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Percentage of contaminated seeds and mycoflora composition of the studied lupin varieties. Methods and accession number of genetic loci used for the characterization of main contaminants are also reported.
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Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01–0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting...
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... most common fungi isolated from the three lupin species were Alternaria alternata, Aspergillus spp., Cladosporium oxysporum, Mucor sp., and Penicillium spp. Overall, fungi of 16 genera and 10 species were recovered from lupin seeds, in addition to one yeast and one sterile mycelium, as shown in Table 1. Fungal colonies developing around the seeds were transferred onto fresh Potato Dextrose Agar (PDA) plates amended with streptomycin before identification, and identification was based on morphological characteristics observed under stereoscopic and optical microscope. ...Context 2
... genomic DNA was successfully extracted from all the fungi listed in Tables 1 and 2. PCR amplification of the ITS region with the primer pair ITS5/ITS4 was performed on non-target species of Colletotrichum spp. ...Context 3
... genomic DNA was successfully extracted from all the fungi listed in Tables 1 and 2. PCR amplification of the ITS region with the primer pair ITS5/ITS4 was performed on non-target species of Colletotrichum spp. (Table 2) and on fungi isolated from the seeds ( Table 1). The ITS region was amplified as positive control for the extracted DNA, thereby excluding false negative results. ...Context 4
... primer pair CLF/CLR was used in the PCR amplification with the DNA template of C. lupini and the Colletotrichum species indicated in Table 2, in addition to the DNA of the isolates of non-target species associated with lupin seeds (Table 1). Sensitivity of the PCR protocol using the primer pair CLF/CLR was performed using 10-fold dilutions ranging between 10 ng and 100 fg of mycelial DNA of C. lupini isolate 70555 ( Figure S3). ...Context 5
... primer pair CLF/CLR was used in the PCR amplification with the DNA template of C. lupini and the Colletotrichum species indicated in Table 2, in addition to the DNA of the isolates of non-target species associated with lupin seeds (Table 1). Sensitivity of the PCR protocol using the primer pair CLF/CLR was performed using 10-fold dilutions ranging between 10 ng and 100 fg of mycelial DNA of C. lupini isolate 70555 ( Figure S3). ...Context 6
... tests were carried out to confirm the specificity of the developed PCR assay by analysing the Colletotrichum species of Table 2 and the fungal strains isolated from lupin seeds of The amplicons were sequenced in both directions and aligned to compare with the sequences used to design the primer pair, and to ensure the availability of the partial IGS region in all the isolates tested. Additional tests were carried out to confirm the specificity of the developed PCR assay by analysing the Colletotrichum species of Table 2 and the fungal strains isolated from lupin seeds of Table 1. The primer pair CLF/CLR proved specific to C. lupini, as no bands were obtained on all the non-target fungi tested (Figures 3 and 4). ...Context 7
... primer pair CLF/CLR proved specific to C. lupini, as no bands were obtained on all the non-target fungi tested (Figures 3 and 4). Table 1. The primer pair CLF/CLR proved specific to C. lupini, as no bands were obtained on all the non-target fungi tested (Figures 3 and 4). ...Context 8
... and L. angustifolius cv. Tango, even at contamination levels as low as 0.01% ( Figure 5; Figure S4; DNA concentrations and absorbance ratios are reported in Table S1). ...Context 9
... and L. angustifolius cv. Tango, even at contamination levels as low as 0.01% ( Figure 5 and Figure S4; DNA concentrations and absorbance ratios are reported in Table S1). ...Context 10
... and L. angustifolius cv. Tango, even at contamination levels as low as 0.01% ( Figure 5; Figure S4; DNA concentrations and absorbance ratios are reported in Table S1). The results were supported by the presence of a signal within the positive control (DNA of pure culture of C. lupini IMI504893), and no-signal within the negative control (not infected seed lots). ...Context 11
... were represented by (i) DNA of C. lupini and other Colletotrichum species (Table 2) and (ii) DNA of isolates of non-target fungi associated with lupin seeds and isolated in this work (Table 1). When more than one species was isolated (e.g., Aspergillus spp.), the most representative was chosen. ...Citations
... Presently, the predominant method for minimizing disease spread involves utilizing diseasefree seeds, subjected to rigorous phytosanitary controls, with PCR methods employed to quantify seed infections [5,17,54,55]. In Australia, this is complemented by additional seed treatments such as seed dressing with foliar fungicides [5]. ...
