Modern Techniques for Detection of Seed Born Fungi.
- 1. MODERN TECHNIQUES FOR DETECTION OF SEED BORNE FUNGAL PATHOGENS Submitted to: Dr Narender Kumar Bharat Submitted by: Paranjay Rohiwala Admission no.: H-2019-40-D
- 2. INTRODUCTION Seed-borne pathogens possess a serious threat to seedling establishment. Close association of the pathogens with seeds facilitates their long-term survival, introduction into new areas and widespread dissemination. Under such conditions, elimination is the most effective disease management strategy accomplished by using seed detection assay. Transboundary spread of pathogens is a major concern today. Precise detection methods are essential for seed-borne pathogens to support seed health strategies. The considerable advancement in molecular biology has facilitated rapid identification/detection of seed-borne pathogens.
- 3. Methods of detection have to fulfil six main requirements: (i) Specificity – the ability to distinguish a particular target organism from others occurring on tested seeds. (ii) Sensitivity – the ability to detect organisms at low incidence in seed stocks. (iii) Speed – less time requirements, to enable prompt action against the target pathogen(s).
- 4. (iv) Simplicity – minimization of a number of examination stages to reduce error and enable testing by a staff not necessarily highly qualified. (v) Cost-effectiveness – costs should determine acceptance to the test. (vi) Reliability – methods must be sufficiently robust to provide repeatable results within and between samples of the same stock regardless who performs the test.
- 5. WHY DETECTION OF SEED-BORNE FUNGAL PATHOGENS IS IMPORTANT? A. Seed-borne fungal pathogens-serious threat to seedling establishment and hence may lead to crop failure. B. Seeds -long-term survival of pathogens and vehicle for their introduction into newer areas and their widespread dissemination. C. May cause catastrophic losses to food crops and hence directly linked to the food security. D. Unlike infected vegetative plant tissues, infested seeds can be asymptomatic, making visual detection impossible. E. Additionally, fungal pathogen’s populations on seeds may be low, and the infested seeds may be non- uniformly distributed within a lot.
- 6. MODERN DETECTION METHODS FOR SEED-BORNE FUNGAL PATHOGENS
- 7. Serology-based assays •Serological seed assays rely on antibodies (polyclonal or monoclonal) generated against unique antigens on the surfaces of plant pathogens (Hampton et al., 1990). •Antibodies bind strongly and specifically to their antigens and can subsequently be detected by the enzymatic digestion of substrates or fluorescent tags. •Serology based seed tests have several formats including the widely applied enzyme linked immunosorbent assay (ELISA) (McLaughlin and Chen, 1990) and immuno fluorescence microscopy (Franken, 1992). Advantages: Serological assays do not require pure isolations of the pathogen and, hence, are applicable to biotrophic and necrotrophic seedborne pathogens.
- 8. ELISA has been used for detection of : Barley seeds using a polyclonal antibody (PAb) raised against Penicillium aurantiogriseum var. melanoconidium in indirect ELISA test. Rice and corn seeds colonizing fungi, viz. Aspergillus parasiticus, Penicillium citrinum and Fusarium oxysporum, were detected by employing double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test. Karnal bunt disease of wheat caused by Tilletia indica detected using SDS-PAGE analysis (Kutilek et al. 2001). Ustilago nuda causes loose smut in barley and it is an internally seed-borne pathogen. Eibel et al. (2005b) employed a DAS-ELISA test with biotinylated detection antibodies to detect loose smut pathogen in naturally infected barley seeds. Phomopsis longicolla causing Phomopsis seed decay of soybean detected using indirect ELISA and a modified immunoblot assay, named as seed immunoblot assay (SIBA),(Gleason et al. 1987).
- 9. Types of ELISA 1) Direct ELISA • A direct ELISA is a plate-based immunosorbent assay. • For the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample. • Simplest and quickest to perform. • In a direct ELISA, antigen is immobilized directly onto the surface of a multi-well microtiter plate and then complexed with an enzyme-labeled primary antibody specific for the antigen.
