Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants: Quantitative PCR and ELISA Accurately Determine Fusarium Biomass

Fungi of the genus Fusarium are worldwide occurring plant pathogens which cause severe damages to numerous cultivable plants (Weiland et al., 2000; Mirete et al., 2004; Youssef et al., 2007; Li et al., 2008) with the highest economical losses upon infection of maize, wheat and barley (Windels et al., 2000; Nganje et al., 2004). Fusarium caused diseases can destroy crops within several weeks and the infection leads to quality losses in two different aspects: besides the reduced yield due to reduced kernel size, the fungus produces various toxic metabolites while colonizing the plant. These mycotoxins heavily impair the quality of the harvest (McMullen et al., 1997). The acute or chronic toxicity of Fusarium released compounds led to the introduction of national or international limits to regulate mycotoxin levels in food and feed (e.g. limits of the European Community since 2006).


Introduction
Fungi of the genus Fusarium are worldwide occurring plant pathogens which cause severe damages to numerous cultivable plants (Weiland et al., 2000;Mirete et al., 2004;Youssef et al., 2007;Li et al., 2008) with the highest economical losses upon infection of maize, wheat and barley (Windels et al., 2000;Nganje et al., 2004).Fusarium caused diseases can destroy crops within several weeks and the infection leads to quality losses in two different aspects: besides the reduced yield due to reduced kernel size, the fungus produces various toxic metabolites while colonizing the plant.These mycotoxins heavily impair the quality of the harvest (McMullen et al., 1997).The acute or chronic toxicity of Fusarium released compounds led to the introduction of national or international limits to regulate mycotoxin levels in food and feed (e.g.limits of the European Community since 2006).
Most Fusarium species are widely distributed in substrates such as soil, on subterranean and aerial plant parts, plant debris, and on dead organic matter.Many Fusarium species have active or passive means to disperse spores or conidia in the atmosphere.The ability to grow on a broad range of substrates combined with their efficient dispersal mechanisms enables a widespread distribution of these fungi (Burgess, 1981).Fusarium species are well adapted to grass hosts (Leonard & Bushnell, 2003) and can colonize on many agricultural commodities such as rice, bean and soybean.According to several studies F. graminearum and F. culmorum are among the most aggressive plant pathogenic fungi known.F. graminearum mainly occurs in temperate and warmer regions of the USA, China and the southern hemisphere (Osborne & Stein, 2007).It is regarded as one of the most vigorous toxin producers and has therefore become the most intensively studied plant pathogen (Goswami & Kistler, 2004).In contrast, F. culmorum predominates in the cooler regions including the U.K., Northern Europe and Canada (Osborne & Stein, 2007) and is also associated with the occurrence of many mycotoxins (Desjardins, 2006).

Toxigenicity and impacts
A worldwide problem of agricultural industry producing wheat, barley and maize is the fungal disease Fusarium head blight (FHB), with maize and wheat as the economically most www.intechopen.comPlant Pathology 204 important host plants.The disease is associated with F. graminearum, F. culmorum, F. poae, F. avenaceum, F. sporotrichoides and Microdochium nivale.Each fungus contributing to FHB has particular biological and environmental requirements, which in part explains the frequency of occurrence in specific locations.This destructive disease was initially described in 1884 in England and was considered a major threat during the early years of the twentieth century (Goswami & Kistler, 2004).Nowadays, Fusarium head blight is a disease of massive economic impact worldwide and has been ranked by the United States Department of Agriculture (USDA) as the worst plant disease to hit the nation (Windels, 2000).The losses due to direct and secondary economic impact of FHB on wheat and barley were estimated at $2.7 billion for the period from 1998 -2000 in the US (Nganje et al., 2002).Epidemics of FHB are strongly influenced by various factors such as local and regional environment, the physiological state and genetic background of the host, as well as pathogen related factors including host adaptation and virulence.
Infected plant debris serves as primary substrate for common FHB pathogens, which are able to survive the winter period as saprophytic mycelium or thick walled chlamydospores.Warm, moist conditions in spring are favorable for the development and maturation of conidia and perithecia, which produce ascospores (Markell & Frankl, 2002).These sticky spores are discharged from the surface of crop debris and dispersed by wind, rain or even insects to the host plants.Infected wheat or maize presents brown, dark purple to black lesions on the exterior.The head of cereals has a characteristically bleached appearance, hence the name Fusarium head blight.During prolonged wet periods infected spikelets, glumes and kernels present pink to salmon-orange spore masses of the fungus.Maize infections occur at the ear apex following colonization of the kernels with white mycelium, which turns pink to red with time.
Fungal colonization is affected by numerous variables such as spike morphology, canopy density, plant height, rainfall, relative humidity, temperature and host plant resistance (Kolb et al., 2001;Rudd et al., 2001).Fusarium infections are increased due to the lack of completely resistant plant material and a frequent application of unsuitable cropping systems.Pathogen survival is favored in reduced tillage systems as residue burial speeds up the decomposition and reduces pathogen reproduction and survival (Khonga et al., 1988;Pereyra et al., 2004).Petcu et al., (1998) investigated the influence of crop rotations on the severity of FHB.Studies show that the economically most lucrative cultivation of alternating maize and wheat cultures turned out to be problematic.The increased production of these Fusarium favorable crops and the dramatic increase in their residues remaining on the soil surface provide a large increase in the amount of niche available to the pathogen.Fusarium non-host plants used as preceding crops or intertillage of wheat and maize are often less profitable and therefore of low interest for farmers.In North and South America erosion causes a dramatic loss of fertile topsoil.To avoid this loss, no-till systems have been established to overcome the drawback of conventional farming.However, Fusarium inoculum density increases in soil in reduced tillage systems compared to plough treated fields (Steinkellner & Langer, 2004).