... Molecular assays stand as the predominant diagnostic tools in plant pathology, with nuclear ribosomal cluster genes, including 18S, 5.8S, and 28S, commonly employed as markers for differentiating plant pathogenic fungi. Due to the limitations of Internal Transcribed Spacer (ITS) regions in distinguishing certain Colletotrichum complexes, the polymorphic intergenic spacer (IGS) has been utilized as a molecular diagnostic assay for C. lupini [54]. To enhance the detection sensitivity, an enrichment step involving the incubation of seeds with amended Yeast Malt Broth was introduced to increase the C. lupini biomass in infected seeds. ...
Anthracnose stands as the primary obstacle to lupin cultivation, impeding development despite the considerable agronomic, ecological, and economic potential of such legume crops. This review explores recent efforts to unravel the complexities of anthracnose in domesticated lupins, focusing on both the plant perspective and the causative pathogenic agent, Colletotrichum lupini. Leveraging cutting-edge technologies has yielded crucial insights into various facets of this devastating disease, encompassing plant and pathogen biology, genetic and molecular regulations of the interaction, fungal diversity and population dynamics, and screening of plant genetic resources for anthracnose resistance. The lack of effective disease control measures, relying primarily on the use of disease-free seeds, highlights the need to develop anthracnose-resistant varieties. However, challenges arise from the intricacy of lupin's response to the disease, influenced by polygenic inheritance, in spite of loci with major effects, and environmental factors. The slow pace of genetic improvement underscores the need for more efficient breeding processes, including biotechnological approaches. This review offers a comprehensive overview of current progress and knowledge gaps, stressing the urgent need to further enhance understanding of C. lupini pathogenic mechanisms and lupin‘s resistance. Integrating advanced technologies and accelerated research efforts is paramount for achieving efficient disease management and sustainable lupin cultivation in the face of anthracnose challenges.
... The established BIO-PCR protocol allowed the detection of C. lupine in Lupinus spp. (Pecchia et al., 2019). The BIO-PCR assay is high sensitivity, elimination of PCR inhibitors and detection of living cells to avoid false positives are the advantages over endpoint PCR techniques. ...
... The established BIO-PCR protocol permitted the detection of C. lupine in Lupinus spp. (Pecchia et al., 2019). The seed-borne fungal pathogens Alternaria alternata, A. radicina, and A. dauci were detected utilizing specific primers of ITS in rDNA with the help of a deep-freeze blotter method during the BIO-PCR assay (Konstantinova et al., 2002). ...
The present study was carried out to detect the proteolytic activity of Bacillus species
isolated from different sources (beef, milk, chicken, egg, and rice). A total of fifty isolated samples were
collected randomly from a public restaurant in Khartoum state, Sudan. Ten samples from each source;
5 were freshly cooked (10 - 30 minutes before sampling) and 5 were raw. The isolation of bacteria has
been carried out perfectly according to the construction of the standards, and identification was done
using primary and secondary biochemical tests. The result revealed that out of 50 samples, 20 were
Bacillus isolates which comprised 40% of the total samples. They were B. circulan 5%, B. cereus 5%,
B. megaterium 10%, B. macerans 10 %, B. licheniformis 5%, B. pamilus 5%, B. subtilis 20%, B.
coagulans 15%, B. laterosporus 5%, and B. amyloliquefaciens 20%. After isolation of Bacillus spp., the
investigation was continued to detect protease production using milk agar medium, the most productive
organism was found to be B. macerans and the lowest one was found to be B. amyloliquefaciens
whereas there was no production by B. circulans. The study concluded that Bacillus species were found
in all food sources, so Bacillus genera consider a major cause of food contamination, as well as cooked
food is considered most contaminated by Bacillus than raw food.