- 10. • Once the enzyme-labeled primary antibody binds to the antigen, the conjugated primary antibody catalyzes a reaction with its respective substrate • Resulting in a visible colorimetric output that is measured by a spectrophotometer or absorbance microplate reader.
- 11. 2) Indirect ELISA • Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. • The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. • In the indirect ELISA test, the sample antibody is between the antigen coated on the plate and an enzyme-labeled, anti-species globulin conjugate.
- 12. •The addition of an enzyme substrate chromogenic reagent causes color to develop. •This color is directly proportional to the amount of bound sample antibody.
- 13. 3) Sandwich ELISA • The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). • The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich. • Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems. • Monoclonal antibodies recognize a single epitope that allows fine detection and quantification of small differences in antigen.
- 14. • A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. • The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA)
- 15. Limitations: 1. The unavailability of species-specific antibodies. 2. The detection thresholds of serology-based assays vary significantly based on the quality of the antibody and the testing format. 3. Finally, with serology-based assays it is possible to detect nonviable pathogens which results in erroneous (false positive) interpretation.
- 16. Nucleic Acid-Based Detection Methods Generally, nucleic acid-based techniques resulting in a high level of sensitivity and specificity are used for species-specific detection of seed-borne pathogens. Through these techniques, very small quantities of samples or tissues are sufficient for the detection of pathogens in seeds of various crops. In recent times nucleic acid-based detection methods have become the preferred choice for detection, identification and quantification of seed-borne fungal pathogens.
- 17. In molecular detection several strategies, viz. 1. Polymerase chain reaction (PCR) i. Bio-PCR, ii. Immuno magnetic separation (IMS) iii. Magnetic capture hybridization-PCR, 2. Loop-mediated isothermal amplification, 3. Rapid Cycle Real-time PCR 4. Multiplex PCR 5. DNA Chip (microarray) technology 6. DNA barcoding 7. Next Generation Sequencing (NGS) are available for the detection and identification of pathogens which involves propagation of putative pathogen propagules on a culture medium and subsequent PCR on washes from the culture plates, often using nested PCR primer pairs and sometimes without DNA extraction.
- 18. 1. Polymerase chain reaction-based seed detection assays Polymerase chain reaction (PCR) is the in-vitro, primer- directed, enzymatic amplification of nucleic acids (Erlich et al., 1988; Saiki et al., 1988). This technique has been used in many diverse applications including diagnosis of plant diseases. For PCR, primers (small oligonucleotide probes) designed to anneal to specific DNA sequences in the target organism’s chromosomal DNA or RNA, hybridize with and direct amplification of millions of copies of the target sequence. This amplified DNA can be visualized after electrophoresis in ethidium bromide stained agarose gels.
- 19. As a result of this great potential, over the past 20 years, many PCR- based assays have been reported for the identification of seedborne pathogens; e.g. Ascochyta lentis in lentil seeds Alternaria radicina in carrot seeds, Alternaria brassicae in cruciferous seeds, Leptosphaeria maculans in canola seeds, and Phoma valerianella in lamb’s lettuce seeds PCR-based methods also provide rapid and unequivocal identification of F. oxysporum f. sp. basilici from basil seeds, unlike the conventional detection methods that cannot distinguish between pathogenic and non-pathogenic F. oxysporum isolates (Baayen, 2000; Chiocchetti et al., 2001).
- 20. Advantages of PCR speed (completed within 2 to 3 h) specificity (DNA probes can be designed to amplify nucleic acids from the desired genus, species, subspecies, race, etc.) sensitivity (single copies of nucleic acids can be detected after amplification) easy and objective result interpretation (the presence of a DNA fragment of specific size indicates the presence of the pathogen).
- 21. Lack of acceptance • Many seed types contain compounds (e.g., tannins, phenolic compounds, phenolic compounds) that inhibit DNA amplification. • This lead to false-negative results when PCR is attempted directly on seed extracts. • To circumvent, elaborate DNA extraction and purification steps are employed. • It reduce assay sensitivity, efficiency and also require potentially harmful chemicals e.g. chloroform and phenol. • Finally, DNA from nonviable cells or tissues can yield false-positive results in seed assays, so it is necessary to confirm positive results by recovering the target organism.
