Next-generation methods for early disease detection in crops

: Plant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. In this review, the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations are presented and discussed. Traditional techniques of diagnosis used for plant pathogen identification are focused typically on the DNA, RNA (when molecular methods), and proteins or peptides (when serological methods) of the pathogens. Serological methods based on mainly enzyme-linked immunosorbent assay (ELISA) are the most common method used for pathogen detection due to their high-throughput potential and low cost. This technique is not particularly reliable and sufficiently sensitive for many pathogens detection during the asymptomatic stage of infection. For non-cultivable pathogens in the laboratory, nucleic acid-based technology is the best choice for consistent pathogen detection or identification. Lateral flow systems are innovative tools that allow fast and accurate results even in field conditions, but they have sensitivity issues to be overcome. PCR assays performed on last-generation portable thermocyclers may provide rapid detection results in situ. The advent of portable instruments can speed pathogen detection, reduce commercial costs, and potentially revolutionize plant pathology. This review provides information on current methodologies and procedures for the effective detection of different plant pathogens. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material

PE0306 Haplotype-Resolved Genome Assemblies and a Pan-Genome of Six Pistachio Cultivars

Trabajo presentado en la International Plant and Animal Genome Conference, celebrada en San Diego, CA (Estados Unidos), del 13 al 18 de enero de 2023Pistachio (Pistacia vera L., 2n =2x = 30) is one of the most economically important long-lived crops, including cashew and mango in the family Anacardiaceae. Although dioecious pistachio trees are xerophytic and highly resilient to abiotic stresses, they require a certain amount of winter rest period to exit bud dormancy and to flower synchronically between male and female trees. The `Kerman¿ (P. vera cv. Kerman) and `Peter¿ (P. vera cv. Peter) have been the dominant pair of female and male cultivars traditionally grown in California for more than 50 years, whereas the understanding of genomic diversity and approaches for pistachio breeding is limited without the reference-quality genome. Genetic diversity, such as structural and gene content variation, is a major source for crop improvement and breeding, which cannot be captured from a single reference genome. In the present study, we report haplotype-resolved chromosome-level reference genomes of six genetically diverse collections of pistachio (`Kerman¿, `Mateur¿, `Sirora¿, Napoletana¿, `Chaparrillo¿, and `T41¿). The diploid assemblies ranged from 568.55 to 608.21 Mb in size with contig N50 of 28 - 37 Mb. More than 98% of conserved orthologs (BUSCO) were represented for all genome assemblies. The dynamics of transposable elements (TEs) were characterized in the genomes where the TEs accounted for about 65 - 68% of the genomes. The gene annotation analyses using Iso-seq data generated from different five tissue types predicted about 38,000 protein-coding genes. Two haplotypes of each genome showed substantial sequence variation, suggesting high heterozygosity in pistachio genomes. Pan-genome construction and analyses characterized remarkable presence-absence gene content variation between cultivars. The reference genomes with fully annotated genes and pan-genome construction serve as rich resources for genomic breeding and improvement and comparative genomics of pistachio

Addition of Olive Leaf Extract (OLE) for Producing Fortified Fresh Pasteurized Milk with an Extended Shelf Life

An olive leaf extract (OLE) has been tested in vitro for its antibacterial activity and ability to inhibit α-glucosidase enzyme. OLE was also evaluated for its potential, when added to pasteurized milk, to preserve nutritional parameters and to limit microbial growth, thus prolonging shelf life. In vitro assays demonstrated a strong antibacterial efficacy of OLE mainly against Bacillus cereus and the capacity to inhibit α-glucosidase enzyme (IC50) when used at 0.2 mg oleuropein/mL. The milk fortification with OLE at 3.6 mg of oleuropein/mL of milk reduced total mesophilic bacteria at undetectable level after 6 d (expiration date) and by 1 log CFU/mL after 10 d. Moreover, OLE addition at 1.44 and 3.6 mg of oleuropein/mL of milk significantly reduced fat and lactose losses up to 10 d. The results motivate the use of the OLE to make a new functional milk with an extended shelf life

Image_4_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.jpeg

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p

Table_2_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.xlsx

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p

Image_2_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.tif

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p

Image_1_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.tiff

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p

Table_1_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.xlsx

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p

Table_4_NGS transcriptomic analysis uncovers the possible resistance mechanisms of olive to Spilocea oleagina leaf spot infection.xlsx

Spilocea oleagina is a dangerous obligate fungal pathogen of olive, feared in the Mediterranean countries, causing Peacock’s eye or leaf spot infection, which can lead to a serious yield loss of approximately 20% or higher depending on climatic conditions. Coping with this disease is much more problematic for organic farms. To date, knowledge on the genetic control of possible mechanisms of resistance/low susceptibility is quite limited. In this work, comparative transcriptomic analysis (RNA-seq) was conducted in leaf tissues of a low susceptible cultivar Koroneiki and a high susceptible cultivar Nocellara del Belice, both tested in the field using the NaOH test, considering two stages—”zero sign of disease” and “evident sign of infection”. Cultivars showed a very large number of differentially expressed genes (DEGs) in both stages. ‘Koroneiki’ showed an extensive hormonal crosstalk, involving Abscisic acid (ABA) and ethylene synergistically acting with Jasmonate, with early signaling of the disease and remarkable defense responses against Spilocea through the over-expression of many resistance gene analogs or pathogenesis-related (PR) genes: non-specific lipid-transfer genes (nsLTPs), LRR receptor-like serine/threonine-protein kinase genes, GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like gene, Mildew resistance Locus O (MLO) gene, glycine-rich protein (GRP), MADS-box genes, STH-21-like, endochitinases, glucan endo-1,3-beta-glucosidases, and finally, many proteinases. Numerous genes involved in cell wall biogenesis, remodeling, and cell wall-based defense, including lignin synthesis, were also upregulated in the resistant cultivar, indicating the possible role of wall composition in disease resistance. It was remarkable that many transcription factors (TS), some of which involved in Induced Systemic Resistance (ISR), as well as some also involved in abiotic stress response, were found to be uniquely expressed in ‘Koroneiki’, while ‘Nocellara del Belice’ was lacking an effective system of defense, expressing genes that overlap with wounding responses, and, to a minor extent, genes related to phenylpropanoid and terpenoid pathways. Only a Thaumatin-like gene was found in both cultivars showing a similar expression. In this work, the genetic factors and mechanism underlying the putative resistance trait against this fungal pathogen were unraveled for the first time and possible target genes for breeding resistant olive genotypes were found.</p