Yield Reduction in Wheat in Relation to Leaf Disease From Yellow (tan) Spot and Septoria Nodorum Blotch

Yellow or tan spot (caused by Pyrenophora tritici-repentis) and septoria nodorum blotch (caused by Phaeosphaeria nodorum) occur together and are a constraint to wheat yields in Australia. Recently, higher crop yields and lower fungicide costs have made fungicides an attractive management tool against these diseases. Yield-loss under different rates of progress of yellow spot and septoria nodorum blotch was examined in four experiments over three years to define the relationship between disease severity and yield. In these experiments, differences in disease were first promoted by inoculations either with P. tritici-repentis-infected stubble or aqueous spore suspensions of P. nodorum. Disease progress was further manipulated with foliar application of fungicide. The pattern of disease development varied in each year under the influence of different rainfall patterns. The inoculation and fungicide treatments produced differences in disease levels after flag leaf emergence. The infection of yellow spot or septoria nodorum blotch caused similar losses in grain yield, ranging from 18% to 31%. The infection by either disease on the flag or penultimate leaf provided a good indication of yield-loss. Disease severity on flag leaves during the milk stage of the crop or an integration of disease as area under the disease progress curve on the flag leaves based on thermal time explained more than 80% variance in yield in a simple regression model. The data provided information towards the development of disease management strategies for the control of septoria nodorum blotch and yellow spot.     

SnToxA,SnTox1, and SnTox3 originated in Parastagonospora nodorum in the Fertile Crescent

The centre of origin of the globally distributed wheat pathogen Parastagonospora nodorum has remained uncertain because only a small number of isolates from the Fertile Crescent were included in earlier population genetic and phylogeographic studies. We isolated and genetically analysed 193 P. nodorum strains from three naturally infected wheat fields distributed across Iran using 11 neutral microsatellite loci. Compared to previous studies that included populations from North America, Europe, Africa, Australia, and China, the populations from Iran had the highest genetic diversity globally and also exhibited greater population structure over smaller spatial scales, patterns typically associated with the centre of origin of a species. Genes encoding the necrotrophic effectors SnToxA, SnTox1, and SnTox3 were found at a high frequency in the Iranian population. By sequencing 96 randomly chosen Iranian strains, we detected new alleles for all three effector genes. Analysis of allele diversity showed that all three effector genes had higher diversity in Iran than in any population included in previous studies, with Iran acting as a hub for the effector diversity that was found in other global populations. Taken together, these findings support the hypothesis that P. nodorum originated either within or nearby the Fertile Crescent with a genome that already encoded all three necrotrophic effectors during its emergence as a pathogen on wheat. Our findings also suggest that P. nodorum was the original source of the ToxA genes discovered in the wheat pathogens Phaeosphaeria avenaria f. sp. tritici 1, Pyrenophora tritici-repentis, and Bipolaris sorokiniana.     

Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead's Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2·5, 2·1 and 0·6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2·5 or 0·5 mm sulphate. Both pathogens infected less frequently at 2·5 mm sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4·5 at 2·5 mm sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance.     

Survey of cereal diseases and petts in the Netherlands. 5. Occurrence of Septoria spp. in winter wheat

Data of the annual surveys of circa 100 commerical winter wheat fields were compiled to describe epidemics ofSeptoria spp. in the Netherlands during 1974–1986. In May, during the first node stage,S. tritici was dominant whileS. nodorum was virtually absent. In July, during ripening,S. tritici on average dominated overS. nodorum, though in the most continental districts of the countryS. nodorum predominated.In May between 1974 and 1984, on average 56% of the fields showed leaf infections bySeptoria spp., while in July between 1975 and 1986, on average 83% of the fields showed leaf infections. Prevalence ofSeptoria spp. has increased during the surveys. Annual intensity ofSeptoria spp. in winter wheat crops was positively correlated with precipitation and negatively with average monthly sunshine duration during the harvest-month August of the previous growing season. The correlation with sunshine during August could indicate that ascospores play a major role in subsequent epidemics; but whether it is a causal relation remains to be answered.Present address: Center for Agrobiological Research (CABO-DLO), PO Box 14, 6700 AA Wageningen, the Netherlands     

Temperature Dependent Seed Transmission of Stagonospora nodorum in Wheat

The transmission of Stagonospora nodorum from four naturally infected winter wheat seedlots was quantified in controlled environment germination chambers at 9, 13, 17, 21, and 25 °C. Seedlings were harvested when the second leaf began to emerge. Coleoptiles and first seedling leaves were examined for the presence of lesions caused by S. nodorum. First leaves were incubated on Bannon's medium for 2 weeks, after which they were examined for pycnidia of S. nodorum. Transmission to the coleoptile occurred at all temperatures, but decreased from 100% to 72% as temperature increased from 9 to 25 °C. Transmission to the first leaf was less, dropping from 37% to 2% as temperature increased from 9 to 25 °C. At least 44% of infected first leaves were symptomless at all temperatures, with 96% of infected leaves showing no symptoms at 25 °C. Transmission to seedling leaves occurred over a broad temperature range. Under the high densities at which wheat is sown, a significant number of infected seedlings per unit area may originate from relatively low initial seed infection levels and transmission efficiencies.     

