Yield and Quality Prediction of Winter Rapeseed — Artificial Neural Network and Random Forest Models

open7siThis research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant numbers 451-03-9/2021-14/200051, 451-03-9/2021-14/200134, 451-03-68/2020-14/ 200032 and 451-03-9/2021-14/200032.As one of the greatest agricultural challenges, yield prediction is an important issue for producers, stakeholders, and the global trade market. Most of the variation in yield is attributed to environmental factors such as climate conditions, soil type and cultivation practices. Artificial neural networks (ANNs) and random forest regression (RFR) are machine learning tools that are used unambiguously for crop yield prediction. There is limited research regarding the application of these mathematical models for the prediction of rapeseed yield and quality. A four-year study (2015–2018) was carried out in the Republic of Serbia with 40 winter rapeseed genotypes. The field trial was designed as a randomized complete block design in three replications. ANN, based on the Broyden–Fletcher–Goldfarb–Shanno iterative algorithm, and RFR models were used for prediction of seed yield, oil and protein yield, oil and protein content, and 1000 seed weight, based on the year of production and genotype. The best production year for rapeseed cultivation was 2016, when the highest seed and oil yield were achieved, 2994 kg/ha and 1402 kg/ha, respectively. The RFR model showed better prediction capabilities compared to the ANN model (the r2 values for prediction of output variables were 0.944, 0.935, 0.912, 0.886, 0.936 and 0.900, for oil and protein content, seed yield, 1000 seed weight, oil and protein yield, respectively).openRajkovic D.; Jeromela A.M.; Pezo L.; Loncar B.; Zanetti F.; Monti A.; Spika A.K.Rajkovic D.; Jeromela A.M.; Pezo L.; Loncar B.; Zanetti F.; Monti A.; Spika A.K

Science Takes Flight: Detection of Black Leg on Turnip Gray Mold on Hemp

Disease detection through traditional techniques such as scouting fields on foot, molecular assays, or morphological identification of plant pathogens is time consuming and costly. Disease diagnosis in the field can be extremely subjective, and largely depends on the experience and knowledge of pathogen identification and disease quantification. This thesis provides an evaluation of remote sensing for assessing disease in agricultural field settings via an unmanned aerial vehicle and machine learning. Case studies are presented on two different fungal diseases important in western Oregon crop production: black leg on turnip (incited by Leptosphaeria spp.) and gray mold on hemp (caused by Botrytis spp.). Both case studies utilized a support vector machine model to classify pixels of digital images collected with a multispectral Micasense RedEdge-M optical sensor. Turnip leaves were imaged at 1.5 m in situ while hemp plant images were collected by an unmanned aerial vehicle with flights at 10 m ex situ. Detection of pixels exemplifying black leg leaf spot symptoms on turnip leaves had an overall accuracy of 97.0% with a model sensitivity of 0.48. The support vector machine model utilized in gray mold detection on hemp incorporated a novel vegetation index, a modified green-red vegetation index along with the triangular greenness index, to identify pixels of diseased hemp inflorescences extracted from background soil and vegetation. The model had an overall accuracy of 95.8% when identifying a hemp inflorescence as diseased or non-diseased. False negatives were found to be high with a sensitivity of 0.70 in the hemp model. Additionally, gray mold disease incidence determined using the support vector machine model was compared with disease assessments collected by scouting on foot and was found to have similar treatment rankings, although the differences in the relative percentages between the two methods were found to be large. The findings of this study provide the foundation for further development of remote sensing techniques for black leg disease assessments in Brassica crops and potential deployment of remote sensing strategies for measuring gray mold in hemp fields

Yield and Quality Prediction of Winter Rapeseed—Artificial Neural Network and Random Forest Models