Plant resistance to Fusarium
Plant resistance can be defined as the relative amount of heritable qualities possessed by a plant that reduces the degree of damage to the plant caused by pathogens.Schroeder and Christensen (Schroeder & Christensen, 1963) described in their work two phenotypic measures of disease resistance; type I resistance operates against initial infection and type II resistance describes the resistance to spreading within infected tissues.A third type of Fusarium resistance (type III) was described by Wang and Miller (1988) as the insensitivity of wheat lines to toxins or the ability of the resistant cultivar to degrade mycotoxins.
Integrated control strategies are essential to prevent Fusarium d i s e a s e s i n m o d e r n agriculture.The use of resistant cultivars is considered necessary for numerous reasons.Firstly, modern agronomic practices such as reduced tillage and cereal and maize rich crop rotations have the tendency to increase the amount of Fusarium related diseases.In addition to this, chemical control measures are only partly effective as only few fungicides are sufficiently active against Fusarium and their application must be performed within a time frame of a few days around the flowering stage of the plants.For the protection of maize cultures no fungicides are commercially available up to now.Understanding Fusarium lifecycle and infection pathways is the first step in preventing disease.Nevertheless, uncontrollable factors such as weather conditions during as well as economic or ecologic interests prevent sustainable success in the reduction of these pathogens.Plant breeders have made significant progress in the development of Fusarium resistant maize and wheat varieties by identifying genetic regions, which are linked to the resistance of plants (Buerstmayer et al., 2003;Draeger et al., 2007).Resistance against Fusarium is a quantitative trait, which is governed by many independent genes distributed on several distinct genetic regions (quantitative trait loci, QTLs) in the plant genome.Therefore an effective approach to investigate FHB resistance is the identification of the QTLs and their mapping (Buerstmayer et al., 2009).The mapping of QTLs is the basis for further efforts towards a straightforward marker assisted resistance breeding strategy.Interestingly, often the best regionally adapted and highly productive crop varieties are susceptible to FHB.For this reason, breeders are faced with the difficult task to combine adaption to certain locations with high yield and Fusarium resistance.
The production of mycotoxins has been identified as a crucial factor for successful infection .Deoxynivalenol is considered the most common occurring mycotoxin involving Fusarium infection.However, DON levels in infected grain vary significantly amongst wheat cultivars (Bai et al., 2001) infected with the same Fusarium isolate.The evaluation of Fusarium resistance of either the parents in current breeding programs or the control of resistance levels of novel lines can be carried out by chemical analysis of the DON accumulation in the seeds.In general DON levels in resistant plants are much lower than those found in more susceptible cultivars.Artificial infection of wheat-ears with Fusarium conidia followed by determination of DON content and visual scoring of the head blight symptoms are the currently established methods for resistance assessment.Nevertheless, the determinations of disease symptoms in combination with the mycotoxin content of grain are both indirect methods to evaluate plant resistance.Furthermore, both methods observe the same effect, as visual disease symptoms are mainly caused by mycotoxin intoxication and not by the growth of mycelium.Studies have shown that the amount of fungal mycelia formed during infection not always correlates well with these parameters (Waalwijk et al, 2004;Hill et al., 2006;Brunner et al., 2009;Kulik et al. 2011).Asymptomatic kernels may contain significant amounts of mycotoxins while symptomatic kernels within the same sample may not.