- 22. •Management decisions based on these types of errors may result in the unnecessary destruction of healthy seedlings and subsequent financial losses for commercial seedling producers. To overcome such problems, several modifications have been developed. These include i. BIO PCR ii. Nested PCR iii. Immuno magnetic separation (IMS) iv. Magnetic capture-hybridization (MCH)
- 23. i. BIO-PCR. Seeds are infested with fungi at very low levels, and consequently the DNA of the pathogen is not sufficient for the subsequent reactions (De Boer et al., 1995). To overcome this problem, Schaad et al. (1995) developed a highly sensitive PCR technique, named BIO-PCR. This consists of a pre-assay incubation step to increase the biomass of the fungal pathogen on the seeds, which is then followed by DNA extraction and amplification by PCR. Target cell enrichment followed by PCR or BIO-PCR improves the efficiency and sensitivity of PCR by allowing target pathogen populations to increase in a pre enrichment phase, before DNA extraction and PCR.
- 24. Selective pre enrichment increases pathogen populations relative to non target microorganisms and Results in higher quantities of target DNA, which ultimately results in higher sensitivity. Additionally, during incubation and enrichment on artificial media, inhibitory compounds are adsorbed or diluted during cell harvest, and do not interfere with DNA amplification. For this technique: in the case of seedborne are incubated under conditions of high relative humidity to increase target fungus mycelial mass before DNA extraction and PCR (Pryor and Gilbertson, 2001).
- 25. BIO-PCR has been developed for the detection of black rot (Alternaria radicina) of carrot (Daucus carota). This technique has been reported to significantly improve the sensitivity and the ease of implementation of PCR, It has detection limits of 2 to 3 cfu/mL (Schaad et al., 1999). In BIO-PCR the target organism must grow on the selective medium before it can be detected by PCR. Bio-PCR has been proven successful in the detection/identification of Tilletia indica teliospores in wheat seed samples (Schaad et al. 1997).
- 26. BIO-PCR has several advantages over traditional PCR: 1. Increased sensitivity, 2. Elimination of PCR inhibitors 3. Detection of viable cells only, thus avoiding false positives due to detection of DNA from dead cells. 4. No need to identify the pathogen based on colony morphology since specific PCR primers are used. Disavantages 1. More expensive than conventional PCR, as selective media are used 2. The disadvantages of BIO-PCR include the need for a semi selective medium for each pathogen. As mentioned earlier, semi selective media require specific knowledge about the nutritional requirements and chemical tolerances of the target organism and usually take time to develop. 3. it usually requires from 5 to 7 days for the fungal growth, which significantly increases the time required for completion of the assays.
- 27. ii. Nested PCR Another way to obviate the low levels of the target fungus on seeds is the use of nested PCR. This procedure can improve the sensitivity and specificity of the assay, thus allowing detection of a target DNA at several-fold lower levels than for conventional PCR (Chiocchetti et al., 2001). Indeed, nested PCR has been used to detect DNA levels of 10 fg for Colletotrichum lindemuthianum in bean seeds (Chen et al., 2007), and of 1 fg for F. oxysporum f. sp. lactucae in lettuce seeds (Mbofung & Pryor, 2010). However, this molecular assay is more labour intensive, more costly, and more prone to contamination than conventional PCR (McCartney et al., 2003; Atkins & Clark, 2004; Tomlinson et al., 2005).
- 28. This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, on pair of primers “outer pair” is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in second PCR after the reaction is diluted with a set of second set “nested or internal” primers whose binding sites are completely or partially different from and located 3’ of each of the primers used in the first reaction.
- 30. Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers. This method can be used for detection of Colletotrichum lindemuthianum in bean and Fusarium oxysporum f. sp. lactucae in the seed of lettuce.