As one of the greatest agricultural challenges, yield prediction is an important issue for producers, stakeholders, and the global trade market. Most of the variation in yield is attributed to environmental factors such as climate conditions, soil type and cultivation practices. Artificial neural networks (ANNs) and random forest regression (RFR) are machine learning tools that are used unambiguously for crop yield prediction. There is limited research regarding the application of these mathematical models for the prediction of rapeseed yield and quality. A four-year study (2015–2018) was carried out in the Republic of Serbia with 40 winter rapeseed genotypes. The field trial was designed as a randomized complete block design in three replications. ANN, based on the Broyden–Fletcher–Goldfarb–Shanno iterative algorithm, and RFR models were used for prediction of seed yield, oil and protein yield, oil and protein content, and 1000 seed weight, based on the year of production and genotype. The best production year for rapeseed cultivation was 2016, when the highest seed and oil yield were achieved, 2994 kg/ha and 1402 kg/ha, respectively. The RFR model showed better prediction capabilities compared to the ANN model (the r2 values for prediction of output variables were 0.944, 0.935, 0.912, 0.886, 0.936 and 0.900, for oil and protein content, seed yield, 1000 seed weight, oil and protein yield, respectively)

Implicación de genes de Pseudomonas simiae PICF7 en endofitismo, control biológico y promoción del crecimiento vegetal