... The established BIO-PCR protocol permitted the detection of C. lupine in Lupinus spp. (Pecchia et al., 2019). The seed-borne fungal pathogens Alternaria alternata, A. radicina, and A. dauci were detected utilizing specific primers of ITS in rDNA with the help of a deep-freeze blotter method during the BIO-PCR assay (Konstantinova et al., 2002). ...
PREFACE
Konya is a province with high agricultural potential. Out of the 5 existing universities in the province, 4 of them have faculties of agriculture where studies on the sustainability and techniques of agriculture are conducted. This congress has been organized with the aim of promoting these studies to a wide audience, facilitating collaboration with researchers from other countries to align with global developments in research and development.
The congress was organized in conjunction with the Konya Agriculture Fair, which provided an opportunity to promote both the city and the university to participants. This will increase the university's visibility and enable the development of international collaborations as well as keeping up with innovations. The fair facilitated the meeting of companies participating in the event with researchers, contributing to the observation of innovations and the emergence of new ideas. It provided a platform for researchers from relevant departments of universities to observe these innovations and establish collaborations with the companies behind them, thus fostering the desired university-industry partnership. Additionally, the visually rich environment of the fair has offered researchers new perspectives.
Companies seek qualified workforce specialized in their respective fields from universities. Having these individuals engage in discussions with companies will broaden their horizons and provide opportunities for researchers to engage in practical work. This situation will present indispensable opportunities for both universities and other research institutions. With such a congress, a precedent has been set, and the meeting of industrialists and researchers has taken place within the fair environment.
The congress was led by the Faculty of Agriculture at Selçuk University and was held in its premises. This has facilitated the contribution of faculty members and students to technology and its practical implementation. The congress saw the participation of 125 individuals from 16 different countries.
... Anthracnose is caused by Colletotrichum spp. in a number of plant species, including herbaceous, woody, and cereal plants [12,13] (Table 1). Among Colletotrichum spp. ...
Pulses have gained popularity over the past few decades due to their use as a source of protein in food and their favorable impact on soil fertility. Despite being essential to modern agriculture, these species face a number of challenges, such as agronomic crop management and threats from plant seed pathogens. This review’s goal is to gather information on the distribution, symptomatology, biology, and host range of seedborne pathogens. Important diagnostic techniques are also discussed as a part of a successful process of seed health certification. Additionally, strategies for sustainable control are provided. Altogether, the data collected are suggested as basic criteria to set up a conscious laboratory approach.
... An advantage is that it contains more polymorphic sites. It was efficiently used for detecting C. lupini in lupins by PCR and can be considered an alternative target for Colletotrichum species [63]. ...
Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.
... This standardized protocol helped in the detection of C. lupine in Lupinus spp. (Pecchia et al. 2019). The seed-borne fungal pathogens like Alternaria alternata, A. radicina, and A. dauci were identified with the help of specific primers of ITS in rDNA using deep-freeze blotter procedure during the BIO-PCR assay (Konstantinova et al. 2002). ...
Higher plants are infected by a large number of plant pathogens. Their effects range from minor symptoms to catastrophic events that result in the destruction of large areas of food crops. Catastrophic plant disease exacerbates the current food supply deficit, which has left at least 800 million people hungry. Plant pathogens' populations are difficult to control because they vary in time, space, and genotype. Most insidiously, they evolve, often overcoming resistance that is the plant breeder's hard-won achievement. It is very necessary to define the problem and explore solutions in order to avoid the losses they cause. The major genera and species of disease-causing organisms can now be quickly and reliably identified, credit goes to recent advances in plant pathogen detection based on immunologi-cal and nucleic acid-based techniques. Monoclonal antibodies or polymerase chain reaction (PCR)-based methods, OMICS techniques, protein-based approaches, and nucleic acid-based approaches are highly sensitive and specific and have the potential to replace traditional technologies.