- 31. iii. IMMUNOMAGNETIC SEPARATION AND PCR (IMS-PCR) Immunomagnetic separation refers to the use of microscopic magnetic beads (IMBs) coated with antibodies produced against a specific microorganism, to selectively sequester target cells from suspensions containing heterogenous cell mixtures (Olsvik et al., 1994; Safarik and Safarikova, 1999). Captured cells can be recovered on selective media or DNA can be extracted from them and used for PCR. This technique has been employed widely for the detection of microorganisms from a variety of backgrounds.
- 32. IMMUNOMAGNETIC SEPARATION AND PCR
- 33. iv. MAGNETIC CAPTURE HYBRIDIZATION PCR (MCH-PCR). Similar in format to IMS-PCR. But MCH-PCR uses single stranded DNA probes to capture and concentrate specific DNA fragments that can then be used as templates for PCR. MCH-PCR is a relatively new technique, first described in 1995 for the detection of Pseudomonas fluorescens in non sterile soil (Jacobsen, 1995). This technique has subsequently been developed for the detection of fungi, viruses and bacteria in water, wood, soil and food, all of which contain PCR-inhibitory compounds (Chen et al., 1998). Recently, a research has been done to develop a MCH-PCR assay for the detection of Botrytis aclada (causal agent of Botrytis neck rot) on onion seed.
- 34. To conduct MCH-PCR, infested onion seeds were crushed and DNA was extracted using a Mini-Beadbeater (Biospec Products Inc., Bartlesville, Okla.). Double-stranded target DNA molecules were denatured by boiling and magnetic beads coated with the capture probe were incubated with the DNA suspensions for 2 h at 62 ºC (143.6 °F). During this time, the capture probe hybridized with singles tranded target DNA fragments. After incubation, the beads were washed and the captured DNA was used for PCR. Development of the MCH-PCRbased seed detection assay is still in progress, however, detection thresholds of 10 conidia/ mL in the presence of PCR-inhibiting onion seed wash can be observed.
- 36. MCH-PCR has been successfully used to detect pathogenic microorganisms in materials that contain PCR inhibitory compounds, e.g. Botrytis aclada in onion seeds (Walcott et al., 2004). Also, a MCH multiplex real-time PCR assay has been developed to detect two different pathogens, Didymella bryoniae and Acidovorax avenae sub sp. citrulli, from watermelon and melon seeds (Ha et al., 2009).
- 37. 2. Loop-mediated isothermal amplification Loop-mediated isothermal amplification (LAMP) of DNA is a recent technology that was developed by Notomi et al. (2000) as a simple, cost- effective and rapid method for specific detection of genomic DNA. LAMP uses a set of four or six primers and a thermophilic DNA polymerase from Geobacillus stearothermophilus that has strand displacement activity to amplify DNA with high specificity and in less than 1 h (Mumford et al., 2006). This technology has several positive features: all of the reactions can be carried out under isothermal conditions; it does not require expensive equipment; and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. Furthermore, as well as being highly specific, the amplification efficiency of LAMP is extremely high, which provides improved sensitivity, and robustness to the inhibitors that usually adversely affect PCR methods (Fu et al., 2011).
- 39. LAMP products can be visualized by gel electrophoresis, by the use of magnesium pyrophosphate, which promotes precipitation of amplified DNA, with a real-time turbidity reader or with the addition of an intercalating dye, such as SYBR Green I, which produces a colour change if the LAMP reaction is positive. LAMP was very effective for the detection of Fusarium graminearum in total genomic DNA isolated from ground wheat grains (Niessen & Vogel, 2010) and from contaminated and germinated wheat seeds (Abd-Elsalam et al., 2011), thus demonstrating its use for early detection of toxigenic Fusarium species in cereals.
- 40. some other fungal pathogens which could be detected by employing LAMP assay 1. Fusarium oxysporum f. sp. ciceris, the incitant of Fusarium wilt, 2. Tilletia indica causing Karnal bunt, 3. Phomopsis longicolla responsible for the deterioration in the seed quality of soybean and 4. Colletotrichum truncatum causal organism of anthracnose in soybean.
