TRACKING A TREE-KILLER: IMPROVING DETECTION AND CHARACTERIZING SPECIES DISTRIBUTION OF \u3cem\u3ePHYTOPHTHORA CINNAMOMI\u3c/em\u3e IN APPALACHIAN FORESTS

Phytophthora cinnamomi is a soil-borne oomycete pathogen causing root rot in susceptible host species. P. cinnamomi is thought to have originated in Southeast Asia, but has since been introduced to many regions around the world, where it causes dramatic declines in many forest tree species. In the eastern US, the primary susceptible tree species of concern are American chestnut (Castanea dentata), white oak (Quercus alba), and shortleaf pine (Pinus echinata). American chestnut, functionally eliminated in the early 1900s by the rapidly acting chestnut blight (Cryphonectria parasitica), has been the subject of decades-long breeding efforts aimed at improving chestnut resistance to chestnut blight. To improve chestnut restoration success, and restoration of other susceptible species, the distribution patterns of P. cinnamomi on a landscape scale must be better understood. This project was initiated to develop an improved method for detecting P. cinnamomi to permit high-throughput screening of forest soils, and to implement the improved detection approach in characterizing the distribution patterns of P. cinnamomi in developing soils on reclaimed surface mines in eastern Kentucky, as well as mature forest soils within an undisturbed watershed in a reference-quality eastern Kentucky forest. We developed an improved detection method using a molecular DNA-amplification approach (PCR), which demonstrated similar sensitivity to traditional culture-based methods, but required less time and space than traditional methods. We used this detection approach to screen soils from a chronosequence of reclaimed surface mines (reclaimed at different points in time) to evaluate whether reclaimed surface mined sites become favorable for P. cinnamomi colonization over time. Our analysis detected P. cinnamomi at the two older sites (reclaimed in 1997 and 2003), but we did not detect P. cinnamomi at the two newer sites sampled (reclaimed in 2005 and 2007). These results suggest that surface mined sites become favorable for P. cinnamomi colonization over time, and should not be considered permanently “Phytophthora-free.” We also collected ~200 samples from a watershed in UK’s Robinson Forest, from plots representing a gradient of topographic position, slope, and aspect. This survey indicated that P. cinnamomi distribution in forests is complex and can be difficult to predict; however, P. cinnamomi was detected in both drier upslope sites and in moister drainage sites

Characterization of Phytophthora cinnamomi from avocado

Species in the genus Phytophthora are widely recognized as some of the most devastating plant pathogens, and are responsible for major losses in crops such as potato, tomato, soybean, pepper and alfalfa, and fruit-and forestry trees. The Irish potato famine of 1845 caused by Phytophthora infestans (Mont.) de Bary is a historic example of the devastation this group of pathogens can cause (DE BARY 1876). More than 100 species of Phytophthora have been identified and described to date, and species of this genus are divided into ten clades (BLAIR et al. 2008; KROON et al. 2011; MARTIN et al. 2014). Research on this group of pathogens is focussed on the identification of host factors interacting with effectors, the impact of each effector on pathogenic fitness and their sub-cellular localization in order to identify proposed roles during infection (HUITEMA et al. 2011). This could lead to the application of knowledge on pathogenicity factors to develop novel control strategies.Dissertation (MSc)--University of Pretoria, 2014.National Research FoundationGeneticsMScUnrestricte

The role of phytophthora in predisposing Corymbia calophylla (marri) to a canker disease