... The method of Bio-PCR consists of a pre-assay incubation step to increase the biomass of the fungal pathogen on the seeds, which is followed by DNA extraction and amplification by PCR. Colletotrichum lupini was diagnosed using the BIO-PCR method from lupin (Lupinus spp.) seeds [68]. Real-time PCR (qPCR) allows quantification of specific DNA targets by means of a fluorescent signal that is proportional to the amount of amplicon produced in each cycle. ...
Seeds can harbor a wide range of microorganisms, especially fungi, which can cause different sanitary problems. Seed quality and seed longevity may be drastically reduced by fungi that invade seeds before or after harvest. Seed movement can be a pathway for the spread of diseases into new areas. Some seed-associated fungi can also produce mycotoxins that may cause serious negative effects on humans, animals and the seeds themselves. Seed storage is the most efficient and widely used method for conserving plant genetic resources. The seed storage conditions used in gene banks, low temperature and low seed moisture content, increase seed longevity and are usually favorable for the survival of seed-borne mycoflora. Early detection and identification of seed fungi are essential activities to conserve high-quality seeds and to prevent pathogen dissemination. This article provides an overview of the characteristics and detection methods of seed-borne fungi, with a special focus on their potential effects on gene bank seed conservation. The review includes the following aspects: types of seed-borne fungi, paths of infection and transmission, seed health methods, fungi longevity, risk of pathogen dissemination, the effect of fungi on seed longevity and procedures to reduce the harmful effects of fungi in gene banks.
... For the present work, a Colletotrichum lupini isolate (RB221, isolated from L. albus in France [29,31]) was used. L. mutabilis LM231 accession was selected as vegetal material due to its high susceptibility to anthracnose [29]. ...
Anthracnose caused by Colletotrichum lupini is the most important disease affecting lupin cultivation worldwide. Lupinus mutabilis has been widely studied due to its high protein and oil content. However, it has proved to be sensitive to anthracnose, which limits the expansion of its cultivation. In this work, we seek to unveil the strategy that is used by C. lupini to infect and colonize L. mutabilis tissues using light and transmission electron microscopy (TEM). On petioles, pathogen penetration occurred from melanized appressoria, subcuticular intramural hyphae were seen 2 days after inoculation (dai), and the adjacent host cells remained intact. The switch to necrotrophy was observed 3 dai. At this time, the hyphae extended their colonization to the epidermal, cortex, and vascular cells. Wall degradation was more evident in the epidermal cells. TEM observations also revealed a loss of plasma membrane integrity and different levels of cytoplasm disorganization in the infected epidermal cells and in those of the first layers of the cortex. The disintegration of organelles occurred and was particularly visible in the chloroplasts. The necrotrophic phase culminated with the development of acervuli 6 dai. C. lupini used the same infection strategy on stems, but there was a delay in the penetration of host tissues and the appearance of the first symptoms.
... 8. Evaluation of pathogen for host plant indexing and certification programs (Weller et al. 2000). 9. Identifying and measuring seed-borne pathogens (Pecchia et al. 2019). ...
There has been a persistent concern that massive crop production loss occurs yearly due to plant pathogens worldwide. Developing and using advanced technologies for timely plant disease detection are vital to reduce crop damage by pathogen infection during crop development, harvesting, and post-harvest processing of the produce, boosting crop productivity and ensuring agriculture sustainability. Among various techniques available to detect plant disease, immunotechnology is broadly recognized as the most effective and powerful disease diagnostic tool. Immunotechnology is generally popular for its quick, highly sensitive, particular features that make it a reliable tool for pathogen detection. However, proper application of such techniques is very challenging as it requires a substantial cost for equipment as well as one has to thoroughly understand the technology to deduce and troubleshoot irregular errors or lab results. In this chapter, we have demonstrated in detail the application of immunotechnology for detecting plant pathogens and highlighted the advantages and limitations of each technique based on immunotechnology.KeywordsDisease diagnostic toolImmunotechnologyDetection of pathogensInoculum quantificationAdvantages and limitations
