Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Effect of host and inoculum patterns on take-all disease of wheat incidence,severity and disease gradient

The importance of the spatial aspect of epidemics has been recognized from the outset of plant disease epidemiology. The objective of this study was to determine if the host spatial structure influenced the spatio-temporal development of take-all disease of wheat, depending on the inoculum spatial structure. Three sowing patterns of wheat (broadcast sowing, line sowing and sowing in hills) and three patterns of inoculum (uniform, aggregated and natural infestation) were tested in a field experiment, repeated over 2 years. Disease (severity, root disease incidence, plant disease incidence and, when applicable, line and hill incidences) was assessed seven times during the course of each season and the spatial pattern was characterized with incidence-incidence relationships. In the naturally infested plots, disease levels at all measurement scales were significantly higher in plots sown in hills, compared to plots sown in line, which were in turn significantly more diseased than plots with broadcast sowing. Disease aggregation within roots and plants was stronger in line and hill sowing than in broadcast sowing. Analysis of the disease gradient in the artificially infested plots showed that the disease intensified (local increase of disease level) more than it extensified (spatial spread of the disease), the effect of the introduced inoculum was reduced by 95% at a distance of 15 cm away from the point of infestation. Yield was not significantly affected by sowing pattern or artificial infestation.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

Spatiotemporal analyses of rhizopus rot progress in peach fruit inoculated with Rhizopus stolonifer

Rhizopus rot, caused by Rhizopus stolonifer, is one of the main postharvest diseases in stone fruits, but there is little known about the processes of disease development during transport and postharvest storage. The objective of this study was to characterize temporal progress and spatial distribution of the disease in peach fruit. Rhizopus rot development was evaluated using two different fruit arrangements. Only one fruit of each arrangement was inoculated with a R. stolonifer spore suspension. Disease incidence and severity were assessed daily for all the fruit. Nonlinear models were fitted to the quantity of fruit and to the area of fruit that became infected over time and distance in relation to the source of inoculum. Disease‐free fruit placed next to the artificially inoculated peaches showed disease symptoms due to pathogen dissemination by mycelial stolons. The disease incidence and severity progress rates varied from 0.33 to 0.53 day^?1 and from 0.30 to 0.49 day^?1, respectively. The spatial spread of the disease followed a dispersive wave pattern with increasing speed over time, but decreasing speed with disease severity. For disease severity y = 0.5, the velocity at day 3 varied from 0.14 to 0.32 fruit diameter day^?1, while it ranged from 0.38 to 1.46 fruit diameter day^?1 at day 12.     

Direct and host‐mediated interactions between Fusarium pathogens and herbivorous arthropods in cereals

Fusarium head blight and fusarium ear rot diseases of cereal crops are significant global problems, causing yield and grain quality losses and accumulation of harmful mycotoxins. Safety limits have been set by the European Commission for several Fusarium‐produced mycotoxins; mitigating the risk of breaching these limits is of great importance to crop producers as part of an integrated approach to disease management. This review examines current knowledge regarding the role of arthropods in disease epidemiology. In the field, diseased host plants are likely to interact with arthropods that may substantially impact the disease by influencing spread or condition of the shared host. For example, disease progress by Fusarium graminearum can be doubled if wheat plants are aphid‐infested. Arthropods have been implicated in disease epidemiology in several cases and the evidence ranges from observed correlations between arthropod infestation and increased disease severity and mycotoxin accumulation, to experimental evidence for arthropod infestation causing heightened pathogen prevalence in hosts. Fusarium pathogens differ in spore production and impact on host volatile chemistry, which influences their suitability for arthropod dispersal. Herbivores may allow secondary fungal infection after wounding a plant or they may alter host susceptibility by inducing changes in plant defence pathways. Post‐harvest, during storage, arthropods may also interact with Fusarium pathogens, with instances of fungivory and altered behaviour by arthropods towards volatile chemicals from infected grain. Host‐mediated indirect pathogen–arthropod interactions are discussed alongside a comprehensive review of evidence for direct interactions where arthropods act as vectors for inoculum.     