Molecular characterization of a Stagonospora nodorum lipase gene LIP1

A triglyceride lipase gene (LIP1) was cloned from Stagonospora nodorum, the causal agent of wheat glume blotch. LIP1 encodes a 561 amino acid preproprotein with a predicted N‐terminal signal peptide. Its expression was up‐regulated during plant infection and in culture media supplemented with saturated fatty glycerides. The recombinant Lip1 protein possessed lipolytic activity against a broad range of lipid substrates. When applied to wheat leaves, recombinant Lip1 decreased the hydrophobicity of the leaf surface, probably by liquefaction of epicuticular wax. Pretreatment of wheat leaves with Lip1 decreased the rate of conidial adhesion from 69·5% to 22·7% and from 58·9% to 28·4% in two independent assays based on different protocols. LIP1 replacement strains showed decreased lipolytic activity on culture media relative to the wild‐type strain, and adhesion of the conidia to the wheat leaf surface was impaired in the gene replacement strains. In two experiments, adhesion rates were 54·3% and 41·6% in the LIP1 replacement strains, as opposed to 77·7% and 66·6%, respectively, in the wild‐type. Collectively, the data demonstrate that the secreted lipase Lip1 is important for the adhesion of S. nodorum infection to wheat leaves.     

Control of Stagonospora nodorum and Septoria tritici in Wheat by Pre-treatment with Drechslera teres, a Non-host Pathogen

A field study is described which explored the possibility of controlling Stagonospora nodorum and Septoria tritici on wheat using a barley pathogen, Drechslera teres. Pre-treatment of wheat cv. Hussar flag leaves with D. teres resulted in a significant reduction in disease caused by S. nodorum and S. tritici, resulting in a significant increase in grain yield. When cv. Brigadier leaves were treated with D. teres prior to inoculation with S. nodorum there was an initial increase in disease expression whilst D. teres had no effect on symptoms produced by S. tritici on cv. Brigadier. There was significantly less disease on leaves of cvs. Hussar and Brigadier pre-treated with D. teres prior to inoculation with an equal mixture of S. nodorum and S. tritici compared to plants pre-treated with water. It is concluded that D. teres and other non-host pathogens show potential as biological control agents for S. nodorum and S. tritici.     

<Emphasis Type="Italic">Fusarium langsethiae</Emphasis> pathogenicity and aggressiveness towards oats and wheat in wounded and unwounded <Emphasis Type="Italic">in vitro</Emphasis> detached leaf assays

In vitro detached leaf assays involving artificial inoculation of wounded and unwounded oat and wheat leaves were used to investigate the potential pathogenicity and aggressiveness of F. langsethiae, which was linked recently to the production of type A trichothecenes, HT-2 and T-2 in cereals in Europe. In the first two experiments, two assays compared disease development by F. langsethiae with known fusarium head blight pathogen species each used as a composited inoculum (mixture of isolates) at 10°C and 20°C and found all fungal species to be pathogenic to oat and wheat leaves in the wounded leaf assay. In the unwounded leaf assay, F. langsethiae was not pathogenic to wheat leaves. Furthermore, there were highly significant differences in the aggressiveness of pathogens as measured by lesion length (P < 0.001). In the second two experiments, pathogenicity of individual F. langsethiae isolates previously used in the composite inoculum was investigated on three oat and three wheat varieties. The wounded leaf assay showed that all isolates were pathogenic to all oat and wheat varieties but only pathogenic towards oat varieties in the unwounded assay. Highly significant differences (P < 0.001) in lesion length were found between cereal varieties as well as between isolates in the wounded assay. Significant differences in lesion lengths (P = 0.014) were also observed between isolates in the unwounded assay. Results from the detached leaf assays suggest that F. langsethiae is a pathogen of wheat and oats and may have developed some host preference towards oats.     