Methods for the identification of Fusarium and quantification of the infection
Quantification and identification of Fusarium species in agriculture or plant pathology is traditionally carried out using culture based methods.Usually single Kernels are spread on petri dishes with appropriate medium and the number of infected kernels is counted after several days.For a further classification of the species a morphological investigation of the grown mycelium is necessary.This kind of species determination is time consuming and requires experience and specific expertise.Furthermore, culture based methods rely on living propagules and the obtained results not always reflects the biological situation.The pathogen detection is only reliable at late stages of the infection when a spread of the disease can no longer be controlled by fungicides (McCArtney et al., 2003).Besides these drawbacks the results are only semi-quantitative as only the number of infected kernels can be determined but not the grade of infection of each kernel.Other conventional methods include instrumental analysis such as ergosterol quantification.Ergosterol is a characteristic compound of fungal cell walls.However this approach is not specific to pathogens or a certain fungal species and the ergosterol amount relative to the fungal mycelia varies within Fusarium species (Pasanen et al., 1999).Finally, the analytical determination of ergosterol is not easier than that of DON, resulting in an alternative method, which is not commercially applicable to determine the Fusarium resistance of plants.
Recent studies aim at developing more direct techniques to quantify Fusarium related diseases which can i) reduce the number of analyses to accurately assess Fusarium infection, ii) eliminate pleiotropy between disease symptomology and mycotoxin production, iii) reduce the error associated with environmental effects and iv) reduce errors related with asymptomatic expression of FHB (Hill et al., 2008).In general, two distinct approaches have become accepted within the last two decades: immunoassays and (quantitative) PCR.The enzyme linked immunosorbent assay (ELISA) has been developed as a direct measure of Fusarium spp.biomass in infested grain samples or plant tissue.ELISA provides at least genus specificity through specific fungal antigens and its ease of sample preparation and low costs make it an interesting novel method.Another approach to overcome the drawbacks of conventional identifications is the development of screening techniques based on DNA identification which are nowadays well established for Fusarium species and a broad range of available literature guarantees to find a suitable PCR assay for many applications.Unlike conventional detection methods, samples can be tested directly with ELISA or PCR -no elaborate isolation and cultivation steps are necessary for a suitable detection or quantification.

Enzyme linked immunosorbent assays (ELISAs)
Enzyme linked immunosorbent assays are based on the specific recognition capabilities of antibodies.These antibodies are usually derived from the immunization of animals (usually rabbits, mice, chicken or goat) with certain immunogens such as culture filtrates or mycelial compounds.After repeated injection of the immunogen blood samples are taken and the serum is used either as a whole or it is applied after certain clean-up steps for the ELISA tests.For the production of the well-defined monoclonal antibodies lymphocytes are isolated from immunized mice and are fused to myeloma cells.The resultant clonal hybridoma cells can be maintained in culture.Soon after the development of the ELISA method in the early 1970s scientists working on agricultural and plant pathology related topics recognized the potential of this analytical tool.Within only a few years the number of annual ELISA based publications in the AGRICOLA database rose from zero to more than 150 and numerous antibodies were developed to detect plant pathogens belonging to different genera.