Virulence and inoculum density‐dependent interactions between clubroot resistant canola (Brassica napus) and Plasmodiophora brassicae

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot‐resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 10³ spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 10⁵ spores mL^?1. At a spore density of 10⁶ spores mL^?1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

Spread and development of quambalaria shoot blight in spotted gum plantations

The aim of this study was to examine the disease development of quambalaria shoot blight, caused by the fungal pathogen Quambalaria pitereka, in plantation‐grown spotted gum (Corymbia citriodora subsp. citriodora, C. citriodora subsp. variegata, C. henryi and C. maculata) in south‐east Queensland, Australia. The results showed that native spotted gums are a primary source of inoculum followed rapidly by the production of secondary inoculum from infected trees in the plantation. The rate of spread and development of Q. pitereka within plantations increased exponentially over time as additional trees became infected and produced secondary inoculum. Spore concentration was shown to play an important role in disease development, with disease severity increasing with increasing disease incidence on individual trees and incidence across the plantation.     

Spatial distribution of Aphanomyces euteiches inoculum in a naturally infested pea field

The main objective of the study was to describe the horizontal and vertical distribution of Aphanomyces root rot in a naturally infested pea field. Measurements of inoculum potential clearly indicated a horizontal distribution of inoculum among several foci in the field, these foci differing in size and disease intensity. A highly significant relationship was observed between disease severity on plants during the cropping season and soil inoculum potential. In terms of the vertical distribution of inoculum in the soil, detection was maximal at a depth of 10 to 40 cm, but inoculum was detected down to a depth of 60 cm. Generally, inoculum potential was lowest for the layers at depths of 0 to 10 and 50 to 60 cm. Inoculum distribution and the value of the methodologies used are discussed in terms of possible use for epidemiology and disease forecasting.     

Contrasted responses of Botrytis cinerea isolates developing on tomato plants grown under different nitrogen nutrition regimes

The nutritional status of a plant is known to influence its susceptibility to pathogens. In the case of Botrytis cinerea, the role of nitrogen fertilization of various host plants on disease development appears to be variable. This study was carried out to characterize possible variability associated with isolates and inoculum density of B. cinerea in its ability to infect leaf‐pruning wounds and to develop stem lesions on tomato plants, as affected by the nitrogen input. Six isolates differing in their aggressiveness to tomato were compared. They all had similar reaction patterns in vitro in response to differential nitrogen levels. In tests on plants grown with contrasted regimes of nitrate fertilization, overall disease severity was lower for all isolates on plants with higher nitrogen inputs, regardless of inoculum concentration. However, differences among isolates were observed in the effect of plant nitrogen nutrition on infection and on lesion expansion. Disease onset was delayed on all plants with higher nitrogen inputs, but the response was greater for isolates with lower aggressiveness on tomato. The highest contrast among isolates was observed with the colonization of stems. The daily rate of stem lesion expansion decreased with increasing nitrogen fertilization levels for the more aggressive isolates, while it increased for the less aggressive isolate. Hypotheses to explain these results are discussed in light of the possible physiological effects of nitrogen fertilization on nutrient availability for the pathogen in the host tissue and of possible production of defence metabolites by the plant.     

Spatial analysis of blackleg‐affected seed potato crops in Scotland

Potato blackleg, caused by Pectobacterium and Dickeya species, is one of the most significant bacterial diseases affecting potato production globally. Although it is generally accepted to be a seedborne disease, the processes underlying the spread of disease largely remain unknown. Spatial point pattern analysis was applied to blackleg occurrence in seed potato crops in Scotland during the period of 2010–2013 (approximately 8000 blackleg‐affected crops), to assess whether its distribution was random, regular or aggregated, and the spatial scales at which these patterns occurred. Blackleg‐affected crops derived from mother stocks with symptoms were omitted from the analyses in order to examine the statistical evidence for horizontal transmission of blackleg. The pair correlation function was used to test for global spatial autocorrelation, and results indicated significant (P < 0·05) clustering of incidence at a wide range of spatial scales. Strength of clustering (degree of aggregation) among blackleg‐affected crops was notably larger at spatial scales of 25 km or less. A hot‐ and coldspot analysis was performed to test for local spatial autocorrelation, and statistically significant clusters of high and low values of disease were found across the country. These analyses provide the first quantitative evidence of localized and large‐scale spatial clustering of potato blackleg. Understanding the mode(s) of inoculum dispersal will be important for developing new management strategies that minimize host–pathogen contacts in potato and numerous other crops affected by pathogenic Pectobacterium and Dickeya species.     