Corymbia calophylla (marri), a keystone tree species in the global biodiversity hot spot of south-western Australia, is suffering decline and mortality due to canker disease caused by the endemic fungus Quambalaria coyrecup. Phytophthora species, fine root oomycete pathogens, are frequently isolated from the rhizosphere of dying C. calophylla, raising the possibility that a Phytophthora infection predisposes C. calophylla to this endemic canker pathogen by compromising its defence mechanisms. Field surveys conducted across the C. calophylla range, found Phytophthora to be present in the rhizosphere of C. calophylla. Five Phytophthora species (P. cinnamomi, P. elongata, P. multivora, P. pseudocryptogea and P. versiformis) were recovered from healthy and cankered C. calophylla. Phytophthora incidence was significantly higher in anthropogenically disturbed areas. Pot infestation trials were conducted where the C. calophylla plants were inoculated with the recovered Phytophthora species. A significant reduction in root volume and even seedling death were observed, demonstrating that Phytophthora can adversely affect C. calophylla health. In a follow-up trial, C. calophylla plants were inoculated with both P. cinnamomi and Q. coyrecup and subjected to a drought stress treatment. Results indicated that neither P. cinnamomi nor the drought stress treatments exacerbated the pathogenic effect of Q. coyrecup on the plants. During these trials, weekly reflectance spectroscopic measurements with a portable high-resolution spectroradiometer, were also taken to investigate its potential to track biochemical changes in the C. calophylla leaves due to these treatments. Reflectance values displayed differences between treatments, as well as a seasonal trend in the leaves. Bandwidths in the visible and shortwave infrared regions of the electromagnetic spectrum were demonstrated to be important regions for characterising C. calophylla response to the Phytophthora, Q. coyrecup, waterlogging and drought stress treatments. More work is required to identify the optimum wavelengths for C. calophylla. Once the optimum bandwidths have been determined, reflectance spectroscopy measurements can be scaled up to canopy level, using unmanned vehicles or fixed-wing aircraft; thus, aiding in the management of this canker disease in C. calophylla

Understanding the Molecular Basis of Fusarium solani Mediated Root Rot in Pisum sativum

Pea (Pisum sativum) is an important cool-season crop, which is gaining renewed prominence due to an increased interest in plant-derived proteins. Its high nutritional value, low production costs, and short life cycle make pea ideal for the plant-derived protein market. However, pea yields are jeopardized by the root rot fungus, Fusarium solani f. sp. pisi (Fsp). Green-seeded cultivars are susceptible to Fsp, while most of the wild, purple-seeded pea genotypes are highly resistant. A time course RNA-sequencing approach was undertaken to identify Fsp-responsive genes in four partially resistant and four susceptible green-seeded pea genotypes. Gene expression analysis resulted in the identification of 42,905 differentially expressed contigs (DECs). Fsp challenge produced a more intense and diverse overexpression of genes in the susceptible genotypes, while the partially resistant genotypes showed fewer changes in the expression of defense-related genes and a faster reset to a basal metabolic state. Genes involved in exocytosis, the anthocyanin synthesis, as well as the DRR230 pathogenesis-related gene were overexpressed in the partially resistant genotypes. Genes involved in endocytosis, sugar transport, salicylic acid synthesis, and cell death were overexpressed in the susceptible genotypes. Analysis of the 42,905 DECs resulted in the identification of 769 predicted Single Nucleotide Polymorphisms (SNPs), which were validated and used to screen two segregating populations and to perform quantitative trait loci (QTL) analyses. A new QTL WB.Fsp-Ps 5.1 explained 14.76% of the resistance to Fsp, while four other QTLs explained 5.26-8.05% of the variance. Lastly, the association between the anthocyanin biosynthesis pathway and Fsp resistance was studied via ectopic overexpression, antisense expression, and CRISPR/Cas9-mediated gene editing of the flavanone 3-hydroxylase gene in a highly resistant purple-seeded line. We report an efficient pea transformation protocol with a mean efficiency of 2.9%. Transgenic events exhibiting a range of variation in petal pigmentation were obtained representing CRISPR/Cas9, overexpression, and silencing constructs. To the best of our knowledge, this is the first demonstration of CRISPR/Cas9 mediated gene editing in pea. The transgenic lines will be used in subsequent pathogen challenge assays to determine if the pea anthocyanin biosynthesis pathway is critical for Fsp resistance

Estudio de la interacción entre oomicetos de podredumbre radical y Quercus ilex L.