ELISAs based methods for the detection and quantification of Fusarium
The first immuno assay for Fusarium was developed at the University of Göttingen in 1989 by J.G. Unger and published in his PhD thesis.The author succeeded in the recognition of F. culmorum exoantigens (compounds secreted by Fusarium during growth) by an antibody.However, after this first success of the ELISA in Fusarium detection almost 15 years passed by until this F. culmorum ELISA was applied to practical applications (Chala et al., 2003).During these field tests the authors found out that the developed antibody is not specific to F. culmorum but binds to antigens from various Fusarium species.Over the years numerous studies on the development of anti-Fusarium antibodies have been published.Although most authors used F. graminearum and/or F. culmorum for immunization, no group succeeded in the production of highly specific antibodies.Gan et al., (1997) immunized chicken with soluble exoantigens and with soluble frations from homogenized mycelia of F. graminarum, F. poae and F. sporotrichioides.The F. graminearum and F. sporotrichioides exoantigen antibodies showed cross-reactivities with almost all common Fusarium species.The antibody against F. poae exoantigens showed absolutely no cross-reactivity against other Fusarium species or against other filamentous fungi.Up to now this is the only speciesspecific antibody for a Fusarium spp.Hill et al., (2006) probably produced the bestcharacterized antibody so far by isolating monoclonal antibodies produced by IF8 cell lines for the detection of Fusarium.These antibodies detected species from different phylogenetic clades and the authors also demonstrated that no cross-reaction -even with closely related ascomycetes -could be observed.Unfortunately, no publication describes the essential analytical parameters like limit of detection, limit of quantification or a linear range of the developed tests.
In general, antibodies detect many different Fusarium species but the specificity of PCR based assays has not yet been obtained by immunoassays.Neither the detection of particular species -except one example for F. poae -nor the detection of a particular toxigenic group (e.g.trichothecene producers or fumonisin producers) is possible with ELISA methods.Furthermore, most antibodies were raised against unknown compounds secreted by Fusarium during growth in liquid medium (exoantigens).As usually the secretion is not a constant attribute but is subjected to complex regulation, the amount of exoantigens secreted during the infection of grains might not always be constant.For this reason, the amount of Fusarium antigen detected in a sample may not always correlate with the fungal biomass.

Applications of Fusarium ELISAs for agriculture and plant pathology
Many of the developed PCR based tests for the determination of Fusarium focus on the screening of harvested grain and frequently the correlation of Fusarium DNA content and toxin accumulation is the central topic.Only few (references) of the many Fusarium PCR publications deal with plant pathology related questions whereas all developed ELISA methods were extensively used in field applications to monitor the efficacy of fungicide www.intechopen.comPlant Pathology 208 application (Chala et al., 2003) or to assess the Fusarium resistance of crop lines (Miedaner et al., 1994, Miedaner et al., 2004, Hill et al., 2006, Slikova 2009, Rohde & Rabenstein 2005).
4.1.2.1 Evaluation of fungicide efficacy with a Fusarium ELISA Chala et al., (2003) presented a comprehensive study on the evaluation of fungicide application to reduce Fusarium infection and DON content in wheat.Winter wheat was planted in four field replications and artificially infected with F. culmorum conidia at flowering stage by spray inoculation.Five different fungicides were tested at different application times alone and in various combinations.The efficiency of the treatment was determined by visual scoring of disease symptoms, Fusarium ELISA, yield a 1000-grain weight.The combination of these different methods allows the evaluation of the results obtained by the immunoassay.The results of the field trials reveal clearly that only one fungicide showed a high efficiency in the reduction of fungal biomass and DON content.All applied methods led to the same conclusion.Interestingly, the application of some fungicides at a later growth stage after flowering did not affect the fungal biomass but slightly increased the production of DON.In general the study demonstrates clearly the potential of the developed Fusarium ELISA to obtain a deeper insight into biomass formation.The main advantage of the ELISA based determination of infection is the enhanced failure-safety.