黄龙病对柑橘叶际微生物组的影响

为明确柑橘叶际微生物组对黄龙病发生的响应规律，采用荧光定量PCR技术检测采集柑橘叶片感染黄龙病菌的情况，通过扩增子测序分析法对PCR检测结果为阴性和阳性叶片的微生物群落进行比较，并通过相关性分析和网络分析对黄龙病菌和叶际微生物的相关性进行解析。结果显示，夏季采集的柑橘叶片中黄龙病菌的检出率为7.5%，而到秋季黄龙病菌的检出率上升到32.3%。黄龙病的发生显著改变了柑橘叶际细菌群落的组成和结构，对于细菌类群的丰度也有一定影响；而对叶际真菌群落的组成和结构无显著影响。黄龙病菌与叶际细菌存在普遍的负相关关系，正相关关系相对较少但相关性更强。黄龙病菌可能通过与厌氧棍状菌Anaerotruncus sp.、梭菌Clostridiales sp.、假单胞菌Pseudomonas sp.、鞘脂单胞菌Sphingomonas sp.、毛螺菌Lachnospiraceae sp.和链球菌Streptococcus sp.的负向互作对柑橘叶际整个细菌群落产生负影响。表明黄龙病发生对柑橘叶际真菌和细菌群落的影响规律不同，同时黄龙病菌可能通过与几种主要细菌的负向互作来实现对叶际微生物组的影响。     

Effect of Fungicides on Septoria nodorum and Wheat Green Leaf Area1

Fungicides applied at different growth stages were compared for the control of wheat glume blotch, caused by Septoria nodorum (Berk.) Berk. In glasshouse and small-scale field tests, 11 commercially used fungicides, when applied to fully emerged wheat ears, cv. Hobbit, gave only partial control of ear infection by spores applied immediately after spraying. They gave better control of infection of leaves by a similar inoculum. Larger field trials, moreover, indicated better foliar and ear disease control from fungicide applications made before ear emergence, compared with those applied later. In another field trial, nine fungicides were applied to replicated plots of winter wheat, cultivars Atou and Hobbit at GS 31, GS 39, GS 55 or GS 71. On five occasions from anthesis (25 June) until 30 July disease was assessed and green leaf area measured. The trial was harvested on 22 August 1980. Significantly greater yields and grain weights were obtained from plots sprayed with fungicide at GS 39 than from those sprayed at any other time or left unsprayed. Treatments made before symptoms of infection by S. nodorum developed (i.e. before GS 55) prolonged the duration of green leaf areas during the post-anthesis period. Grain weight and yield increases were related closely to the duration of green leaf area above 75 96, but not to the amount of green leaf area at any single assessment nor to the subsequent degree of disease development. The data on which this abstract is based form part of a continuing research programme, the results of which will be published in full at a later date. Full details on the 1980 results referred to above appear in the Annual Report of Long Ashton Research Station (1980).     

Mating Relationships between Isolates of Phaeosphaeria nodorum, (anamorph Stagonospora nodorum) from Geographical Locations

Mating types of Phaeosphaeria nodorum isolates (from North Africa, North America, Australia, Europe and Near East) were determined in laboratory conditions. Although both mating types were found, MAT1-1 and MAT1-2 were not evenly distributed among the isolates. Sexual compatibility pairings with standard mating type testers revealed that 56 of 101 isolates could be assigned to MAT1-1 and 5 to MAT1-2. Although the teleomorph occurred in different countries, the two mating types were observed only among isolates collected from France, Great Britain, Germany and Morocco. For Morocco, where no P. nodorum pseudothecia have been reported, this is the first report of the existence of the two opposite mating types. The remaining 40 isolates could not be designated to a specific mating type. However, in 17 crosses, pseudothecial initials or sterile pseudothecia were observed on wheat straw after two months. The implications of the predominance of one mating type are discussed.     

Magnaporthe oryzae conidia on basal wheat leaves as a potential source of wheat blast inoculum

Wheat blast caused by Magnaporthe oryzae Triticum causes significant losses on wheat during outbreak years in several South American countries. Despite reports of wheat blast leaf lesions on some wheat cultivars, the importance of inoculum originating from leaves in severely affected commercial fields is disputed. It is generally considered that leaf lesions and/or sporulation on leaves do not usually appear before the occurrence of spike blast in wheat. The purpose of this study was to (i) determine the occurrence of wheat blast on basal leaves, (ii) estimate the number of conidia produced on these leaves, and (iii) determine the impact of current fungicide application practices on inoculum produced from sporulating lesions on basal wheat leaves. Inoculations at the three‐leaf stage showed that certain cultivar and isolate combinations caused more disease on old wheat leaves than young expanding leaves. Under optimum conditions, M. oryzae had the potential to produce tens to hundreds of thousands of conidia on small amounts of wheat basal leaves. A mean of 1 669 000 conidia were produced on 1 g dry basal leaves of a highly susceptible cultivar under optimum conditions for sporulation. Conidia production on leaves coincided with spike emergence under both greenhouse and field conditions. When field studies were conducted under natural epidemic conditions, foliar fungicide applications reduced the amount of M. oryzae conidia on basal leaves by 62–77% compared to non‐sprayed controls. An earlier application of foliar fungicides might reduce inoculum if conidia from basal leaves contribute to wheat spike blast development.     

