Effects of temperature and rainfall on date of release of ascospores of Leptosphaeria maculans (phoma stem canker) from winter oilseed rape (Brassica napus) debris in the UK

Data from a controlled environment experiment investigating effects of temperature on maturation of Leptosphaeria maculans pseudothecia were used to derive equations describing the times until 30% or 50% of pseudothecia were mature as a function of temperature. A wetness sensor was developed to estimate the oilseed rape debris wetness and operated with debris exposed in natural conditions in 2000 and 2001. The maturation of L. maculans pseudothecia on debris and concentrations of airborne L. maculans ascospores were observed from 1999 to 2004. There were considerable differences between years, with the first mature pseudothecia observed in September in most years. There were linear relationships between the first date when 10% of maximum ascospore release was observed and the dates when 30% or 50% of pseudothecia were mature. By summing the daily temperature‐dependent rate of pseudothecial maturation for days after 1 August with rainfall >0.5 mm, the dates when 30% or 50% of pseudothecia were mature were predicted. There was good agreement between predicted and observed dates when 30% or 50% of pseudothecia were mature. These equations for predicting the timing of L. maculans ascospore release could be incorporated into schemes for forecasting, in autumn, the severity of phoma stem canker epidemics in the following spring/summer in the UK.     

Occurrence of Didymella fabae,the Teleomorph of Ascochyta fabae,on Faba Bean Straw in Spain

Pseudothecia of Didymella fabae, the teleomorph of Ascochyta fabae, were first observed on faba bean (Vicia faba) debris in Spain during autumn 1995. Most pseudothecia were mature by December–February. The ascospores gave rise to typical cultures of A. fabae, and conidia from these cultures caused ascochyta blight symptoms on inoculated faba bean plants. Placing straw‐bearing pseudothecia over the plants to allow ascospore discharge also resulted in typical ascochyta blight symptoms. Pseudothecia maturity and discharge of ascospores from the infested faba bean straw overlapped with the vegetative stage of the faba bean crop, which occurs in southern Spain during winter as the crop is sown in autumn and harvested in spring. These observations indicate that ascospores may serve as primary inoculum for the disease.     

Field and laboratory studies of the effect of urea on ascospore production of Venturia inaequalis (Cke.) Wint

Applications of urea after harvest but before leaf-fall restricted perithecial production by Venturia inaequalis. Immersion of detached leaves in urea appeared to be the most effective method of preventing perithecial formation, although spraying attached leaves was equally effective when leaf abscission occurred within a week of treatment. A high nitrogen content within the leaf was one of the major factors contributing to suppression. Urea-treated leaves decomposed rapidly, thus destroying the overwintering substrate for the fungus. When apple plants (clone M. 111) were sprayed in autumn with 5 % urea, followed by a second (pre-bud-burst) application at 2 %, ascospore production in the spring was suppressed. The second treatment appeared to prevent the release of ascospores from mature perithecia.     

Analyses of air samples for ascospores ofLeptosphaeria maculans andL.biglobosa by light microscopy and molecular techniques

Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is important in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region ofLeptosphaeria maculans andL. biglobosa — the causal organisms of phoma stem canker and stem lesions ofBrassica spp., including oilseed rape — were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA fromL. maculans andL. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensitive than traditional PCR, especially in years with low ascospore numbers.     

Survival of A-group and B-group Leptosphaeria maculans (phoma stem canker) ascospores in air and mycelium on oilseed rape stem debris

Mycelium of Leptosphaeria maculans survived on oilseed rape stem base debris buried in sand for 2,4, 6, 8,10 or 12 months and produced pseudothecia after subsequent exposure on the surface of the ground under natural conditions for 2–4 months, but did not survive on upper stem debris buried for 2 months. Only A‐group L. maculans ascospores were produced on the stem base debris which had been buried; no B‐group ascospores were produced. Mycelium of L. maculans survived on both stem base and upper stem debris exposed on the sand surface for 2, 4, 6, 8, 10 or 12 months and pseudothecia with viable ascospores were observed at the time of sampling. Both A‐group L. maculans (predominant on stem bases) and B‐group L. maculans (predominant on upper stems) ascospores were produced on unburied stem base and upper stem debris. Thus B‐group L. maculans survived longer on unburied debris than on buried debris. A‐group ascospores which were exposed in dry air in darkness at 5–20°C survived longer than B‐group ascospores; 10–37% of A‐group ascospores, compared with 2–31% of B‐group ascospores, survived after 35 days.     