Evaluation of Fusarium resistance with ELISA tests
The comparison of the resistance of different plant varieties is often a challenge for breeders.The increase in Fusarium resistance of novel crop lines is just marginal and for some plants like durum or triticale disease symptoms are usually not obvious.Furthermore, other fungal diseases like Microdochium nivale or Septoria nodurum also lead to similar symptoms as Fusarium head blight and the result of the visual assessment might be misinterpreted.Besides the PCR based methods described above the ELISA tests represent the only available tool to determine fungal biomass and so the application for resistance tests is self-evident.
The first field test for Fusarium resistance based on ELISA analysis was performed in the early 1990s (Miedaner et al., 1994).Winter rye was infected with F. culmorum colonized wheat flour in November when the plants have reached the three-leaf status.The colonization of the host plant started early after inoculation and increased continuously till full maturity.Interestingly, different genotypes showed the highest variance in Fusarium protein content during medium growth states.In adult states almost no difference in Fusarium infection between resistant and susceptible plants was found.The disappearance of the discrepancy results in saprophytic growth of F. culmorum on rye during ripening and therefore the medium growth stages are the optimal date to discriminate resistant from susceptible varieties.The same group also investigated the F. culmorum resistance of wheat and triticale cultivars in six different environments and demonstrated the performance of ELISA biomass determination for two more cereal crops (Miedaner et al., 2004).However, the correlation of the ELISA based Fusarium protein content to the DON content in a sample was not better than the correlation between DON and visual disease symptom assessment.Another ELISA test based on monoclonal antibodies was optimized for the quantification of Fusarium in barley (Hill et al., 2006) and was used to study the influence of the applied analytical method on the results of resistance tests (Hill et al., 2008).A mapping population was grown in two environments and breeding lines were grown at four different locations and disease data were collected by visual disease assessment, determination of the DON accumulation and the Fusarium ELISA.The obtained data were subjected to statistical analyses and the authors calculated a model for the prediction of different replications and different environments necessary to certainly identify differences in Fusarium resistances.Interestingly, the determination of DON was identified as the factor that is least feasible for resistance studies.The quantification of the Fusarium biomass by ELISA required less different locations and fewer field replicates per location to classify barley varieties according to their Fusarium resistance than visual disease assessment or toxin measurements.
Often the correlation between visual disease scoring and DON content is better than between ELISA results and DON content.This fact has also been demonstrated in a Slovakian study (Slikova et al., 2009) for the infection of winter wheat with F. culmorum.As visual assessment just records the damage caused by the fungal toxins the correlation between symptoms and the DON content is obvious.The fact that visual symptoms and DON content are not independent features makes the combined use of only these two factors for resistance determination questionable.Therefore, Slikova et al. ( 2009) recommend the application of the Fusarium ELSIA to get more reliable results for plant resistance than with the two other methods.

Detection and quantification of Fusarium by PCR methods
The extensive application of DNA based identification technologies has increased the knowledge on suitable diagnostic DNA fragments of Fusarium species such as ITS (internal transcribes spacer) or IGS (intergenic spacer) sequences (Gagkaeva & Yli-Mattila, 2004, Jurado et al., 2006, Konstantinova & Yli-Mattila, 2004, Kulik 2008, Yli-Mattila, 2004), mitochondrial DNA (Laday et al., 2004), the -tubulin encoding gene (Yli-Mattila, 2004, Mach et al., 2004, Reischer et al., 2004), the translation elongation factor gene (Knutsen et al., 2004) and the calmodulin gene (Mule et al., 2004) which were sequenced from numerous Fusarium spp.As a result highly specific PCR primers could be developed for Fusarium detection.In order to distinguish between producers and non-producers of certain toxins the genes from biosynthesis pathways for mycotoxins were accurately studied.The sequence information gained throughout numerous molecular taxonomic investigations allows not only qualitative applications to detect particular species but also quantitative measurement of fungal biomass.Different techniques like DGGE, RFLP and AFLP can be applied for the identification of adequate PCR targets for a review see Brunner & Mach, (2010), for quantitative detection methods however, only the real-time PCR has been shown to be applicable.Molecular diagnosis of plant pathogenic fungi has been proven to be highly specific, very sensitive and fast and relatively insensitive to microbial backgrounds and nontarget organisms (McCartney et al., 2003).
Within the last decade numerous PCR based assays have been published for the detection of most agriculturally important Fusarium species.An intensive literature study reveals that all such publications can be separated into two distinct groups.Either the authors focus on highly specific test systems to allow the differentiation of Fusarium species or the focus is the comprehensive detection of a whole group sharing a common feature.The latter assays usually use a key-gene for a biosynthetic pathway -e.g. for mycotoxin synthesis -as a target for PCR.This system allows the detection or even the quantification of all isolates, which are able to produce a certain class of toxins even if they belong to different species or to different genera.

Species-specific PCR assays
Species-specific assays allow the determination of one particular Fusarium species.This method focuses on high selectivity in order to quantify a target species even in a background of highly similar isolates.Several TaqMan based PCR assays have been developed for the quantification of some of the predominant species associated with head blight in Europe including F. graminearum, F. poae, F. culmorum, F. sporotrichioides, F. verticillioides or F. avenaceum .The authors clearly demonstrated the advantage of quantitative real time PCR systems combined with a high-throughput DNA extraction protocol over morphologic based methods.Unlike conventional agar-plating techniques, the quantification of different fungal species is possible directly from infested material (grain or plant tissue) and the entire procedure is less time consuming than microbiological methods (for a review see Brunner & Mach, 2010).