Genetic linkage map of <Emphasis Type="Italic">Phaeosphaeria nodorum</Emphasis>, the causal agent of stagonospora nodorum blotch disease of wheat

A genetic linkage map of the fungal pathogen Phaeosphaeria nodorum, the causal agent of stagonospora nodorum blotch disease of wheat, was created. A total of 152 ascospore-derived progeny from a single pseudothecium, which resulted from a cross of two opposite mating type isolates, Sn37-1 and S-81-B13B, was analysed with AFLP, RAPD, ISSR, expressed sequence tag (EST)-derived microsatellite primers and sequence tagged site markers developed from specific genes. The genetic linkage map consisted of 276 molecular markers, and included markers developed from five genes [Glyceraldehyde 3-phosphate dehydrogenase (gpd), malate synthase (Mls1), mannitol 1-phosphate dehydrogenase (Mpd1), mating type (MAT1) and RNA polymerase II (RPB2)], which were assigned to 21 major linkage groups (LGs). The total length of the 21 major LGs was 1,932.1 centiMorgans (cM) with an average spacing of 6.88 cM between loci. The idiomorph mating type gene (MAT1) loci was placed in LG 2 and was closely linked to RAPD marker A4-680. On the other hand, 24 molecular markers and four gene loci [β-glucosidase (bgl1), histidinol dehydrogenase (Hdh2), mannitol 1-phosphate dehydrogenase (Mpd2), and xylanase (Xyl 10-2)] were dispersed in 11 minor LGs. The segregation ratio of the xylanase (Xyl 10-1) locus was distorted and not mapped. This is the first genetic linkage map reported for this important foliar pathogen of wheat. In combination with the genomic sequence of P. nodorum strain SN15 (), the availability of a genetic linkage map of this organism would be an important tool to investigate quantitative trait loci (QTL) of biologically important phenotypes and for positional cloning.     

Attachment and adhesion of conidia of Stagonospora nodorum to natural and artificial surfaces

Attachment and adhesion of conidia of a wheat-isolate of Stagonospora nodorum to leaf and artificial surfaces was studied. Attachment of conidia was a non-viable process, separate from adhesion, that occurred rapidly and irreversibly. Attachment involved conidial-surface carbohydrates and was partially influenced by surface hydrophobicity. The subsequent adhesion, via the secretion of extracellular matrix from conidia, was a viable process that induced the complete cover of conidia in response to wheat leaf surface components containing epi-cuticular wax and to a lesser extent to barley but inducing only partial covering on glass. Results suggest that specific surface components from the compatible host promote rapid attachment and adhesion of S. nodorum conidia.     

Frequency of Phaeosphaeria nodorum, the Sexual Stage of Stagonospora nodorum, on Winter Wheat in North Carolina

ABSTRACT Ascocarps of Phaeosphaeria nodorum, which causes Stagonospora nodorum blotch (SNB) of wheat, have not been found by others in the eastern United States despite extensive searches. We sampled tissues from living wheat plants or wheat debris in Kinston, NC, each month except June from May to October 2003. Additional wheat samples were gathered in Kinston, Salisbury, and Plymouth, NC, in 2004 and 2005. For the 3 years, in all, 2,781 fruiting bodies were dissected from the wheat tissues and examined microscopically. Fruiting bodies were tallied as P. nodorum pycnidia or ascocarps, "unknown" (not containing spores, potentially P. nodorum or other fungi), or "other fungi." In the 2003 sample, asco-carps of P. nodorum were present each month after May at a frequency of 0.8 to 5.4%, and comprised a significantly higher percentage of fruiting bodies from wheat spikes than of those from lower stems and leaves. Ascocarps also were found at frequencies <10% in some wheat debris samples from 2004 and 2005. Analysis of the nucleotide sequences of internally transcribed spacer regions of 18 genetically distinct North Carolina isolates from 2003 suggested that all were P. nodorum, not the morphologically similar P. avenaria f. sp. triticea. Neither the 18 isolates from 2003 nor a set of 77 isolates derived from 2004 Kinston leaf samples gave reason to suspect a mating-type imbalance in the larger P. nodorum population (P >/= 0.4). We conclude that, in North Carolina, sexual reproduction plays a role in initiation of SNB epidemics and the creation of adaptively useful genetic variability, although its relative importance in structuring this population is uncertain.     