Biphasic diurnal cycle of ascus development of Taphrina maculans Butler

Taphrina maculans Butler inciting the brown leaf spot disease of turmeric (Curcuma longa L.) produces cuboid ascogenous cells several layers in depth in the subcuticular interspaces of the epidermis. The ascogenous cells germinate to produce asci centrifugally maturing toward the periphery of the stroma, expelling octosporous microcolonies of asco-blastospores on the leaf surface inciting secondary infection in favorable environment. Occurrence of a rhythmic cycle of ascus development and ascospore discharge giving 2 peaks of ascospore discharge each day has been demonstrated in Taphrina maculans. The cycle is directly affected by atmospheric temperature, availability of free moisture on the leaf surface and sunlight. Free moisture on a leaf surface appropriately soaks the infection spots (ascogenous cells) and induces ascus elongation and ascospore discharge, when a suitable atmospheric temperature is reached. Sunlight may adversely affect the cycle by increasing the temperature and lowering humidity in the atmosphere.     

Temporal Patterns of Ascospore Release in Leptosphaeria maculans Vary Depending on Geographic Region and Time of Observation

Diurnal patterns of spore release have been observed in a number of fungal pathogens that undergo wind-assisted dispersal. The mechanisms that drive these patterns, while not well understood, are thought to relate to the ability of dispersing spores to survive their journey and infect new hosts. In this paper, we characterise the diurnal pattern of ascospore release by a Western Australian population of Leptosphaeria maculans. Although L. maculans has been previously shown to exhibit diurnal patterns of ascospore release, these patterns appear to vary from region to region. In order to characterise the pattern of release in the Mediterranean climate of Western Australia, we analysed historical data describing the bi-hourly count of airborne ascospores at Mt Barker, Western Australia. Results of this analysis showed diurnal patterns that differ from those previously observed in other countries, with ascospore release in our study most likely to occur in the afternoon. Furthermore, we found that the time of peak release can shift from month to month within any one season, and from year to year. In explaining the hourly pattern of spore release over an entire season, time since rainfall, time since last release, temperature, hour and month were all shown to be significant variables.     

Influence of meteorological parameters on Leptosphaeria maculans and L. biglobosa spore release in central and eastern Poland

Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date.     

Evaluation of fungicides for control of South American leaf blight of Hevea brasiliensis

Of 43 fungicides tested in vitro, 19 showed strong, seven moderate and 17 weak inhibition of germination of conidia and ascospores of Microcyclus ulei. The formation of lesions on Hevea brasiliensis leaf discs was also suppressed by the first category of fungicides as well as by the five adjuvants tested. Ascospores were not released when perithecia were treated with urea, thiabendazole or alcoholic mercury chloride at 10.00, 0.10, 1.00 g/1 respectively; other fungicides had no such inhibitory effect. In field trials, thiophanate methyl (0.07% a.i.) and benomyl (0.025% a.i.) were most effective in controlling leaf infection, followed by chlorothalonil (0.15% a.i.) and mancozeb (0.32% a.i.). Benomyl suppressed conidial sporulation, whereas one application of thiophanate methyl (0.14% a.i.) to perithecia inhibited ascospore release; half of this concentration applied to conidial lesions or pycnidia caused the perithecia formed subsequently to abort. Thiophanate methyl thus shows promise for SALB control and elimination and benomyl may be valuable as a supplement in later rounds of spraying to control conidial sporulation. After 6 days of showery rain (2 mm for 17 min per day), water collected from sprayed leaves still gave complete inhibition of spore germination. However, inhibition was markedly reduced after 6 days of heavy rain (over 8 mm for 24 min per day).     