Coincidence of maximum severity of powdery mildew on grape leaves and the carbohydrate sink‐to‐source transition

Grapevine leaves infected with powdery mildew are a source of inoculum for fruit infection. Leaves emerging on a single primary shoot of Vitis vinifera cv. Cabernet Sauvignon were exposed to average glasshouse temperatures of 18°C (0·23 leaves emerging/day) or 25°C (0·54 leaves emerging/day). All leaves on 8–10 shoots with approximately 20 leaves each were inoculated with Erysiphe necator conidia to assess disease severity after 14 days in the 25°C glasshouse. Two photosynthetic ‘source’ leaves per shoot on the remaining 8–10 shoots were treated with ¹⁴CO₂ to identify, by autoradiography, the leaf position completing the carbohydrate sink‐to‐source transition. There was a clear association between the mean modal leaf position for maximum severity of powdery mildew (position 3·7 for 18°C; position 4·4 for 25°C) and the mean position of the leaf completing the sink‐to‐source transition (position 3·8 for 18°C; position 4·7 for 25°C). The mean modal leaf position for the maximum percentage of conidia germinating to form secondary hyphae was 4·2 for additional plants grown in the 25°C glasshouse. A higher rate of leaf emergence resulted in a greater proportion of diseased leaves per shoot. A Bayesian model, consisting of component models for disease severity and leaf ontogenic resistance, had parameters representing the rate and magnitude of pathogen colonization that differed for shoots developing in different preinoculation environments. The results support the hypothesis that the population of leaves in a vineyard capable of supporting substantial pathogen colonization will vary according to conditions for shoot development.     

Effect of planting density of maize on the progress and spread of northern leaf blight fromExserohilum turcicum infested residue source

Effect of plant density (37,037, 44,444 and 55,555 plants/ha) on the increase of northern leaf blight in time and space on two maize cultivars planted at spacings of 90, 75 and 60 between rows and 30 cm within rows was investigated.Exserohilum turcicum infested maize residue was used as an inoculum source. Maize density did not significantly affect the disease indices, but significantly influenced the grain yield of the two cultivars. In contrast, the two cultivars differed significantly (P0.01) in disease severity, but not grain yield. Higher disease severities and grain yields were associated with higher plant densities, whereas the apparent infection rate was lower in higher plant density. Distance from the maize residue (inoculum source) significantly influenced disease severity. The percentage leaf area blighted, area under disease progress curve and disease progress curve intercept decreased with distance from the maize residue area, but the apparent infection rate on EV8429-SR appeared to increase with distance. Disease gradients (b) were higher in closely planted maize and flattened with time in one location only.     

A reconsideration of natural dispersal distances of the pine pathogen Dothistroma septosporum

The natural spread of Dothistroma septosporum, the causal agent of a foliar disease of pines, was investigated at three sites in the south of England using trap plants. The pathogen is considered to be primarily rain‐splash dispersed, but this study shows that it can be spread many hundreds of metres from an inoculum source, demonstrating that dispersal is not solely via rain splash. The maximum distance the pathogen was recorded from any infection source was in excess of 1400 m, over five times the distance defined in the only previous work of this kind. Consequently, a reassessment of forest and production nursery management practices is called for, as these assume that the pathogen only spreads naturally over limited distances. Detection of the pathogen on trap plants over 100 m from the inoculum source was, in most cases, only possible using quantitative real‐time PCR diagnosis. The entire diagnostic procedure, from DNA extraction to amplification, was able to detect a minimum of approximately 17 D. septosporum cells in a pine needle sample, assuming only a moderate DNA extraction efficiency of 30%.     