The olive (Olea europaea L.) is the most emblematic tree in the Mediterranean basin, since 98% of its world´s cultivated area is found in this region. The morphological characteristics of this plant, its excellent adaptation to the typical dry and hot summers of this geographical area and the multiple uses of the cultivated and wild olive, make this tree of a huge economic, social and ecological importance. Spain leads the world´s production of olive oil and table olives. However, a/biotic factors such as the presence of pathogens and pests which are difficult to eradicate, erosion and soil loss, and the foreseeable effects of climate change are major threats to the crop. Currently, Verticillium wilt of olive (VWO), a disease caused by the hemibiotrophic fungus Verticillium dahliae Kleb. is considered one of the most devastating diseases affecting olive trees, and constitutes a limiting factor for the olive oil and table olives production. Moreover, recent studies have predicted an increase in temperature and prolonged periods of drought in the Mediterranean basin, which could lead to restrictions in the use of fresh water for irrigation, making it necessary to use saline or reclaimed water. To date, individual control measures employed against VWO have been ineffective. Consequently, integrated control methods combining preventive and palliative measures are considered the best strategy to manage the disease as well as to mitigate the spread of the causal pathogen. Within the integrated management strategy, biological control represents a sustainable and environmentally friendly approach and an alternative to the traditional chemical fungicides. Previous studies have demonstrated that Pseudomonas simiae PICF7 (formerly P. fluorescens PICF7), an indigenous inhabitant of olive roots, is an effective biological control agent (BCA) against VWO. In addition, its ability to promote growth in olive and barley, as well as in the model plant Arabidopsis thaliana, has been demonstrated. So far, strain PICF7 is perhaps the best characterized BCA against VWO. Nevertheless, the mechanisms involved in disease control and its endophytic root colonization ability in several hosts (i.e. olive, barley and wheat) remain largely unknown. Efforts to unravel the keys to the success of strain PICF7 as a BCA have even been made even by studying its interaction with other pathogens. So far, it has only been demonstrated the ability of strain PICF7 to induce systemic resistance to Botrytis cinerea in A. thaliana. Moreover, it is essential that PICF7 and V. dahliae share the same ecological niche (roots, rhizosphere) for the biocontrol of VWO, since an effective suppression of this pathogen has not been observed when both BCA and fungus are spatially separated ("split-root" system). Similarly, no disease control was observed when the phytopathogenic bacterium Pseudomonas savastanoi pv. savastanoi (causal agent of olive knot disease) and PICF7 were inoculated in different parts of the plant (BCA in the roots and pathogen in the stem). However, it was detected a transient reduction of the pathogen population and changes in the morphology of the associated tumors were detected when both microorganisms were applied to the same point (stem). Previous work has also shown that colonization of olive and banana roots by the strain PICF7 results in changes in the expression of genes involved in defensive responses, both locally (roots) and systemically (aerial part). Finally, it has been studied whether bacterial phenotypes traditionally associated with biological control and root colonization (e.g. siderophore production or motility) could be involved in VWO control. Interestingly, the results showed that PICF7 mutants altered in these phenotypes were not compromised in their ability to control the disease. In order to untangle the molecular and genetic bases underlying the effective biocontrol of VWO exerted by strain PICF7, as well as its ability to endophytically colonize olive roots, the present Thesis evaluated the possible contribution of three new phenotypes: 1) biofilm formation, which is traditionally associated with colonization and endophytism; 2) copper tolerance, a metal present in most commercial agricultural antifungals, which could contribute to greater adaptation and survival in soils; and 3) production of phytase, an enzyme involved in phosphorus mobilization and bioavailability, thus promoting plant growth. For this purpose, more than 5.500 Tn5-TcR (Tetracycline-resistance) insertion mutants (transposants) of a pre-existing random transposon insertion mutant bank were screened. Subsequently, for the identification of the Tn5-TcR insertion sites and, consequently, of the altered genes in the selected transposants, nested PCR was used. Finally, the mutants designated as Bfm8 and Bfm9 (unable to form biofilms), Cop1 and Cop33 (altered in copper tolerance) and Phy17 and Phy18 (deficient in phytase activity) were selected to carry out the olive root colonization and VWO biocontrol assays. By using confocal microscopy, it has been shown that all mutants affected in the aforementioned phenotypes were able to superficially colonize olive roots, although analysis of longitudinal sections of these roots revealed that mutants defective in biofilm formation (Bfm8 and Bfm9) did not colonize them internally. None of the studied phenotypes seemed to be involved in the biocontrol ability