Group specific assays
Group specific assays were designed for the simultaneous quantification of all strains, which produce certain toxins such as trichothecenes, fumonisins or enniatin, based on key-genes involved in the mycotoxin biosynthesis.In contrast to the species-specific assays, a whole group of Fusarium spp., regardless of taxonomic origin, that is able to produce a certain class of mycotoxins can be quantified in a single run.A correlation between qPCR determined fungal biomass and DON, fumonisin or enniatin content in cereals could be shown using these group specific assays.

Applications of the Fusarium PCR in agriculture and plant pathology
In contrast to the practical applications of the developed ELISA tests, the PCR assays are only rarely used to address typical questions related to plant pathology.Some methods were developed to identify the chemotype of different Fusarium strains on a molecular basis.These assays are designed to differentiate between nivalenol, 3-and 15acetyl-deoxynivalenol chemotypes of F. graminearum, F. culmorum, and F. cerealis.The biosynthetic pathway of trichothecene production is highly conserved between different Fusarium species and the genes within the trichothecene gene cluster are well investigated.Based on this knowledge three genes were chosen in all studies to identify the chemotypes: tri3, tri5, tri7 and tri13 or regions between these genes.The developed assays were applied to Fusarium monitoring programs in numerous countries of Europe and Nortehrn America (Chandler et al., 2003;Jennings et al. 2004;Lia et al., 2005;Quarta et al., 2006;Stepien et al., 2008;).
A tri5 based assay was used to evaluate the efficiency of fungicides against F. graminearum and F. culmorum (Edwards et al., 2001.Three chemical fungicides were applied alone in various concentrations and in combinations.Interestingly, the authors could demonstrate that different pathogenic fungi require different fungicides for the optimal control of the disease.The study revealed a good correlation between the DON content of samples and the Fusarium DNA.Furthermore, the ration between DON and the fungal DNA was not altered by the application of fungicides.This fact is of particular interest as previous in vitro studies postulated the enhanced toxin production of Fusarium after the treatment with antifungal compounds. Most of the published studies focus on correlations between accumulated toxins and the PCR determined fungal DNA concentration in harvested grain and the PCR is postulated as a screening method for applications in food and feed safety.For some of these studies species specific assays were used to assess the biomass of various Fusarium species (Schnerr et al., 2000;Waalwijk et al., 2004;Brandfass & Karlovsky, 2006;Yli-Mattila et al. 2008) or group specific assays were used to identify of quantify certain toxin producers (Schnerr et al. 2001(Schnerr et al. , 2002;;Bluhm et al. 2004;Waalwijk et al., 2008;Kulik et al., 2011).
Although the sensitive PCR methods represent an optimal tool to monitor even minor amounts of Fusarium during colonialization of a host these applications are rare.Reischer et al. (2004) developed a TaqMan based quantitative PCR assay to quantify F. graminearum directly from infected plant material.A central spikelet of wheat heads was artificially infected with conidia and the increase of fungal biomass was monitored by real-time PCR and the disease symptoms were also assessed visually.Due to the superior sensitivity of the PCR assay the disease could be detected and quantified the first day after inoculation, whereas the first visual symptoms became obvious five days later.Nicholson et al. (1998) used PCR tests for F. culmorum and F. graminearum to address the fungal spread in wheat.
The authors found out that the severity of Fusarium stem rot caused by F. culmorum correlates not only with the inoculums load but also with the time point the inoculums is applied.The earlier in the season the conidia are applied the more fungal DNA could be measured in the stem.The influence of trichothecene production on successful infection was tested in the same study with toxigenic and atoxigenic F. graminearum isolates.The toxin producers colonized grain better than non-producers, supporting the theory of trichothecens as a virulence factor.However, non-producing strains could also infect the wheat heads but the amount of detected fungal DNA was only 1 to 10% of the DNA amount found in plants infected with toxin producing strains.The infected wheat heads were also subjected to visual assessment of disease symptoms.Although the conservative method led to the same results the difference between toxigenic and atoxigenis strains was less pronounced.The above described assay for F. culmorum was also used to monitor natural stem rot in wheat in the U.K. (Nicholson et a., 2002), The authors found out that usually other pathogens than F. culmorum were the causal agent of this disease as almost no quantifiable DNA of Fusarium was present in the samples.The same F. culmorum assay was again used in combination with a F. poae assay to evaluate fungicide efficiency for Fusarium head blight control in a greenhouse experiment (Doohan et al., 1999).Under all tested conditions the applied fungicides reduced the disease severity between 20% and 80% and in general the results could be confirmed by visual monitoring of the disease symptoms.
A Canadian Fusarium monitoring program integrated PCR tests for F. avenaceum, F. culmorum, F. crookwellense, F. poae, F. sporotrichioides, F. equiseti, F. pseudograminearum, and F. graminearum to compare results with the traditional agar plating method (Demeke et al., 2005).For 83% of the tested grain samples the two methods led to the same results.However, the agar plate method not always gave positive results when DNA was found in samples.This leads to the assumption that PCR based methods provide enhanced sensitivity.Additionally, F. graminearum, F. pseudograminearum and F. crookwellense could not be distinguished by morphological analysis whereas PCR could clearly differentiate these species.This comprehensive study clearly demonstrated the power of DNA based methods if integrated into Fusarium monitoring programs.PCR allows the quantification (if applied as real-time PCR) and classification of numerous strains within a few hours of analysis.Classical methods need some days to weeks to give the same results and quantification is difficult as only the number of infected kernels can be determined.
An LNA-TaqMan based assays was used to investigate the differences in Fusarium resistance of twenty novel wheat lines ranging from highly susceptible to highly resistant (Brunner et al., 2009).The wheat-ears were inoculated with F. graminearum and F. culmorum in two consecutive years and the formation of mycotoxins, the accumulation of Fusarium DNA and the visual disease symptoms were recorded.This study demonstrated a certain discrepancy between visual scoring and quantitative PCR results.In accordance to other studies the visible symptoms matched perfectly the DNA content in medium to low resistant lines.In contrast, highly resistant lines with low Fusarium biomass -but nevertheless high amount of toxins -did sometimes not show any disease symptoms.The authors addressed this effect to the detoxification mechanism of high resistant plants as some toxins can be "masked" by the plant.So they are converted to less plant-toxic metabololites which do not damage the wheat-ears.

