Preliminary Studies on Detection of Fusarium Basal Rot Infection in Onions and Shallots Using Electronic Nose.

The evaluation of crop health status and early disease detection are critical for implementing a fast response to a pathogen attack, managing crop infection, and minimizing the risk of disease spreading. Fusarium oxysporum f. sp. cepae, which causes fusarium basal rot disease, is considered one of the most harmful pathogens of onion and accounts for considerable crop losses annually. In this work, the capability of the PEN 3 electronic nose system to detect onion and shallot bulbs infected with F. oxysporum f. sp. cepae, to track the progression of fungal infection, and to discriminate between the varying proportions of infected onion bulbs was evaluated. To the best of our knowledge, this is a first report on successful application of an electronic nose to detect fungal infections in post-harvest onion and shallot bulbs. Sensor array responses combined with PCA provided a clear discrimination between non-infected and infected onion and shallot bulbs as well as differentiation between samples with varying proportions of infected bulbs. Classification models based on LDA, SVM, and k-NN algorithms successfully differentiate among various rates of infected bulbs in the samples with accuracy up to 96.9%. Therefore, the electronic nose was proved to be a potentially useful tool for rapid, non-destructive monitoring of the post-harvest crops.

Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens.

KEY MESSAGE: We demonstrate genetic variation for quantitative resistance against important fungal pathogens in lettuce and its wild relatives, map loci conferring resistance and predict key molecular mechanisms using transcriptome profiling. Lactuca sativa L. (lettuce) is an important leafy vegetable crop grown and consumed globally. Chemicals are routinely used to control major pathogens, including the causal agents of grey mould (Botrytis cinerea) and lettuce drop (Sclerotinia sclerotiorum). With increasing prevalence of pathogen resistance to fungicides and environmental concerns, there is an urgent need to identify sources of genetic resistance to B. cinerea and S. sclerotiorum in lettuce. We demonstrated genetic variation for quantitative resistance to B. cinerea and S. sclerotiorum in a set of 97 diverse lettuce and wild relative accessions, and between the parents of lettuce mapping populations. Transcriptome profiling across multiple lettuce accessions enabled us to identify genes with expression correlated with resistance, predicting the importance of post-transcriptional gene regulation in the lettuce defence response. We identified five genetic loci influencing quantitative resistance in a F6 mapping population derived from a Lactuca serriola (wild relative) × lettuce cross, which each explained 5-10% of the variation. Differential gene expression analysis between the parent lines, and integration of data on correlation of gene expression and resistance in the diversity set, highlighted potential causal genes underlying the quantitative trait loci.

Identification and Expression of Secreted In Xylem Pathogenicity Genes in Fusarium oxysporum f. sp. pisi.

Fusarium oxysporum is a soilborne fungal plant pathogen responsible for causing disease in many economically important crops with "special forms" (formae speciales) adapted to infect specific plant hosts. F. oxysporum f. sp. pisi (FOP) is the causal agent of Fusarium wilt disease of pea. It has been reported in every country where peas are grown commercially. Disease is generally controlled using resistant cultivars possessing single major gene resistance and therefore there is a constant risk of breakdown. The main aim of this work was to characterise F. oxysporum isolates collected from diseased peas in the United Kingdom as well as FOP isolates obtained from other researchers representing different races through sequencing of a housekeeping gene and the presence of Secreted In Xylem (SIX) genes, which have previously been associated with pathogenicity in other F. oxysporum f. spp. F. oxysporum isolates from diseased United Kingdom pea plants possessed none or just one or two known SIX genes with no consistent pattern of presence/absence, leading to the conclusion that they were foot-rot causing isolates rather than FOP. In contrast, FOP isolates had different complements of SIX genes with all those identified as race 1 containing SIX1, SIX6, SIX7, SIX9, SIX10, SIX11, SIX12, and SIX14. FOP isolates that were identified as belonging to race 2 through testing on differential pea cultivars, contained either SIX1, SIX6, SIX9, SIX13, SIX14 or SIX1, SIX6, SIX13. Significant upregulation of SIX genes was also observed in planta over the early stages of infection by different FOP races in pea roots. Race specific SIX gene profiling may therefore provide potential targets for molecular identification of FOP races but further research is needed to determine whether variation in complement of SIX genes in FOP race 2 isolates results in differences in virulence across a broader set of pea differential cultivars.