of PICF7 against VWO, since all selected mutants behaved similar to the wild-type strain PICF7, even though phytase defective mutants (Phy17 and Phy18) were impaired in their ability to antagonize V. dahliae in in vitro assays on one of the culture media used. This fact highlights the importance of contrasting in vitro observations with in planta assays, where the olive-Verticillium-BCA tripartite interaction becomes more complex. This is because there are numerous interactions with and among the remaining plant´s microbiota, as well as with several environmental and pedological factors. Pseudomonas sp. strain PICF6, another effective BCA against VWO, was included in some experiments performed in this Thesis for comparative purposes in order to accomplish some of the proposed objectives. In contrast to strain PICF7, there is virtually no information concerning the control of phytopathogens or the plant growth promotion capabilities of the beneficial strain PICF6, also originating from the olive rhizosphere. To overcome this lack of knowledge, both BCA and the Bfm and Phy mutants of PICF7, were tested in in vitro experiments against several pathogens of great agroeconomic importance in different crops. Strains PICF6 and PICF7, along with all selected mutants derived from the latter, only exhibited the ability to antagonize Verticillium longisporum ELV25, despite the inhibition rates of PICF7 mutants defective in phytase activity being significantly lower. The ability of these rhizobacteria to promote plant growth and colonize oilseed rape roots, host plant of V. longisporum, was also evaluated. Although none of the strains tested promoted the growth of this plant, belonging to the Brassicaceae family, all of them were able to colonize the root surface of oilseed rape seedlings. However, no evidence of endophytic colonization was found. Production of volatile organic compounds (VOC) has been described amongst the possible mechanisms by which beneficial microorganisms are able to inhibit the growth of phytopathogens or promote plant growth. A recent study has shown that strain PICF7 is able to promote plant growth of A. thaliana, not only through direct contact between the BCA and plant roots, but also through the emission of VOC without any physical contact. Nonetheless, it is unknown whether the ability of strains PICF6 and PICF7 to reduce V. dahliae growth could be attributed to this mechanism. In this context, the characterization of PICF6 and PICF7 volatilomes, as well as of the Bfm and Phy mutants, using Gas Chromatography-Mass Spectrometry (GC-MS), allowed the identification of several compounds described in the literature for their antimicrobial potential or plant growth-promoting activity. Conversely, in vitro VOC inhibition assays, using TCVA (Two Clamp VOC Assay) methodology, showed that none of the assayed strains were able to antagonize V. dahliae. Nevertheless, the compounds identified in the volatilome of these rhizobacteria, used individually and at a higher concentration, could represent a new avenue to explore substances with antagonistic capacity against V. dahliae and other relevant pathogens, as well as to evaluate their potential for plant growth promotion. As mentioned above, drought and salinity (abiotic stresses), along with to pathogen infections (biotic stresses), constitute another major threat to olive crop. Several microorganisms have shown high potential to reduce the effects of these stresses in plants. For this reason, the present Thesis has also been focused on studying the role of Pseudomonas sp. PICF6 and P. simiae PICF7 in the alleviation of the symptoms in plants caused by water scarcity of water or by salt accumulation in the soil. Numerous studies have attributed the beneficial effect of certain rhizobacteria on plants subjected to salt or drought stress to the production of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACD). These stresses induce the biosynthesis of ethylene (ET), a phytohormone that, among the numerous processes in which it is involved, causes a negative effect on the plant at high concentrations. ACD catalyzes the conversion of ACC, the immediate precursor of ET, into α-ketobutyrate and ammonia. Therefore, ACD-producing microorganisms contribute to reduce ET levels and, consequently, the stress caused by the accumulation of this phytohormone in the plant. Furthermore, the plant can also develop different defense strategies to mitigate the negative effects caused by this type of stress, including changes in stem water potential (Ψ) and stomatal conductance (gs), or in chlorophyll (Chl), flavonoid (Flv) or proline contents. Experiments were carried out to compare the physiological parameters mentioned above in plants inoculated with an ACD-producing rhizobacteria (Pseudomonas sp. PICF6) and in plants inoculated with a non ACD-producing rhizobacteria (P. simiae PICF7). The results obtained showed that, under the experimental conditions here used, Flv values were influenced by the presence of the bacteria. However, Ψ, gs and the Chl and proline contents remained unchanged upon inoculation of the different strains. Overall, the assayed rhizobacteria, regardless of whether or not they possess ACD activity, did not alleviate the stress produced by drought or salinity in olive plants under the experimental conditions. Finally, as a consequence of the work carried out in this section, it was also