Forest decline is nowadays a major challenge to management and sustainability of natural ecosystems worldwide. This syndrome is a multifactorial disease influenced by several biotic and abiotic agents such as alien invasive pathogens, changes in land use and management policies, population dynamics driven by economic and politic changes, and climatic perturbations. All these factors changing due to anthropogenic influence, together with others, conformed the so-called global change. Since the 1990’s decade, the oak decline has been identified as one of the most important ecological problems in Europe, affecting deciduous and evergreen Quercus species from the continental forests on Central and North Europe, to temperate forests of the Mediterranean basin. In the case of Iberian Peninsula, holm oak (Quercus ilex L.) and cork oak (Quercus suber L.) decline has been detected since the 1980’s decade. These two species covered most of the forest surface of the south and central part of the Iberian Peninsula, mainly through “dehesas” and “montados” formations. This area is considered to be one of the worst affected regions in the world by climate change, worsening the effects and the consequences of oak decline in “dehesas”. “Dehesas” are Mediterranean savanah-like ecosystems, which provide several economic yields and ecological services. In turn, the holm oak is the most representative tree in the Iberian Peninsula, and the main species conforming the tree layer on the Spanish “dehesas”. The loss of this tree layer is a major ecological and economical constraint. The holm oak decline is mainly associated to the action of soil-borne pathogens, especially Phytophtora cinnamomi. Despite the influence of other factors in the decline, there is a strong association between root rot caused by oomycetes and the death of trees. Phytophtora cinnamomi is an aggressive alien plant pathogen widely widespread worldwide, which has been associated with the disease, die-off and death in a large list of different plant hosts. It is able to change trophic relationships with their hosts, becaming biotroph in asymptomatic hosts and hemibiotroph or necrotroph in susceptible hosts. It is considered that holm oak is the most susceptible Quercus sp. to the action of this pathogen. Additionally, other Phytophthora spp. and Pythium spp. have been recorded associated with the holm oak decline in Spain, Portugal, Italy and France. Many scientific efforts have focused to study this important hostpathogen system, obtaining great results and increasing the knowledge of the causes and effects of the interaction, improving management techniques to limit the spread and the symptoms. However, most of the reviewed works are based on empirical approaches, being the underlying mechanisms regulating the interaction between both species mostly unknown. This PhD Thesis covers part of this lack of basic knowledge, focusing on the host-pathogen interaction at histological and physiological level, and exploring the influence of the soil biota in the severity of the disease symptoms. For this purpose, the work was structured in seven chapters. Chapter 1 provides the framework in which the present PhD Thesis has been developed, and the general and specific objectives. Chapter 2 presents the methodology developed to evaluate the colonization and infection of Phytophthora cinnamomi in Quercus ilex seedling through the semi-automated quantification of pathogen structures present in histological sections of fine roots. A workflow was tuned-up testing different fixing solutions, embedding substances and staining methods, and the results allowed the clear differentiation of pathogen structures from host tissues. Furthermore, different indices based on structure location, host tissue classification and specificity of pathogen structures were evaluated to find the easiest and statistically robust indices that are able to explain the progress of the oomycete into the root. In chapter 3, inoculation experiments in growth chamber were carried out to describe the pathogenesis of the Quercus ilex-Phytophthora cinnamomi interaction. Longitudinal sections were analysed for epidermal, cortex, parenchymatous tissue of central cylinder and vascular tissue after 1, 3, 7 and 14 days after inoculation. Total oomycete structures area, intracellular structures area, extracellular structures area, and specific structures area of the pathogen were quantified. The analysis of these data results in the description of the colonization/infection cycle of the pathogen, classified in three different stages related with their trophic behaviour. Moreover, histological changes of the root tissues as a result of the presence of defence responses and the action of the pathogen were described. In Chapter 4, the differential responses of holm oak seedlings to the inoculation with P. cinnamomi, the acute drought and combination of both stressors were assessed. Six-months old seedlings were inoculated and mock-inoculated, and half of each inoculation treatment plans were subjected to acute drought meanwhile the others were well irrigated. Photosynthesis, stomatal conductance and