- 41. 3. RAPID-CYCLE REAL-TIME PCR Recent advances in PCR, in the form of rapid-cycle real-time PCR promise to eliminate many of these barriers and make PCR more accessible for seed detection. With real-time PCR, DNA amplification is coupled with the production of a fluorescent signal that increases proportionally with the numbers of amplicons produced (Kurian et al., 1999; Cockerill and Smith, 2002). The fluorescent signal is monitored on a computer in real-time and provides an indirect visual representation of DNA amplification. Detection of amplified DNA can be accomplished by staining with SYBR Green I (Molecular Probes Inc., Eugene Ore.) that binds double-stranded DNA indiscriminately or with the use of specific reporter probes like TaqMan (Taylor et al., 2002).
- 42. Advantages that potentially make it more acceptable for use in routine seed testing: 1) Rapid cycling which reduces DNA amplification time significantly. 2) Closed system which reduces the risk of cross-contamination. 3) No need for time consuming post-PCR electrophoresis to determine PCR results 4) The use of different dyes and probes can allow for mutliplex PCR, by which multiple pathogens can be detected in the same reaction (Wittwer et al., 2001) 5) Real-time PCR can allow quantification of template DNA which may be of use in determining levels of seed infestation. 6) Real-time PCR is able to generate a specific fluorescent signal detected by an integrated fluorometer to provide real-time analysis of reaction kinetics and so allows quantification of specific DNA targets
- 43. On the other hand, there are some key factors that may prevent the immediate adoption of this technology for seed detection (Disadvantages) 1. Real-time PCR requires thermal cyclers that are equipped to detect fluorescence. 2. These thermal cyclers are significantly more expensive than conventional thermal cycler. 3. Taq Man probes used here are costly. 4. Real-time PCR is subject to many of the problems that hamper conventional PCR , including inhibition by seed-derived compounds.
- 44. For example, Quantitative real-time PCR has been used to detect and quantify i. Verticillium dahliae on spinach seeds (Duressa et al., 2012), ii. A. brassicae on cruciferous seeds (Guillemette et al., 2004), iii. Botrytis spp. on onion seeds (Chilvers et al., 2007), and iv. C. lindemuthianum on dry bean seeds (Chen et al., 2013). v. Didymella bryoniae, an incitant of gummy stem blight of cucurbits (Ling et al. 2010). More recently, a multiplex TaqMan real-time PCR assay was developed for the detection of spinach seed-borne pathogens, viz. 1. Peronospora farinosa f. sp. spinaciae, 2. Stemphylium botryosum, 3. Verticillium dahliae and 4. Cladosporium variabile, that cause economically important diseases on spinach (Feng et al. 2014).
- 45. 4. Multiplex real-time PCR Multiplex real-time PCR uses multiple primers (together with probes in the TaqMan assay) in the same reaction, to reduce costs and labour. To ensure adequate specificity and sensitivity, and comparable amplification efficiency of different pathogens in real-time PCR assays, it is critical to choose the appropriate target DNA fragments for the design of the primers and probes. Since multiplex PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of different sizes that are specific to different DNA sequences, it is capable of detecting several seed-borne pathogenic fungi simultaneously with high sensitivity.
- 47. Multiplex PCR was employed to differentiate members of two groups belonging to Aspergillus flavus. The first Aspergillus flavus group encloses A. flavus and A. parasiticus as aflatoxin producers, and the second group includes A. oryzae and A. sojae which are best known for their capability to ferment soybean to prepare various food products. Multiplex PCR could successfully detect Fusarium species within Fusarium head scab complex (Waalwijk et al. 2003) and Rhynchosporium secalis, a seed-borne fungal incitant causing economically important leaf blotch disease of barley (Fountaine et al. 2007).
- 48. 5. DNA Chip (microarray) technology DNA chips or microarrays represent another DNA-based detection assay that may be applied to test seeds for pathogens. This relatively new technology relies on the unique ability of nucleic acid molecules to hybridize specifically with molecules with complementary sequences (Lemieux et al., 1998; Vernet, 2002). With DNA chip technology, oligonucleotide probes are attached to small (approximately 1 cm sq.) glass or silica-based surfaces (chips). The power of this technique lies in the fact that hundreds to thousand of oligonucleotides can be attached to specific, locations on each chip. These oligonucleotides can be complementary to DNA sequences that are unique to certain microorganisms and hence, can be used detect pathogens in seed samples.