demonstrated that the colonization pattern of olive roots by Pseudomonas sp. PICF6 was similar to that observed for PICF7, including its endophytic capacity. In summary, the results obtained in this Thesis allow us to conclude that the three phenotypes of P. simiae PICF7 here examined (phytase activity, biofilm formation and copper tolerance) are not involved in the ability of this BCA to control VWO, although in the particular case of mutants altered in biofilm formation no evidence of endophytic colonization of olive roots was found. Therefore, VWO biocontrol by strain PICF7 does not seem to require inner colonization of this organ. Additinally, strains PICF6 and PICF7 did not show the ability to alleviate symptoms caused by salt or drought stress in olive plants, ruling out the involvement of the ACD activity, at least under the conditions analyzed. Colonization pattern studies for both strains showed that the two olive rhizobacteria superficially colonize the root of this model plant. However, neither PICF6 nor PICF7 promoted the growth of this plant. Yet, the characterization of PICF6 and PICF7 volatilomes allowed for the identification of several VOC with a reported antimicrobial activity. Even though the involvement of VOC in the inhibition of V. dahliae growth in vitro was not observed, the collected information could pave the way for further study of these molecules individually and against different pathogens.El olivo (Olea europaea L.) es el árbol más emblemático de la cuenca mediterránea, concentrándose en esta región el 98% de superficie cultivada de este árbol a nivel mundial. Las características morfológicas de esta planta, su excelente adaptación a los veranos secos y cálidos propios de esta área geográfica, además de los múltiples usos del olivo cultivado y silvestre, hacen que este posea una enorme importancia económica, social y ecológica. España lidera la producción mundial de aceite de oliva y aceituna de mesa. Sin embargo, factores a/bióticos como la presencia de patógenos y plagas de difícil erradicación, la erosión y pérdida de suelo o los previsibles efectos del cambio climático, suponen importantes amenazas para el cultivo. Enfermedades como la verticilosis del olivo (VO), causada por el hongo hemibiotrófico Verticillium dahliae Kleb. y considerada como una de las afecciones más devastadoras del olivo, constituyen un factor limitante para la producción de aceite de oliva y aceitunas de mesa. Asimismo, estudios recientes prevén un aumento de la temperatura y episodios prolongados de sequía en la cuenca mediterránea, lo que podría conducir a restricciones en el uso de agua dulce para irrigación, haciendo necesaria la utilización de agua salada o regenerada y comprometiendo la producción. En lo que respecta a la VO, los diferentes métodos de control de la enfermedad empleados de manera individual han sido ineficaces. Es por ello que el control integrado, combinando medidas preventivas y paliativas de diversa índole, se plantea como la mejor estrategia para manejar la enfermedad y mitigar la dispersión del patógeno que la provoca. Una de las medidas dentro de esta estrategia integrada es el control biológico, herramienta sostenible y respetuosa con el medio ambiente que surge como alternativa a los tradicionales fungicidas químicos. Estudios previos demostraron que Pseudomonas simiae PICF7 (anteriormente P. fluorescens PICF7), bacteria aislada de raíces de olivo (cultivar Picual), es un eficaz agente de control biológico (ACB) frente a la VO. Además, se ha comprobado su capacidad para promover el crecimiento en olivo y cebada, así como en la planta modelo Arabidopsis thaliana. La cepa PICF7 es quizás el ACB mejor caracterizado contra la VO, aunque los mecanismos implicados en el control de la enfermedad y en la capacidad de colonización endofítica de raíces en diversos huéspedes (olivo, cebada y trigo) continúan siendo bastante desconocidos. Las claves del éxito de la cepa PICF7 como ACB se han intentado desvelar incluso estudiando su interacción con otros patógenos. Hasta el momento solo se ha demostrado que induce resistencia sistémica frente a Botrytis cinerea en A. thaliana. Por otra parte, en el caso del control de la VO, es necesario que PICF7 y V. dahliae compartan el mismo nicho ecológico (raíces, rizosfera), pues no se ha observado un control efectivo de este patógeno cuando ACB y hongo se inoculan en raíces separadas en distintos compartimentos (sistema “split-root”). De igual manera, no se observó control cuando la bacteria fitopatógena Pseudomonas savastanoi pv. savastanoi (agente causal de la tuberculosis del olivo) y PICF7 se inoculaban en órganos diferentes de la planta (ACB en las raíces y patógeno en el tallo), pero sí se detectó una reducción transitoria de la población del patógeno y un cambio en la morfología de los tumores cuando ambos microorganismos eran aplicados en el mismo lugar (tallo). Trabajos anteriores también han demostrado que la colonización de las raíces de olivo y platanera por la cepa PICF7 desencadena cambios en la expresión de genes implicados en respuestas defensivas tanto a nivel local (raíces) como sistémico (parte aérea). Finalmente, se ha estudiado si fenotipos bacterianos tradicionalmente asociados con el control biológico y colonización de las raíces (p. ej., producción de sideróforos o motilidad) podían influir en el control de la VO, evidenciándose que mutantes de PICF7 alterados en estos fenotipos