fluorescence were measured weekly, and total biomass and biomass allocation parameters were quantified at the end of the experiment. The resulting data showed differences in the response of seedlings to drought and inoculation, and the influence of the additive effect of both stressors in the seedlings die-off. In Chapter 5, soil samples of “dehesas” were collected and total DNA was extracted and analysed through metabarcoding techniques, to evaluate the specific composition and diversity of the fungal and oomycete communities, and to study their relationship with the disease symptoms. The fungal community included a wide range of pathogens and abundance of ectomycorrhizal key taxa. Phytophthora spp. dominated the oomycete community, but the species related to root rot did not appear as the most abundant, nor were they related directly to defoliation levels. A particular Operational Taxonomic Unit (OTU) belonging to the genus Trichoderma was strongly correlated with the scarcity of pathogenic Phytophthora spp. The differences in defoliation were related to changes in the functionality of soil microbiota and diversity levels of pathogenic species. Chapter 6 presents the general discussion of the Thesis, including some aspects limiting the results of the works carried out, ad new work lines deriving from this Thesis, and finally Chapter 7 contains the conclusions of the work. Changes in roots as a consequence of P. cinnamomi inoculation, including defence responses, and the differential response identified with pathogen colonization/inoculation, leads to new insights about the causes of tree death. Holm oak responds to the attack of the pathogen, and their physiological changes differ from the ones caused by water stress, allowing the recovery of plants if no additional stress is present. Moreover, the study of soil microbiome in declined “dehesas” showed the influence of the microbial diversity in the health status of trees, and also presented new species of oomycetes and fungi that must be considered in the management of holm oak decline in Andalusian “dehesas”.El decaimiento forestal es hoy en día uno de los mayores desafíos para el manejo y la sostenibilidad de los ecosistemas naturales en todo el mundo. Dicho síndrome es una enfermedad multifactorial en la que intervienen diversos agentes bióticos y abióticos como los patógenos invasores, los cambios en los usos del territorio y las políticas de recursos, o las perturbaciones climáticas. El cambio producido en todos estos factores debido a la influencia del hombre entre otros motivos es lo que se ha dado en llamar cambio global. Desde la década de los 90 del siglo XX, el decaimiento de los robles se ha identificado como uno de los problemas ecológicos más relevantes en Europa, afectando a masas de Quercus caducifolios y perennifolios desde los bosques continentales de Centro Europa y Norte Europa, hasta los bosques templados de la cuenca mediterránea. En el caso de la Península Ibérica, el decaimiento de la encina (Quercus ilex L.) y el alcornoque (Quercus suber L.) se identificó en los años 80 del siglo XX. Estas dos especies cubren la mayor parte de la superficie forestal del sur y centro de la Península Ibérica, principalmente formando sistemas de dehesa y “montados”. Dicha área geográfica está considerada como una de las regiones a nivel mundial que se verán peor afectadas por el cambio climático, lo que agravaría las consecuencias y los efectos del decaimiento de la encina en las dehesas. Las dehesas son ecosistemas mediterráneos semejantes a la sabana, que proveen de diversos beneficios económicos y servicios ambientales. Por su parte, la encina es el árbol más representativo de la Península Ibérica, y la especie principal del estrato arbóreo de las dehesas. La pérdida de este estrato es un problema ecológico y económico de gran relevancia. El decaimiento de la encina está asociado principalmente a la acción de patógenos de suelo, especialmente Phytophthora cinnamomi. Sin olvidar la influencia de otros factores en el síndrome, existe una fuerte asociación entre la podredumbre radicular causada por los oomicetos y la muerte del arbolado. Phytophthora cinnamomi es un patógeno invasor muy agresivo, ampliamente distribuido por todo el mundo, que ha sido asociado con la enfermedad, la decadencia y la mortalidad de una larga lista de diferentes especies vegetales. Es una especie capaz de cambiar su relación trófica con el huésped, comportándose como un organismo biótrofo en huéspedes asintomáticos, y como hemibiótrofo ó necrótrofo en huéspedes susceptibles. Se considera que la encina es la especie del género Quercus más susceptible a la acción del patógeno. De forma adicional, otras especies de los géneros Phytophthora y Pythium se han encontrado asociadas con el decaimiento de la encina en España, Portugal, Italia y Francia. Para estudiar este sistema patógeno-huésped se han llevado a cabo grandes esfuerzos científicos, que han obtenido resultados muy meritorios y han incrementado el conocimiento de las causas y los efectos de la interacción, mejorando las técnicas de gestión para limitar la dispersión y los síntomas del decaimiento. Sin