The effect of post‐harvest storage conditions on the development of black dot (Colletotrichum coccodes) on potato in crops grown for different durations

The effects of post‐harvest curing and storage temperature on severity of black dot, caused by Colletotrichum coccodes, were investigated for potato crops grown for different crop durations (days from 50% emergence to harvest) in soils that posed a low, medium and high risk of disease. In field trials over four growing seasons (2005–8), black dot severity at harvest increased with increasing crop duration, within the range 103–146 days from 50% emergence to harvest (P < 0.05). In field trials over three growing seasons (2006–8), black dot severity on tubers at harvest increased significantly with increasing soil inoculum in each year, within the range 43–4787 pg C. coccodes DNA/g soil (P < 0.05). Storage trials were conducted to measure the influence of accumulated post‐harvest temperature on black dot. In 2005, no difference in black dot severity was observed on tubers stored for 20 weeks at 2.5 and 3.5 °C. In 2006 (but not 2007), increasing the duration of curing after harvest from 4 to 14 days increased black dot severity on tubers from 8.9 to 11.2% (P < 0.01) in long duration crops (>131 days after 50% emergence) grown under high (>1000 pg C. coccodes DNA/g soil) soil inoculum. The number of days of curing did not affect disease severity for shorter duration crops grown at high soil inoculum, or on crops grown at medium or low (100–1000 and <100 pg C. coccodes DNA/g soil, respectively) soil inoculum concentrations. Soil inoculum and crop duration together provided a reasonable prediction of black dot severity at harvest and after a 20‐week storage period.     

Loop‐mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot (LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape (Brassica napus) crops in the UK. The pathogen is spread by both asexual splash‐dispersed conidia and sexual wind‐dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time‐consuming laboratory‐based processing. This study describes two loop‐mediated isothermal amplification (LAMP) assays, targeting internal transcribed spacer (ITS) or β‐tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species (B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, or other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30 minutes, although the β‐tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100 ng–10 pg), enabling quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the β‐tubulin sequence‐based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in parts of leaves either with or without visible LLS symptoms. The P. brassicae LAMP assays described here could have multiple applications, including detection of symptomless host infection and automated real‐time monitoring of pathogen inoculum.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Temporal dynamics of root and foliar severity of soybean sudden death syndrome at different inoculum densities

Temporal dynamics of soybean sudden death syndrome (SDS) root and foliar disease severity were studied in growth chamber experiments on susceptible plants exposed to different inoculum densities (0, 10⁰, 10¹, 10², and 10³ conidia g⁻¹ soil) of Fusarium virguliforme. The monomolecular model provided the best fit to describe the progress of root and foliar disease severity over time. Disease severity and area under disease progress curve (AUDPC) both increased in response to increasing inoculum density (P < 0.01), particularly for foliar symptoms. Rate of disease progress increased as inoculum densities increased for both root and foliar disease severities. The incubation period for root and foliar disease severity ranged from 9 to 18 and 15 to 25 days, respectively. Significant differences in root rot severity were most easily detected during the early stages of infection, whereas root rot and foliar severities were only weakly correlated when both were assessed simultaneously at later stages of disease development. Root rot severity assessments performed 15 to 20 days after inoculation (DAI) were most highly correlated (r > 0.9, P < 0.01) with foliar disease severity assessments performed 30 to 50 DAI. Root biomass was reduced by up to 67% at the three highest inoculum densities, indicating the aggressiveness that F. virguliforme possesses as a root rot pathogen on soybeans.     

Transmission of rice blast from seeds to adult plants in a non‐systemic way

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a serious threat to rice production worldwide. In temperate regions, where rice is not cultivated for several months each year, little is known about the initial onset of the disease in the field. The main overwintering and primary inoculum sources reported are infested residues and seeds, but the subsequent steps of the disease cycle are largely unknown, even though a systemic infection has been proposed but not demonstrated. The present work follows rice blast progression in infected seeds from germination to seedling stage, with direct and detailed microscopic observations under both aerobic conditions and water seeding. With the use of GFP‐marked M. oryzae strains, it was shown that spores are produced from contaminated seeds, infect emerging seedling tissues (coleoptile and primary root) and produce mycelium that colonizes the newly formed primary leaf and secondary roots. Using different rice cultivars exhibiting distinct levels of resistance/susceptibility to M. oryzae at the 2/4‐leaf stage, it was observed that resistance or susceptibility of a considered genotype is already established at the seedling stage. The results also showed that when plants are inoculated either at ripening stage (mature panicles), heading stage (flowering/immature panicles) or even before heading (flag leaf fully developed), they produce infested seeds. These seeds produce contaminated seedlings that mostly die and serve as an inoculum source for healthy neighbouring plants, which gradually develop disease symptoms on leaves. The possible rice blast disease cycle was reconstructed on irrigated rice in temperate regions.