Epidemiology of Leptosphaeria maculans in relation to forecasting stem canker severity on winter oilseed rape in the UK

In the UK, ascospores of Leptosphaeria maculans first infect leaves of oilseed rape in the autumn to cause phoma leaf spots, from which the fungus can grow to cause stem cankers in the spring. Yield losses due to early senescence and lodging result if the stem cankers become severe before harvest. The risk of severe stem canker epidemics needs to be forecast in the autumn when the pathogen is still in the leaves, since early infections cause the greatest yield losses and fungicides have limited curative activity. Currently, the most effective way to forecast severe stem canker is to monitor the onset of phoma leaf spotting in winter oilseed rape crops, although this does not allow much time in which to apply a fungicide. Early warnings of risks of severe stem canker epidemics could be provided at the beginning of the season through regional forecasts based on disease survey and weather data, with options for input of crop-specific information and for updating forecasts during the winter. The accuracy of such forecasts could be improved by including factors relating to the maturation of ascospores in pseudothecia, the release of ascospores and the occurrence of infection conditions, as they affect the onset, intensity and duration of the phoma leaf spotting phase. Accurate forecasting of severe stem canker epidemics can improve disease control and optimise fungicide use.     

Quantitative PCR analysis of abundance of airborne propagules of Leptosphaeria species in air samples from different regions of Poland

When airborne propagules of Leptosphaeria maculans and L. biglobosa were collected in Poland at three ecologically different sites from 1 September to 30 November in 2004 to 2008, using a Hirst-type seven-day volumetric spore trap, there were fluctuations in timing of ascospore release and diverse ratios between airborne propagules of both species depending on season, field location and weather conditions. The detection was done using the microscope as well as quantitative PCR with species-specific primers targeted against fragments of β-tubulin genes and quantified with a dual-labelled fluorescent probe approach. This detection chemistry is described for the first time for L. maculans and L. biglobosa. Its advantage over the previous ITS-based SYBR-Green chemistry resides in improved sensitivity and the virtual absence of false positives in the detection of these fungi. There were significant, positive correlations between data obtained using visual assessment of ascospore numbers and DNA concentrations that were measured by qPCR. Climatic differences between the oilseed rape growing regions could have significantly affected the biological processes of pseudothecial maturation and ascospore development of the pathogens. The data suggest that regular rain events of intermediate intensity recorded in the Maritime region favoured the maturation of the pathogen more than the drier weather recorded in the Silesia or Pomerania regions. It was observed that the number of rainy days was of greater importance than the cumulative rainfall to obtain the generative sporulation of the pathogen. Accurate detection of airborne inoculum of pathogenic Leptosphaeria spp. facilitates improved targeting of disease management decisions for oilseed rape protection against phoma stem canker and stem necrosis diseases.     

Individual and Combined Effects of Certain Pesticides on Rhizoctonia solani, Sheath Blight Pathogen of Rice

The effect of different pesticides (fungicide, herbicide and insecticide) on the mycelial growth and formation and germination of sclerotia of sheath blight pathogen of rice, Rhizoctonia solani Kühn was studied in nutrient medium and in natural soil. Among the herbicides and insecticides, fenitrothion (insecticide) and thiobencarb (herbicide) were the most effective in inhibiting the mycelial growth and sclerotial formation. These pesticides effected significant inhibition of both mycelialgrowth and sclerotial formation at 7.5 to 10μg/ml concentration while fungicides carbendazim and tolclofosmethyl effected almost complete inhibition at 2.5 μg/ml. Interestingly, a synergistic increase in the toxicity to mycelial growth, sclerotial formation and sclerotial germination was noticed when the fungicide, mancozeb and the herbicide, thiobencarb were applied at subtoxic concentration in combination. Such synergistic interactions between pesticides in a combination leading to increased toxicity suggest that under the current practice of applying fungicides, herbicides and insecticides either simultaneously or in rotation, certain combinations may help in reducing the dose of a fungicide in disease control.     