Conclusions
Visual assessment of disease caused by Fusarium species is frequently insufficient to identify the causal agent or to quantify the plant pathogen.In natural systems pathogenic fungi often occur in a combination of species, which can induce similar symptoms and a visual discrimination might be impossible.Other methods like agar plate assays rely on the fact that viable propagules are present in a sample.Especially in harvested grain this is not www.intechopen.com Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants: Quantitative PCR and ELISA Accurately Determine Fusarium Biomass 213 always the case as long dry periods during ripening in combination with intense UV radiation can reduce the survival of fungal mycelium or even of conidia.Furthermore, the morphologic discrimination of grown mycelium is difficult and experienced personal is indispensible.Novel methods for the detection and quantification of fungal biomass based on marker molecules (e.g.antigens or DNA) have the potential to revolutionize the field of pathogen monitoring in plant pathology.Within the last twenty years immunoassays and PCR methods found their way into this scientific field.ELISA test for different Fusarium species have been developed intensely in the 1990s and were applied in numerous field studies.However, the specificity of the produced antibodies leaded a lot to desire.Although particular species were used for the immunization of animals the resulting antibodies did detect many different Fusarium species.Only one group succeeded in the production of polyclonal antibodies which were specific to a single species, namely to F. poae .Furthermore, the re-production of antibodies is difficult as immunization is a complex procedure.The resulting immunoglobulins can vary in their features and therefore a careful characterization must be performed each production cycle.Interestingly with the availability of affordable real-time PCR cyclers on the market published studies using Fusarium ELISA almost disappeared.Most probably this tendency can be ascribed to two crucial advantages of PCR methods: i) these methods can be performed in high throughput and are easily transferred to other laboratories.Primers are synthesized commercially, are cheap and they are available within a few days.ii) PCR assays are highly specific and can be used to detect either genera or species and even distinctive isolates can be differentiated.
Although quantitative PCR provides numerous advantages to study Fusarium in its environment only a few studies have been published to address biological problems.This might be due to the fact that nowadays food safety related questions are of high relevance and so the comparison between fungal DNA and mycotoxins is estimated as more important than other applications.On the other hand, the barrier for traditional plant pathologist might still be high to enter a technical field like quantitative PCR.
An investigation of the NCBI pubmed database reveals that the published studies applying Fusarium PCR methods in field-trials increase continuously.Although less novel assays are developed, more and more previously published methods are included in current studies, which might indicate the changeover from PCR based method development to PCR applications.

Table 1 .
Overview of frequently applied PCR assays.This table cites the original method development papers (qualitative and quantitative) but not potentially following publications demonstrating the practical application of these assays.