Assembly and characterisation of a unique onion diversity set identifies resistance to Fusarium basal rot and improved seedling vigour.

KEY MESSAGE: A unique, global onion diversity set was assembled, genotyped and phenotyped for beneficial traits. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers. Conserving biodiversity is critical for safeguarding future crop production. Onion (Allium cepa L.) is a globally important crop with a very large (16 Gb per 1C) genome which has not been sequenced. While onions are self-fertile, they suffer from severe inbreeding depression and as such are highly heterozygous as a result of out-crossing. Bulb formation is driven by daylength, and accessions are adapted to the local photoperiod. Onion seed is often directly sown in the field, and hence seedling establishment is a critical trait for production. Furthermore, onion yield losses regularly occur worldwide due to Fusarium basal rot caused by Fusarium oxysporum f. sp. cepae. A globally relevant onion diversity set, consisting of 10 half-sib families for each of 95 accessions, was assembled and genotyping carried out using 892 SNP markers. A moderate level of heterozygosity (30-35%) was observed, reflecting the outbreeding nature of the crop. Using inferred phylogenies, population structure and principal component analyses, most accessions grouped according to local daylength. A high level of intra-accession diversity was observed, but this was less than inter-accession diversity. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers, confirming the utility of the diversity set for discovering beneficial traits. The onion diversity set and associated trait data therefore provide a valuable resource for future germplasm selection and onion breeding.

Draft Genome Sequence of an Onion Basal Rot Isolate of Fusarium proliferatum.

Fusarium proliferatum is a component of the onion basal rot disease complex. We present an annotated F. proliferatum draft genome sequence, totaling 45.8 Mb in size, assembled into 597 contigs, with a predicted 15,418 genes. The genome contains 58 secondary metabolite clusters and homologs of the Fusarium oxysporum effector SIX2.

Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes.

The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

Basal Rot of Narcissus: Understanding Pathogenicity in Fusarium oxysporum f. sp. narcissi.

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae.

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

Shifts in diversification rates and host jump frequencies shaped the diversity of host range among Sclerotiniaceae fungal plant pathogens.

The range of hosts that a parasite can infect in nature is a trait determined by its own evolutionary history and that of its potential hosts. However, knowledge on host range diversity and evolution at the family level is often lacking. Here, we investigate host range variation and diversification trends within the Sclerotiniaceae, a family of Ascomycete fungi. Using a phylogenetic framework, we associate diversification rates, the frequency of host jump events and host range variation during the evolution of this family. Variations in diversification rate during the evolution of the Sclerotiniaceae define three major macro-evolutionary regimes with contrasted proportions of species infecting a broad range of hosts. Host-parasite cophylogenetic analyses pointed towards parasite radiation on distant hosts long after host speciation (host jump or duplication events) as the dominant mode of association with plants in the Sclerotiniaceae. The intermediate macro-evolutionary regime showed a low diversification rate, high frequency of duplication events and the highest proportion of broad host range species. Our findings suggest that the emergence of broad host range fungal pathogens results largely from host jumps, as previously reported for oomycete parasites, probably combined with low speciation rates. These results have important implications for our understanding of fungal parasites evolution and are of particular relevance for the durable management of disease epidemics.

Population Structure of Sclerotinia subarctica and Sclerotinia sclerotiorum in England, Scotland and Norway.