no veían mermada su capacidad para controlar la enfermedad. Con el objetivo de desentrañar las bases moleculares y genéticas que subyacen y explican el control efectivo de la VO ejercido por la cepa PICF7, así como su capacidad para colonizar endofíticamente raíces de olivo, en la presente Tesis se evaluó la posible contribución de tres nuevos fenotipos: 1) formación de biopelículas, fenotipo tradicionalmente asociado a colonización y endofitisimo; 2) tolerancia al cobre, metal presente en la mayoría de antifúngicos agrícolas comerciales, que podría contribuir a una mayor adaptación y supervivencia en suelos; y 3) producción de fitasa, enzima que ayuda a incrementar la movilización y disponibilidad del fósforo promoviendo el crecimiento vegetal. Para ello, se llevó a cabo el escrutinio de más de 5.500 mutantes procedentes de una mutateca preexistente generada mediante inserciones al azar del transposón Tn5-TcR, el cual confiere a los transposantes resistencia al antibiótico tetraciclina. Posteriormente, para la identificación de los lugares de inserción de Tn5-TcR y, consecuentemente, de los genes alterados en los transposantes seleccionados, se empleó la técnica de PCR anidada (“nested”-PCR). Finalmente, los mutantes denominados Bfm8 y Bfm9 (incapaces de formar biopelículas), Cop1 y Cop33 (alterados en tolerancia al cobre) y Phy17 y Phy18 (deficientes en actividad fitasa) se seleccionaron para llevar a cabo los ensayos de colonización de la raíz del olivo y biocontrol de la VO. En cuanto a colonización, el uso de microscopía confocal demostró que todos los mutantes afectados en los fenotipos mencionados fueron capaces de colonizar superficialmente las raíces de olivo, aunque el análisis de secciones longitudinales de dichas raíces reveló que los mutantes defectivos en formación de biopelículas (Bfm8 y Bfm9) no las colonizaron internamente. En lo que respecta a capacidad de biocontrol, ningún fenotipo pareció estar involucrado en esta habilidad, ya que todos los mutantes seleccionados controlaron la enfermedad al mismo nivel que la cepa parental PICF7, aun cuando los mutantes defectivos en actividad fitasa (Phy17 y Phy18) vieron mermada su capacidad de antagonizar V. dahliae in vitro en uno de los medios de cultivo empleados. Este hecho pone de manifiesto la crucial importancia de contrastar las observaciones efectuadas in vitro con los ensayos in planta, donde la interacción tripartita olivo-Verticillium-ACB se torna más compleja al entrar en juego las interacciones con y entre el resto de la microbiota de la planta, así como de diversos factores ambientales y pedológicos. La cepa Pseudomonas sp. PICF6, otro ACB eficaz frente a la VO, se incluyó en esta Tesis a efectos comparativos para abordar algunos de los objetivos planteados. Al contrario de lo que ocurre con PICF7, la información sobre esta bacteria beneficiosa originaria de la rizosfera de olivo en lo que respecta al control de fitopatógenos o a la promoción del crecimiento vegetal es prácticamente nula. Para soslayar esta deficiencia, ambos ACB, así como los mutantes Bfm y Phy de PICF7, se enfrentaron en experimentos in vitro a varios patógenos de gran importancia agroeconómica en diferentes cultivos. Las cepas PICF6 y PICF7, así como todos los mutantes derivados de esta última, sólo mostraron capacidad de antagonizar a Verticillium longisporum ELV25, aunque los índices de inhibición de los mutantes PICF7 defectivos en actividad fitasa fueron significativamente inferiores. También se evaluó la capacidad de estas rizobacterias de promover el crecimiento vegetal y de colonizar las raíces de colza, planta huésped de V. longisporum. Aunque ninguna de las cepas ensayadas promovió el crecimiento de esta brasicácea, todas fueron capaces de establecerse superficialmente sobre sus raíces. Sin embargo, no se encontró evidencia de colonización endófita. Entre los posibles mecanismos por los cuales microorganismos beneficiosos son capaces de inhibir el crecimiento de fitopatógenos o promover el crecimiento de las plantas se ha descrito la producción de compuestos orgánicos volátiles (COV). Un estudio reciente ha demostrado que la cepa PICF7 es capaz de promover el crecimiento vegetal de A. thaliana, no solo a través del contacto directo entre el ACB y las raíces de la planta, sino también a través de la emisión de COV sin mediar contacto físico. Sin embargo, se ignora si la capacidad de las cepas PICF6 y PICF7 para reducir el crecimiento de V. dahliae podría deberse a este mecanismo. La caracterización del volatiloma de las rizobacterias PICF6 y PICF7, así como de los mutantes Bfm y Phy de esta última, mediante el uso de Cromatografía de Gases-Espectrometría de Masas (GC-MS), permitió identificar numerosos compuestos descritos en la bibliografía por su potencial actividad antimicrobiana o promotora del crecimiento vegetal. Aun así, los ensayos de inhibición in vitro por COV, en los que se usó la metodología TCVA (de sus siglas en inglés “Two Clamp VOC Assay”), mostraron que ninguna de las cepas analizadas era capaz de antagonizar frente V. dahliae. A pesar de todo, los compuestos identificados en el volatiloma de estas rizobacterias, usados de forma individual y a una concentración mayor, podrían suponer una nueva vía para explorar sustancias con poder antagonista frente a V. dahliae y otros patógenos relevantes, así como para evaluar su potencial en la promoción del crecimiento de plantas. Como se menc

