on germination of P. farinosa conidia. Stimulation of spore germination on the surfaces of buds and leaves did not seem, therefore, to be an important factor in determining resistance or susceptibility to downy mildew in these stocks.     

The influence of foliar applications of sugars on the susceptibility of sugar beet to downy mildew

Spraying sugar-beet seedlings in the glasshouse, with 1% or 10% solutions of sucrose, 24 h before inoculation with Peronospora farinosa, significantly reduced their susceptibility to downy mildew. The proportion of inoculated plants that became infected was reduced by spraying with sucrose but the main effect was the inhibition of sporulation. Applications of glucose or fructose also increased the resistance of beet seedlings to P. farinosa. Spraying with sucrose 1 or 2 days before inoculation was much more effective than was spraying shortly before inoculation, or 24 h afterwards, or adding sucrose to the inoculum. Washing sucrose-sprayed seedlings with distilled water 1–2 h before inoculation removed only part of the effect of sucrose on sporulation. Although the mechanism by which applications of sugars affected susceptibility to downy mildew is not understood, the results suggest that the main effects occurred inside the host plant rather than externally. The possible significance of these results, in breeding for resistance to downy mildew and in the control of this disease in the field, is discussed.     

Extraction from Plant Tissue and Germination of Oospores of Peronospora viciae f.sp. pisi

A protocol was developed to extract oospores of Peronospora viciae f.sp. pisi from plant tissue and control bacterial contamination in a germination assay. Oospores were extracted by comminuting infected leaves and pods in water, sonicating the suspension and sieving it through mesh sizes 53 and 20 pm, respectively. Germination of oospores was negatively influenced by addition of chloramphenicol and penicillin. A combination of ampicillin and rifampicin strongly inhibited bacterial growth at 10 C, and did not negatively affect germination. Washing oospores in water or 0.02% Tween-80, and sonication did not influence germination. Treating oospore suspensions with cellulase buffered at pH 4.6 for 2 h digested most plant tissue but did not influence germination. Incubation in 0.05 m acetate buffer delayed germination. Germination was unaffected when oospores were incubated in 0.05 m citrate buffer.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Propagule bank buildup of Chara aspera and its significance for colonization of a shallow lake

Several aspects of the propagule bank dynamics including germination, burial and storage characteristics, and the relationship between the distribution of the propagule bank and cover of an expanding Chara vegetation (dominated by C. asperaDeth. ex Willd.) was studied in the shallow lake Veluwemeer. The density of oospores in the sediment was positively correlated with the number of years that Chara was present at that particular site. After six years of Chara presence, at least 1.7 × 10⁶ oospores m^–2 had accumulated in the sediment. Oospores in sediment were evenly distributed in the 15 cm top layer, which was very similar to the foraging depth of Bewick's Swans (Cygnus columbianus bewickii Yarr). Burial in the sediment may be an important mechanism by which oospores are stored. On the other hand, at shallow areas about 50% of the biomass was consumed enhancing the potential dispersal of oospores by water birds. In a laboratory experiment, 100% of the tested bulbils of C. aspera emerged and thus may be important for short time survival of established vegetation. In contrast, oospore germination varied between 1 and 15% depending on light level and burial depth. The low germination and the high accumulation of oospores suggest that oospores are adapted to long time survival in a dormant state. Charophytes colonized Veluwemeer step by step in the course of about nine years. Not all the sites with suitable light conditions were colonized at the same speed. C. aspera established a dense vegetation only at sites with high oospores densities (> c. 1 × 10⁴ m^–2). The results indicate that the high density of oospores needed for attaining complete vegetation cover may be an important limiting factor during colonization.