- 49. Fungal cell
- 50. To apply DNA chip technology, DNA or RNA must be extracted from the sample being tested and amplified. The amplified DNA is digested into smaller fragments that are then labelled with fluorescent markers and hybridized with oligonucleotides fixed to the DNA chip. After hybridization, the chip is washed thoroughly and fluorescence, which is directly proportional to the amount of nucleotide retained, is measured. If the DNA from the pathogen of interest in present in the seed sample, then the oligonucleotide probe at the position on the chip that corresponds to that pathogen will display fluorescence.
- 51. Advantages of this technology include simultaneous detection of a wide range of pathogens and rapid completion time (6 h). However, since DNA-chip technology relies in part, on DNA amplification, it has similar limitations as those described for conventional PCR.
- 52. 6. DNA Barcoding DNA barcoding is a taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to a particular species (Hebert et al. 2003). The nuclear ribosomal internal transcribed spacer (ITS) region is a recently proposed DNA barcode marker for fungi (Schoch et al. 2012). Identification of universal barcoding regions is important to detect seed-borne fungi. Internal transcribed spacer (ITS) has been used as the primary barcode marker for fungi based on its ability to successfully identify inter- and intraspecific variation among a wide range of fungi.
- 54. An ideal barcoding gene should be sufficiently conserved to be amplified with wide range of primers, however divergent enough to identify closely related species.
- 55. 7. Next-Generation Sequencing (NGS) Next-generation sequencing (NGS), also known as high- throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such revolutionised the study of genomics and molecular biology. Such technologies include:
- 56. Illumina (Solexa) sequencing Illumina sequencing works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain. Roche 454 sequencing This method is based on pyrosequencing, a technique which detects pyrophosphate release, again using fluorescence, after nucleotides are incorporated by polymerase to a new strand of DNA. Ion Torrent: Proton / PGM sequencing Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase and therefore differs from the previous two methods as it does not measure light.
- 57. • After its first application in basic biological research, NGS technologies have been extended to other fields of application, which have included plant disease diagnosis. • Valuable technique for the rapid identification of disease- causing agents from infected plants, • Can also be applied to the detection of fungal pathogens in seeds. • Has been applied to study the mycobiome of wheat seed, using 454 pyrosequencing, allowing the identification of several fungal genera (Nicolaisen et al. 2014).
- 58. Other Newly Developed Diagnostic Techniques a. Biospeckle Laser Technique A recently applied tool that can reveal the presence of pathogenic fungi on seeds is known as the ‘biospeckle’ laser technique. This technique is based on the optical phenomenon of interference that is generated by a laser light that interacts with the seed coat. Examination of seeds under laser light allows the identification of areas with different activities (Braga et al. 2005; Rabelo et al. 2011). As fungi present on the seeds have biological activity, this method can detect their presence on seeds.
- 60. b. Videometer Lab Instrument One more recently developed tool called videometer lab instrument that can distinguish infected seeds from healthy seeds is a multispectral vision system also useful to determine the colour, texture and chemical composition of seed surfaces (Boelt et al. 2018). The combinations of the features from images captured by visible light wavelengths and near-infrared wavelengths were worthwhile in the separation of healthy spinach seeds from seeds infected by Stemphylium botryosum, Cladosporium spp., Fusarium spp., Verticillium spp. or A. alternata (Olesen et al. 2011).
- 61. • Seed quality of castor (Ricinus communis L.) based on seed coat colour was predicted employing multispectral imaging technology using VideometerLab instrument. • This technology was able to distinguish viable seeds from dead seeds with 92% accuracy, suggesting its utility for seed deterioration caused by fungal pathogens (Olesen et al. 2015).
- 62. Features of seed detection assays including time required for completion, sensitivity, ease of application, specificity and applicability for the detection of fungi on seed