embargo, la mayoría de los trabajos revisados se basaron en aproximaciones empíricas, desconociéndose gran parte de los mecanismos subyacentes que controlan la interacción entre las dos especies. Esta Tesis cubre parte de esta falta de conocimiento básico, centrándose en la interacción entre el huésped y el patógeno a nivel histológico y fisiológico, así como explorando la influencia de la biota del suelo en la severidad de los síntomas de la enfermedad. Para dicho propósito, el trabajo se estructuró en 7 capítulos. El Capítulo 1 proporciona el marco teórico en el que se desarrolla esta Tesis Doctoral, así como los objetivos generales y específicos. El Capítulo 2 muestra la metodología desarrollada para evaluar la colonización e infección de Phytophthora cinnamomi en plántulas de Quercus ilex a través de la cuantificación semiautomática de las estructuras del patógeno presentes en secciones histológicas de raíces finas. El flujo de trabajo fue puesto a punto probando diferentes soluciones de fijación, sustancias de inclusión y métodos de tinción, y los resultados permitieron la diferenciación clara de las estructuras del patógeno y de los tejidos del huésped. Además, distintos índices basados en la localización y especificidad de las estructuras del patógeno y en el tejido del huésped, se evaluaron con el fin de buscar la manera más sencilla y estadísticamente robusta de explicar el progreso del oomiceto en la raíz a través de índices. En el capítulo 3, se llevaron a cabo experimentos en cámara de crecimiento para describir la patogénesis de la interacción entre P. cinnamomi y Q. ilex. Se analizaron secciones longitudinales de epidermis, córtex, tejido parenquimático del cilindro central y tejido vascular, a los 1, 3, 7 y 14 días después de la inoculación. Se cuantificó el área total de estructuras, estructuras intracelulares, estructuras extracelulares y estructuras específicas del patógeno. El análisis de estos datos proporcionó una descripción del ciclo de colonización/infección del patógeno, clasificada en tres etapas diferentes relacionadas con su comportamiento trófico. Asimismo se describieron los cambios histológicos resultantes de la presencia del patógeno o de las respuestas desencadenadas por la planta. En el capítulo 4 se analizó la respuesta diferencial de plántulas de encina ante la inoculación con P. cinnamomi, ante la sequía severa y ante ambos estreses combinados. Plántulas de seis meses de edad fueron inoculados y sometidos a falsa inoculación, y la mitad de cada uno de estos tratamientos fue sometida a sequía severa, mientras que el resto fue regada de manera óptima. Semanalmente se midieron los valores de fotosíntesis, conductancia estomática y fluorescencia, y la biomasa total así como la compartimentación de la biomasa fueron cuantificadas al final del experimento. Los datos resultantes mostraron la existencia de diferencias en la respuesta de las plántulas ante la sequía y la inoculación, así como el efecto aditivo de ambos estreses en la muerte de las plántulas. En el Capítulo 5 se colectaron muestras de suelo de dehesas y se extrajo el ADN total, que se analizó a través de técnicas de metabarcoding, con el fin de evaluar la composición específica y la diversidad de las comunidades fúngica y de oomicetos, y para estudiar sus relaciones con los síntomas de la enfermedad. La comunidad fúngica presentó una gran variedad de patógenos y abundancia de taxones clave de ectomicorrizas. Phytophthora spp. apareció como el taxón dominante dentro de la comunidad de oomicetos, pero las principales especies relacionadas con la podredumbre radicular no fueron las más abundantes, ni presentaron relación directa con los niveles de defoliación. Una unidad taxonómica operacional (OTU) particular, perteneciente al género Trichoderma, presentó correlaciones significativas con la escasez de especies patógenas de Phytophthora spp. Las diferencias en defoliación se correlacionaron con cambios en la funcionalidad de la microbiota del suelo y con los niveles de diversidad de las especies patógenas. El Capítulo 6 presenta la discusión general de la Tesis, incluyendo algunos aspectos que limitan los resultados de los trabajos realizados, y nuevas líneas de trabajo que se derivan de esta Tesis, y finalmente, el Capítulo 7 contiene las conclusiones del trabajo. Los cambios que se producen en la raíz a consecuencia de la inoculación con P. cinnamomi, incluyendo las respuestas defensivas, así como la respuesta diferencial identificada con la colonización/infección, conducen a nuevas apreciaciones sobre la causa de la muerte del arbolado. La encina responde al ataque del patógeno, presentando variaciones en la fisiología diferentes de las causadas por el estrés hídrico, las cuales permiten la recuperación de las plantas si no se superpone un estrés adicional a la inoculación. Adicionalmente, el estudio del microbioma del suelo en dehesas con decaimiento del encinar mostró la influencia de la diversidad microbiana en el estado sanitario del arbolado, así como mostró nuevas especies de oomicetos y hongos que deben tenerse en consideración en el manejo del decaimiento de las dehesas de encina en Andalucía