Sclerotinia species are important fungal pathogens of a wide range of crops and wild host plants. While the biology and population structure of Sclerotinia sclerotiorum has been well-studied, little information is available for the related species S. subarctica. In this study, Sclerotinia isolates were collected from different crop plants and the wild host Ranuculus ficaria (meadow buttercup) in England, Scotland, and Norway to determine the incidence of Sclerotinia subarctica and examine the population structure of this pathogen for the first time. Incidence was very low in England, comprising only 4.3% of isolates while moderate and high incidence of S. subarctica was identified in Scotland and Norway, comprising 18.3 and 48.0% of isolates respectively. Characterization with eight microsatellite markers identified 75 haplotypes within a total of 157 isolates over the three countries with a few haplotypes in Scotland and Norway sampled at a higher frequency than the rest across multiple locations and host plants. In total, eight microsatellite haplotypes were shared between Scotland and Norway while none were shared with England. Bayesian and principal component analyses revealed common ancestry and clustering of Scottish and Norwegian S. subarctica isolates while English isolates were assigned to a separate population cluster and exhibited low diversity indicative of isolation. Population structure was also examined for S. sclerotiorum isolates from England, Scotland, Norway, and Australia using microsatellite data, including some from a previous study in England. In total, 484 haplotypes were identified within 800 S. sclerotiorum isolates with just 15 shared between England and Scotland and none shared between any other countries. Bayesian and principal component analyses revealed a common ancestry and clustering of the English and Scottish isolates while Norwegian and Australian isolates were assigned to separate clusters. Furthermore, sequencing part of the intergenic spacer (IGS) region of the rRNA gene resulted in 26 IGS haplotypes within 870 S. sclerotiorum isolates, nine of which had not been previously identified and two of which were also widely distributed across different countries. S. subarctica therefore has a multiclonal population structure similar to S. sclerotiorum, but has a different ancestry and distribution across England, Scotland, and Norway.

Identification of pathogenicity-related genes in Fusarium oxysporum f. sp. cepae.

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non-pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non-pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.

Discrete lineages within Alternaria alternata species group: Identification using new highly variable loci and support from morphological characters.

The Alternaria alternata species group is ubiquitous in the environment acting as saprotrophs, human allergens, and plant pathogens. Many morphological species have been described within the group and it is unclear whether these represent re-descriptions of the same species or discrete evolutionary taxa. Sequencing of five loci identified three major lineages within the A. alternata species group. These loci included three new phylogenetic loci (TMA22, PGS1, and REV3) identified as highly variable based on publically available genome sequence data for Dothideomycete species. Lineages were identified as A. alternata ssp. arborescens, A. alternata ssp. tenuissima, and A. alternata ssp. gaisen in accordance with the placement of reference isolates. The phylogenetic results were supported by morphological analysis, which differentiated strains in A. alternata ssp. arborescens and A. alternata ssp. tenuissima and also aligned with previous morphological species descriptions for A. arborescens and A. tenuissima. However, phylogenetic analysis placed the morphologically described species A. alternata and A. mali within the A. alternata ssp. tenuissima and did not support them as discrete taxa. As A. alternata are of phytosanitary importance, the molecular loci used in this study offer new opportunities for molecular identification of isolates by national plant protection organizations.

Automatic detection of diseased tomato plants using thermal and stereo visible light images.

Accurate and timely detection of plant diseases can help mitigate the worldwide losses experienced by the horticulture and agriculture industries each year. Thermal imaging provides a fast and non-destructive way of scanning plants for diseased regions and has been used by various researchers to study the effect of disease on the thermal profile of a plant. However, thermal image of a plant affected by disease has been known to be affected by environmental conditions which include leaf angles and depth of the canopy areas accessible to the thermal imaging camera. In this paper, we combine thermal and visible light image data with depth information and develop a machine learning system to remotely detect plants infected with the tomato powdery mildew fungus Oidium neolycopersici. We extract a novel feature set from the image data using local and global statistics and show that by combining these with the depth information, we can considerably improve the accuracy of detection of the diseased plants. In addition, we show that our novel feature set is capable of identifying plants which were not originally inoculated with the fungus at the start of the experiment but which subsequently developed disease through natural transmission.