Fungitoxicity of bromo- and iodo-3,5-dinitrobenzoates

Esters of 2-bromo-, 4-bromo-, 2-iodo- and 4-iodo- 3,5-dinitrobenzoic acids show considerable antifungal activity in spore germination tests against Alternaria brassicicola, Botrytis cinerea, Septoria nodorum and Uromyces fabae. They act as protectant fungicides against Erysiphe graminis, Puccinia recondita and Septoria nodorum on wheat and Botrytis fabae on broad bean. The most active compounds are methyl and ethyl 4-bromo-3,5-dinitrobenzoates which give better control of wheat rust than oxycarboxin and methyl 4-iodo-3,5-dinitrobenzoate which is at least as effective as captan against Septoria leaf spot on wheat. Several of the esters are superior to captan in controlling chocolate spot on broad bean.     

Hyperspectral signal decomposition and symptom detection of wheat rust disease at the leaf scale using pure fungal spore spectra as reference

This study establishes a method to detect and distinguish between brown rust and yellow rust on wheat leaves based on hyperspectral imaging at the leaf scale under controlled laboratory conditions. A major problem at this scale is the generation of representative and correctly labelled training data, as only mixed spectra comprising plant and fungal material are observed. For this purpose, the pure spectra of rust spores of Puccinia triticina and P. striiformis, causal agents of brown and yellow rust, respectively, were used to serve as a spectral fingerprint for the detection of a specific leaf rust disease. A least-squares factorization was used on hyperspectral images to unveil the presence of the spectral signal of rust spores in mixed spectra on wheat leaves. A quantification of yellow and brown rust, chlorosis and healthy tissue was verified in time series experiments on inoculated plants. The detection of fungal crop diseases by hyperspectral imaging was enabled without pixel-wise labelling at the leaf scale by using reference spectra from spore-scale observations. For the first time, this study shows an interpretable decomposition of the spectral reflectance mixture during pathogenesis. This novel approach will support a more sophisticated and precise detection of foliar diseases of wheat by hyperspectral imaging.     

Fungal communities in organically grown winter wheat affected by plant organ and development stage

The fungal community on the roots, stem bases, stems and grains of organically grown winter wheat was analysed using terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and sequencing of the ITS region. The changes in the composition of fungi in different plant parts and over time as well as interactions between fungi were also investigated. Among 58 fungal taxa found the most common were Davidiella macrospora, Cladosporium spp., Tetracladium maxilliforme, Didymella exitialis, Microdochium nivale and an unidentified species within Ascomycetes. Several potential wheat pathogens were found: Fusarium spp. including F. poae and G. avenacea (F. avenaceum), Microdochium nivale, Oculimacula yallundae, Parastagonospora nodorum and Zymoseptoria tritici and most of them were present on all plant parts. Plant part affected the most the fungal colonization of wheat as was shown both by multivariate analysis of the whole fungal community as well as the analysis based on the identified species. The composition of fungal communities in different parts changed during the growing season but no pattern common for the whole crop could be observed. The most dynamic and significant changes were found among yeasts. Both positive and negative significant interactions between pairwise combinations of pathogens were observed. Positive significant associations were also found between pathogens and other fungi.     

Antagonism of yeastlike phyllosphere fungi against Septoria nodorum on wheat leaves

Aureobasidium pullulans, Sporobolomyces roseus, andCryptococcus laurentii var.flavescens, added to the inoculum, reduced the superficial mycelial growth ofSeptoria nodorum and the infection of wheat leaves by 50% or more. The mycelial growth was affected similarly in vitro, on slides covered with water agar. The antagonistic effect on germination was slight. The concentration of the saprophytes on the leaves after inoculation was comparable to population densities occurring on field-grown wheat.Samenvatting Aureobasidium pullulans, Sporobolomyces roseus enCryptococcus laurentii var.flavescens toegevoegd aan een conidiënsuspensie vanSeptoria nodorum verminderden de oppervlakkige myceliumgroei vanSeptoria en de infectie, van de bladeren tot de helft of meer (Tabel 1). Het effect op de sporekieming was gering. In vitro, op zgn. agarglaasjes, werd de myceliumgroei op vergelijkbare wijze geremd (Tabel 2). De concentrative van de saprofyten na inoculatie kwam overeen met in het veld voorkomende populatiedichtheden (Tabel 3).     

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.