Metabolitos secundarios microbianos como alternativa de control frente a fitopatógenos del aguacate (Persea americana Mill.)

Serratia marcescens ARP5.1 se seleccionó a partir de una colección de 667 aislados bacterianos, gracias a su capacidad para producir compuestos inhibitorios frente a patógenos del aguacate. Para mejorar la producción de metabolitos bioactivos, se evaluaron combinaciones de velocidades de agitación y velocidades de aireación en un biorreactor de tanque agitado. Se realizaron ensayos in vivo del extracto metabólico de S. marcescens ARP5.1 para evaluar el control de enfermedades postcosecha en frutos de aguacate y en plántulas infectadas por Phytophthora cinnamomi. Se utilizó un enfoque bioguiado, con P. cinnamomi y Colletotrichum gloeosporioides como indicadores de la actividad, para aislar, purificar y elucidar estructuralmente tres compuestos a partir S. marcescens ARP5.1: serratamolide A, prodigiosin y haterumalide NA. Se llevó a cabo un proceso de optimización del rendimiento de haterumalide NA en fermentaciones líquidas. Posteriormente, se realizó una aproximación al modo de acción del compuesto sobre especies de Phytophthora. Los resultados mostraron que el oxígeno y la tensión de cizallamiento fueron factores fundamentales a escala de biorreactor. El extracto demostró niveles de control de las enfermedades similares a los productos disponibles comercialmente. Tres de las 9 variables (maltosa, magnesio y NaCl) se seleccionaron, optimizaron y validaron, mostrando un rendimiento en el punto óptimo de 31 veces (129.0 ± 2.6 mg / L a las 48 h) en el biorreactor. Se evidenció una alta actividad inhibitoria de haterumalide NA frente a P. cinnamomi y dos especies adicionales de Phytophthora (P. nicotianae y P. palmivora). Sin embargo, no se observaron efectos evidentes en ninguna de las etapas asexuales evaluadas en las especias de Phytophthora, a excepción del hinchamiento hifal de P. cinnamomi en la prueba de estructuras de resistencia; por lo tanto, no es posible concluir sobre un posible blanco de haterumalide NA. Los experimentos futuros deberán centrarse en evaluar blancos moleculares intracelulares específicos en los patógenos.Abstract: Serratia marcescens ARP5.1 was selected from a collection of 667 bacterial strains due to its promising capacity to produce inhibitory compounds against avocado pathogens. To enhance bioactive metabolite production, combinations of agitation speeds and aeration rates were evaluated on a stirred tank bioreactor. In vivo assays of the metabolic extract of S. marcescens ARP5.1 were performed to assess the control of postharvest diseases in avocado fruits and on infected seedlings by Phytophthora cinnamomi. A bioguided approach was used, with Phytophthora cinnamomi and Colletotrichum gloeosporioides as bioactivity indicators, to isolate, purify and structurally elucidate three compounds from S. marcescens ARP5.1: serratamolide A, prodigiosin and haterumalide NA. An optimization process of the yield of haterumalide NA in liquid fermentation was carried out. Subsequently, an approach to the mode of action of the compound on Phytophthora species was performed. Results showed that oxygen and shear stress were crucial factors at bioreactor scale. The extract demonstrated control levels of the diseases similar to commercially available products. Three out of 9 variables (maltose, magnesium and NaCl) were selected, optimized and validated, showing a 31-fold yield optimization (129.0 ± 2.6 mg/L at 48 h) in a bioreactor. High inhibitory activity of haterumalide NA, compared with commercially available products, was evidenced against P. cinnamomi, and two additional Phytophthora species (P. nicotianae and P. palmivora). However, no evident effects on any of the assessed asexual stages of the Phytophthora species were observed, except for hyphal swelling of P. cinnamomi in the resistant structures test; therefore, evidence was not conclusive on a possible target for haterumalide NA. Further experiments should be focused in assessing specific intracellular molecular target of the pathogens.Doctorad