A model for Sclerotinia sclerotiorum infection and disease development in lettuce, based on the effects of temperature, relative humidity and ascospore density.

The plant pathogen Sclerotinia sclerotiorum can cause serious losses on lettuce crops worldwide and as for most other susceptible crops, control relies on the application of fungicides, which target airborne ascospores. However, the efficacy of this approach depends on accurate timing of these sprays, which could be improved by an understanding of the environmental conditions that are conducive to infection. A mathematical model for S. sclerotiorum infection and disease development on lettuce is presented here for the first time, based on quantifying the effects of temperature, relative humidity (RH) and ascospore density in multiple controlled environment experiments. It was observed that disease can develop on lettuce plants inoculated with dry ascospores in the absence of apparent leaf wetness (required for spore germination). To explain this, the model conceptualises an infection court area containing microsites (in leaf axils and close to the stem base) where conditions are conducive to infection, the size of which is modified by ambient RH. The model indicated that minimum, maximum and optimum temperatures for ascospore germination were 0.0, 29.9 and 21.7°C respectively and that maximum rates of disease development occurred at spore densities >87 spores cm-2. Disease development was much more rapid at 80-100% RH at 20°C, compared to 50-70% RH and resulted in a greater proportion of lettuce plants infected. Disease development was also more rapid at 15-27°C compared to 5-10°C (85% RH). The model was validated by a further series of independent controlled environment experiments where both RH and temperature were varied and generally simulated the pattern of disease development well. The implications of the results in terms of Sclerotinia disease forecasting are discussed.

Development of modelling method selection tool for health services management: from problem structuring methods to modelling and simulation methods.

BACKGROUND: There is an increasing recognition that modelling and simulation can assist in the process of designing health care policies, strategies and operations. However, the current use is limited and answers to questions such as what methods to use and when remain somewhat underdeveloped. AIM: The aim of this study is to provide a mechanism for decision makers in health services planning and management to compare a broad range of modelling and simulation methods so that they can better select and use them or better commission relevant modelling and simulation work. METHODS: This paper proposes a modelling and simulation method comparison and selection tool developed from a comprehensive literature review, the research team's extensive expertise and inputs from potential users. Twenty-eight different methods were identified, characterised by their relevance to different application areas, project life cycle stages, types of output and levels of insight, and four input resources required (time, money, knowledge and data). RESULTS: The characterisation is presented in matrix forms to allow quick comparison and selection. This paper also highlights significant knowledge gaps in the existing literature when assessing the applicability of particular approaches to health services management, where modelling and simulation skills are scarce let alone money and time. CONCLUSIONS: A modelling and simulation method comparison and selection tool is developed to assist with the selection of methods appropriate to supporting specific decision making processes. In particular it addresses the issue of which method is most appropriate to which specific health services management problem, what the user might expect to be obtained from the method, and what is required to use the method. In summary, we believe the tool adds value to the scarce existing literature on methods comparison and selection.

Forecasting Sclerotinia Disease on Lettuce: A Predictive Model for Carpogenic Germination of Sclerotinia sclerotiorum Sclerotia.

ABSTRACT A predictive model for production of apothecia by carpogenic germination of sclerotia is presented for Sclerotinia sclerotiorum. The model is based on the assumption that a conditioning phase must be completed before a subsequent germination phase can occur. Experiments involving transfer of sclerotia from one temperature regime to another allowed temperature-dependent rates to be derived for conditioning and germination for two S. sclerotiorum isolates. Although the response of each isolate to temperature was slightly different, sclerotia were fully conditioned after 2 to 6 days at 5 degrees C in soil but took up to 80 days at 15 degrees C. Subsequent germination took more than 200 days at 5 degrees C and 33 to 52 days at 20 degrees C. Upper temperature thresholds for conditioning and germination were 20 and 25 degrees C, respectively. A predictive model for production of apothecia derived from these data was successful in simulating the germination of multiple burials of sclerotia in the field when a soil water potential threshold of between -4.0 and -12.25 kilopascals (kPa) was imposed. The use of a germination model as part of a disease forecasting system for Sclerotinia disease in lettuce is discussed.