THE EFFECTS OF SELECTED HERBICIDES ON ZYGOSPORE GERMINATION AND GROWTH OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE,VOLVOCALES)11

A laboratory study was conducted, to examine and compare the sensitivity of vegetative cells and zygospores of Chlamydomonas moewusii Gerloff to 20 different herbicides. Under the culture conditions employed, both vegetative growth and zygospore germination were affected by certain herbicides and not by others. Over a concentration range from 1.0–80.0 μM, growth was inhibited to various degrees by herbicides containing ametryne, paraquat, endothall, diquat, diuron, linuron, propanil, dinoseb, ioxynil, atrzine, prometon, and alachlor. Zygospore germination was inhibited significantly by herbicides containing dinoseb, endothall, parquet, diquat, propanil, linuron, ioxynil, ametryne, fenac and picloram at 80.0 μM concentrations. Comparisons of the results obtained indicate that concentrations of herbicides which affect growth may or may not effect zygospore germination and vice versa. Zygospores may be more resistant than vegetative cells to some but not all herbicides.     

Enzymatic Preparation of Aromatic Ethylamines from Aromatic L-Amino Acids

The characteristic mode for the herbicidal action of 2-amino-3-chloro-1,4-naphthoquinone (ACN) was investigated by using autotrophic green microalgae (Scenedesmus acutus). The effects of ACN on the growth, chlorophyll content, protoporphyrin-IX accumulation, and ethane production in Scenedesmus cells were measured in comparison with three reference herbicides, i.e., ioxynil, dinoseb, and chlorophthalim. ACN appeared to have a different mechanism for action from these three herbicides. S. acutus cells grown with ACN produced ethane, but the mechanism for its production is considered to have been different from that of chlorophthalim. ACN may inhibit porphyrin biosynthesis at a different stage from that by chlorophthalim.     

Effect of climate change on sporulation of the teleomorphs of <Emphasis Type="Italic">Leptosphaeria</Emphasis> species causing stem canker of brassicas

Leptosphaeria maculans and L. biglobosa are closely related sibling fungal pathogens that cause phoma leaf spotting, stem canker (blackleg) and stem necrosis of oilseed rape (Brassica napus). The disease is distributed worldwide, and it is one of the main causes of considerable decrease in seed yield and quality. Information about the time of ascospore release at a particular location provides important data for decision making in plant protection, thereby enabling fungicides to be used only when necessary and at optimal times and doses. Although the pathogens have been studied very extensively, the effect of climate change on the frequencies and distributions of their aerially dispersed primary inoculum has not been reported to date. We have collected a large dataset of spore counts from Poznan, located in central-west part of Poland, and studied the relationships between climate and the daily concentrations of airborne propagules over a period of 17 years (1998–2014). The average air temperature and precipitation for the time of development of pseudothecia and ascospore release (July–November), increased during the years under study at the rates of 0.1 °C and 6.3 mm per year. The day of the year (DOY) for the first detection of spores, as well as the date with maximum of spores, shifted from 270 to 248 DOY, and from 315 to 265 DOY, respectively. The acceleration of the former parameter by 22 days and the latter by 50 days has great influence on the severity of stem canker of oilseed rape.     