Boosting precision crop protection towards agriculture 5.0 via machine learning and emerging technologies: A contextual review

Crop protection is a key activity for the sustainability and feasibility of agriculture in a current context of climate change, which is causing the destabilization of agricultural practices and an increase in the incidence of current or invasive pests, and a growing world population that requires guaranteeing the food supply chain and ensuring food security. In view of these events, this article provides a contextual review in six sections on the role of artificial intelligence (AI), machine learning (ML) and other emerging technologies to solve current and future challenges of crop protection. Over time, crop protection has progressed from a primitive agriculture 1.0 (Ag1.0) through various technological developments to reach a level of maturity closelyin line with Ag5.0 (section 1), which is characterized by successfully leveraging ML capacity and modern agricultural devices and machines that perceive, analyze and actuate following the main stages of precision crop protection (section 2). Section 3 presents a taxonomy of ML algorithms that support the development and implementation of precision crop protection, while section 4 analyses the scientific impact of ML on the basis of an extensive bibliometric study of >120 algorithms, outlining the most widely used ML and deep learning (DL) techniques currently applied in relevant case studies on the detection and control of crop diseases, weeds and plagues. Section 5 describes 39 emerging technologies in the fields of smart sensors and other advanced hardware devices, telecommunications, proximal and remote sensing, and AI-based robotics that will foreseeably lead the next generation of perception-based, decision-making and actuation systems for digitized, smart and real-time crop protection in a realistic Ag5.0. Finally, section 6 highlights the main conclusions and final remarks

Peanut leaf spot disease identification using pre-trained deep convolutional neural network

Reduction of quality and quantity of agricultural products, particularly peanut or groundnut, is usually associated with disease. This could be solved through automatic identification and diagnoses using deep learning. However, this technology is not yet explored and examined in the case of peanut leaf spot disease due to some aspects, such as the availability of sufficient data to be used for training and testing the model. This study is intended to explore the use of pre-trained visual geometry group–16 (VGG16), visual geometry group–19 (VGG19), InceptionV3, MobileNet, DenseNet, Xception, InceptionResNetV2, and ResNet50 architectures and deep learning optimizers such as stochastic gradient descent (SGD) with Momentum, adaptive moment estimation (Adam), root mean square propagation (RMSProp), and adaptive gradient algorithm (Adagrad) in creating a model that can identify leaf spot disease by using a total of 1,000 images of leaves captured using a mobile camera. Confusion matrix was used to assess the accuracy and precision of the results. The result of the study shows that DenseNet-169 trained using SGD with momentum, Adam, and RMSProp attained the highest accuracy of 98%, while DenseNet-169 trained using RMSProp achieved the highest precision of 98% among pre-trained deep convolutional neural network architectures. Furthermore, this result could be beneficial in agricultural automation and disease identification systems for peanut or groundnut plants

Phytophthora parasitica and lupin (Lupinus angustifolius) interactions: changes in gene expression during infection and after phosphite treatment