Development of MILIONCAST, an Improved Model for Predicting Downy Mildew Sporulation on Onions.

The effects of temperature and relative humidity on Peronospora destructor sporulation on onion (Allium cepa) leaves were studied under controlled environmental conditions. Sporangia were produced most rapidly at 8 to 12°C after 5 h of high humidity during dark periods. The greatest number of sporangia was produced at 100% relative humidity (RH), and sporulation decreased to almost nil when humidity decreased to 93% RH. A model, named MILIONCAST (an acronym for MILdew on onION foreCAST), was developed based on the data from these controlled environment studies to predict the rate of sporulation in relation to temperature and relative humidity. The accuracy of prediction of sporulation was evaluated by comparing predictions with observations of sporulation on infected plants in pots outdoors. The accuracy of MILIONCAST was compared with the accuracy of existing models based on DOWNCAST. MILIONCAST gave more correct predictions of sporulation than the DOWNCAST models and a random model. All models based on DOWNCAST were more accurate than the random model when compared on the basis of all predictions (including positive and negative predictions), but they gave fewer correct predictions of sporulation than the random model. De Visser's DOWNCAST and ONIMIL improved their accuracy of prediction of sporulation events when the threshold humidity for sporulation was reduced to 92% RH. The temporal pattern of predicted sporulation by MILIONCAST generally corresponded well to the pattern of sporulation observed on the outdoor potted plants at Wellesbourne, UK.

Forecasting sclerotinia disease on lettuce: toward developing a prediction model for carpogenic germination of sclerotia.

ABSTRACT The feasibility of developing a forecasting system for carpogenic germination of Sclerotinia sclerotiorum sclerotia was investigated in the laboratory by determining key relationships among temperature, soil water potential, and carpogenic germination for sclerotia of two S. sclerotiorum isolates. Germination of multiple burials of sclerotia to produce apothecia also was assessed in the field with concurrent recording of environmental data to examine patterns of germination under different fluctuating conditions. Carpogenic germination of sclerotia occurred between 5 and 25 degrees C but only for soil water potentials of >/=-100 kPa for both S. sclerotiorum isolates. Little or no germination occurred at 26 or 29 degrees C. At optimum temperatures of 15 to 20 degrees C, sclerotia buried in soil and placed in illuminated growth cabinets produced stipes after 20 to 27 days and apothecia after 27 to 34 days. Temperature, therefore, had a significant effect on both the rate of germination of sclerotia and the final number germinated. Rate of germination was correlated positively with temperature and final number of sclerotia germinated was related to temperature according to a probit model. Thermal time analysis of field data with constraints for temperature and water potential showed that the mean degree days to 10% germination of sclerotia in 2000 and 2001 was 285 and 279, respecttively, and generally was a good predictor of the observed appearance of apothecia. Neither thermal time nor relationships established in the laboratory could account for a decline in final percentage of germination for sclerotia buried from mid-May compared with earlier burials. Exposure to high temperatures may explain this effect. This, and other factors, require investigation before relationships derived in the laboratory or thermal time can be incorporated into a forecasting system for carpogenic germination.

Ascospore release and survival in Sclerotinia sclerotiorum.

The release and survival of ascospores of a UK Sclerotinia sclerotiorum isolate were studied. Apothecia placed in a spore clock apparatus with different lighting regimes at 15 degrees C released ascospores continuously with an increasing rate for the duration of experiments (72-84 h). Spore release was not confined to light or dark periods in alternating regimes and occurred in continuous dark or light. Ascospores were released in both saturated air (90-95% rh) and at 65-75% rh. High temperature and rh were detrimental to ascospore survival but spore viability was maintained for longer periods than previously reported. The significance of these results in relation to disease control is discussed.