Environmental Factors Influencing the Dispersal of Venturia inaequalis Ascospores in the Orchard Air

A 6-year study was carried out in an apple-growing region of North Italy by trapping airborne ascospores of Venturia inaequalis with a volumetric spore trap operated continuously during the ascospore season, with the aim of better defining the weather conditions that allow ascospores both to discharge and to disperse into the orchard air. A total of more than 60 ascospore trapping events occurred. Rain events were the only occurrences allowing ascospores to become airborne (a rain event is a period with measurable rainfall ≥0.2 mm/h – lasting one to several hours, uninterrupted or interrupted by a maximum of two dry hours); on the contrary, dew was always insufficient to allow ascospores to disperse into the air at a measurable rate, in the absence of rain. In some cases, rain events did not cause ascospore dispersal; this occurred when: (i) rain fell within 4–5 h after the beginning of a previous ascospore trapping; (ii) rain fell at night but the leaf litter dried rapidly; (iii) nightly rainfalls were followed by heavy dew deposition that persisted some hours after sunrise. Daytime rain events caused the instantaneous discharge and dispersal of mature ascospores so that they became airborne immediately; for night-time rainfall there was a delay, so that ascospores became airborne during the first 2 h after sunrise. This delay did not always occur, and consequently the ascospore trapping began in the dark, when: (i) the cumulative proportion of ascospores already trapped was greater than 80% of the total season's ascospores; (ii) more than one-third of the total season's ascospores was mature inside pseudothecia and ready to be discharged.     

Comparative inhibition ofAcetobacterium wieringae by two non-ionic surfactants: Tween 20 and ATPlus 258

Summary The effect of two non-ionic surfactants, Tween 20 and ATPlus 258 was assayed on the growth ofAcetobacterium wieringae on fructose. Both surfactants proved to inhibit the growth ofA. wieringae, already at 100 mg per litre. At 1000 mg per litre, the growth ofA. wieringae was inhibited by 41% by Tween 20 and completely by ATPlus 258. The production of the fermentation end-metabolite, acetic acid, followed the same inhibition pattern.     

Identification of a 32–34-kilodalton polypeptide as a herbicide receptor protein in Photosystem II

Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32–34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a ¹⁴C-labeled photoaffinity triazine.In Photosystem II particles depleted of the 32–34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.     

Studies on taphrina maculans butler,inciting leaf spot of turmeric (curcuma longa L.). I. Isolation of the pathogen

Summary T. maculans inciting leaf spot of turmeric is difficult to be isolated into a pure culture. The best stage of the spot development on turmeric which yielded successful isolations was either the initial or the middle stage expressing yellowish necrotic areas. The successful course of development of asci, ascospore and conidia has been worked out. By streaking ascospore and conidial suspensions, the spot bearing portions of leaves on the inner side of the lid thus placing them in hanging position above the plates poured with cleared and acidified potato dextrose agar or turmeric leaf decoction agar or by streaking sterile water washings from the chocolate brown spots on these media, it was possible to isolate successfully the pathogenic culture ofT. maculans at 20° C.Condensed from the thesis submitted by the senior author to the University of Poona for M. Sc. (Agri.) under the guidance of the junior author. Respectively Assistant Professor of Plant Pathology, College of Agri. Poona and Wheat Rust Mycologist, Mahabaleshwar, India.     

Influence of nutritional supplementation on solid-substrate fermentation of wheat straw with an alkaliphilic white-rot fungus (Coprinus sp.)

Summary The solid-substrate fermentation of wheat straw with an alkaliphilic white-rot fungus (Coprinus sp.) was found to be influenced by the levels of nitrogen, phosphorus+sulphur and free carbohydrates, in terms of biodegradation of straw ingredients, microbial protein production and changes in in-vitro dry matter digestibility (IVDMD). Nitrogen and Phosphorus+Sulphur compounds favoured the bioconversion and their optimum levels were (g/100 g DM): urea (sterile): 1.5, urea (unsterile): 3.0; superphosphate: 1.0. The addition of free carbohydrates as molasses and whey had detrimental effect on biodegradation of lignin as also on organic matter degradation and digestibility. However, the protein production was enhanced in the supplemented straw. The optimized laboratory fermentation was also extended to 4 kg-(sterile and unsterile) and 50 kg-(unsterile) fermentation.