Phytophthora species are Oomycete pathogens that cause highly destructive diseases in a variety of agricultural and horticultural crops, and natural ecosystems. An understanding of the key biological processes that occur during development and infection of hosts is important for the development of effective Phytophthora control mechanisms. An infection assay model system was developed for P. parasitica based on lupin (Lupinus angustifolius) seedlings. The progress of lesion development and colonisation of P. parasitica in inoculated root tissues was assessed macroscopically and using light microscopy of sectioned material. At 24 hours post inoculation (hpi), a few hyphae were observed in the epidermal and outer cortical cells in the region of the root that had been at the surface of the zoospore suspension during the inoculation period. As root infection progressed, the hyphae grew both towards the vascular tissue at the centre of the root and longitudinally along the root. At 42 hpi, P. parasitica hyphae developed haustoria within root cortical cells. No evidence of callose deposition, a typical plant defence response, by the lupin root cells was observed after infected roots stained with aniline blue. Development of the model lupin-P. parasitica infection assay system facilitated ensuing studies of this plant-pathogen interaction, including the cellular and molecular basis of plant infection. The model assay system was used to examine levels of resistance of different lupin cultivars following inoculation with P. parasitica and to analyse temporal patterns of P. parasitica gene expression using quantitative real-time PCR (qPCR) during lupin root infection. One crucial component of Phytophthora pathogenicity is the digestion of the plant cell wall to allow penetration of the plant surface and colonisation within the plant tissues. Plant cell walls are complicated structures that are composed of a wide range of complex polysaccharides (i.e. cellulose, hemicelluloses and pectins) and proteins and they constitute an effective barrier that impedes the entry of many potential pathogens. In order to penetrate the plant cell wall, pathogens secrete a diverse array of cell wall degrading enzymes (CWDEs). The identity and timing of the expression of genes encoding P. parasitica CWDEs was analysed using qPCR. It is believed that pathogens secrete cascades of CWDEs during the infection process and evidence supporting this hypothesis was obtained from the lupin-P. parasitica data. One management strategy used in the control of Phytophthora diseases is the application of the chemical phosphite. Our understanding of the mechanism(s) underlying phosphite inhibition of Phytophthora diseases in plants is limited. Phosphite is known to have effects on both host plants and Phytophthora pathogens. In the present study, RNA-Seq was used to investigate the effects of phosphite on P. parasitica gene expression in vitro and in planta. Phosphite treatment was found to induce extensive changes in the expression of many pathogen genes both in vitro and in planta. One of the exciting results was the discovery that there was a general tendency for phosphite to up-regulate the expression of genes that are normally expressed early in lupin infection (30-36 hpi) and to down-regulate the expression of genes that are normally expressed during late infection (54-60 hpi). This was exemplified in particular by P. parasitica genes encoding pectinase and cellulase CWDEs and RxLR effectors. In conclusion, the research described in this thesis has developed a new and robust model infection assay for use in studies of plant infection by P. parasitica and, potentially, by other Phytophthora species. The research also presents the results of using this assay in transcriptomic studies of pathogen gene expression during plant infection. The results that have been obtained provide a better understanding of Phytophthora pathogenicity mechanisms and should aid the future development of improved methods of controlling Phytophthora diseases

Integrated management of Phytophthora stem and root rot of soybean and the effect of soil-applied herbicides on seedling disease incidence

Soybean seedling diseases and Phytophthora stem and root rot (PSRR; caused by Phytophthora sojae) are two of the most economically important diseases in North Central U.S. Remarkable differences in disease incidence occur each year, which demonstrate that abiotic and biotic factors must interact for disease onset and development. During 2017 and 2018, field studies were conducted to (i) address the efficacy of seed treatment and genetic resistance for PSRR management on soybean population, canopy coverage (CC), and yield, and (ii) investigate potential interactions between pre-emergence (PRE) herbicides and the incidence of seedling diseases in alluvial soils in Nebraska. Despite field history, PSRR developed in only four of six environments studied. Commercial seed treatment had a positive effect on plant population density, CC, and yield in at least three environments. Compared to non-treated control, seed treatment increased emergence between 11,600 to 53,700 plants ha–1 and early-season CC between 0.7 to 1.2%. Under high disease pressure, management programs using moderately resistant cultivars improved yields when compared to moderately susceptible cultivars. By contrast, minimum yield differences were detected between Rps1k and Rps1c genotypes, except in one environment. While a weak to moderate correlation was observed between CC and incidence of P. sojae symptomatic plants, a moderate to strong association was found between CC and yield. Across multiple environments, PRE herbicides chlorimuron-ethyl, metribuzin, saflufenacil, sulfentrazone, and flumioxazin had no impact on seedling root rot (disease severity index; DSI) when compared to the non-treated control. Similarly, no significant differences between PRE herbicides were detected on plant population, plant height, and yield. Community composition depicting primary pathogenic genera Fusarium, Phytophthora, Pythium, and Rhizoctonia did not occur at random but rather varied across environments and DSI classes. In two of the three environments, Phytophthora structured approximately 22% of primary pathogenic genera, whereas, Rhizoctonia recovery was low (\u3c5.5%). These results suggest compatibility of PRE herbicides programs in late-planted soybeans with a history of seedling diseases. Collectively, the research presented in this thesis furthers our knowledge on the management of soilborne pathogens in soybeans and offers insights into new avenues of research. Advisor: Loren. J. Giesle