A review of neural networks in plant disease detection using hyperspectral data

© 2018 China Agricultural University This paper reviews advanced Neural Network (NN) techniques available to process hyperspectral data, with a special emphasis on plant disease detection. Firstly, we provide a review on NN mechanism, types, models, and classifiers that use different algorithms to process hyperspectral data. Then we highlight the current state of imaging and non-imaging hyperspectral data for early disease detection. The hybridization of NN-hyperspectral approach has emerged as a powerful tool for disease detection and diagnosis. Spectral Disease Index (SDI) is the ratio of different spectral bands of pure disease spectra. Subsequently, we introduce NN techniques for rapid development of SDI. We also highlight current challenges and future trends of hyperspectral data

Early Detection of Magnaporthe oryzae-Infected Barley Leaves and Lesion Visualization Based on Hyperspectral Imaging

Early detection of foliar diseases is vital to the management of plant disease, since these pathogens hinder crop productivity worldwide. This research applied hyperspectral imaging (HSI) technology to early detection of Magnaporthe oryzae-infected barley leaves at four consecutive infection periods. The averaged spectra were used to identify the infection periods of the samples. Additionally, principal component analysis (PCA), spectral unmixing analysis and spectral angle mapping (SAM) were adopted to locate the lesion sites. The results indicated that linear discriminant analysis (LDA) coupled with competitive adaptive reweighted sampling (CARS) achieved over 98% classification accuracy and successfully identified the infected samples 24 h after inoculation. Importantly, spectral unmixing analysis was able to reveal the lesion regions within 24 h after inoculation, and the resulting visualization of host–pathogen interactions was interpretable. Therefore, HSI combined with analysis by those methods would be a promising tool for both early infection period identification and lesion visualization, which would greatly improve plant disease management

Volatile organic compounds associated with neonectria ditissima infection in apples (Malus pumila cv Gala)

Postharvest diseases in apples during long term storage result in loss and waste. This is mainly caused by fungal pathogens. Fungal contamination and rot can change some of the volatile organic compounds (VOC) emitted by apple fruits. In this study, disease free Gala apples were inoculated with Neonectria ditissima. The aim was to identify VOCs associated with N. ditissima infection in gala apples. The inoculated apples were placed in 5L glass flask, sealed, and incubated at 20oC for one hour after which a charcoal filtered airflow of 1 L/min was maintained for one hour through the Volatile Capture Trap (VCT) with volatile emissions captured on a porapak-Q absorbent filter. Captured volatiles were eluted using 1 mL of dichloromethane (DCM) into a standard Agilent 1.5 mL HPLC vial. Eluted volatiles were analysed using Gas Chromatography coupled with Mass Spectrometry (GC/MS). Volatiles were capture in three replicates for both inoculated and healthy control groups at 2 days, 8 days, 14 days, 21 days, 28 days, 35 days, and 42 days post-inoculation. The N. ditissima discriminatory volatile were identified/discriminated qualitatively based on the unique volatile compounds detected and quantitatively based on variation in peak area of certain combinations of volatile compounds. Some of the discriminatory volatiles such as dodecyl hexanoate, 9-decen-1-yl hexanoate, hexyl butanoate and pentyl acetate were detected in the early stages of the infection. Styrene, terpinene-4-ol, ethyl hexanoate, ethyl butanoate, ethyl pentanoate and 2-methylpentyl formate constituted the main VOCs emitted during apple fruit decay. Other compounds such as alpha Farnesene and hexyl acetate were common to both healthy and inoculated apples but the peak areas in the healthy apples were well above the peak areas in the inoculated apples. However, these compounds expressed a decline in peak area over time. Apples are stored commercially in sealed stores for months making visual observation for early detection of disease almost impossible. Disease of stored apples are most times only detected at advanced stages when it has become nearly impossible to prevent losses. These discriminatory volatile metabolites detected at early stages of infection are important for early non-visual detection of N. ditissima in stored apples. Further research is recommended to the use of these compounds in early detection of the disease caused by N. ditissima

Detección presintomática y no destructiva de enfermedades causadas por patógenos de suelo en maíz (Marchitez tardía) y en girasol (Jopo) mediante medidas térmicas y de fluorescencia multicolor

Algunas enfermedades de cultivos originadas por patógenos de suelo se caracterizan por la tardía aparición de síntomas. Este es el caso de la Marchitez tardía del maíz (causada por el hongo Harpophora maydis) y del Jopo del girasol (causado por la planta parásita de raíz Orobanche cumana). Harpophora maydis se refirió por vez primera en la Península Ibérica en 2010 y durante los últimos años se ha convertido en una preocupación importante para los productores de maíz. Por el contrario, los ataques de O. cumana son frecuentes en los cultivos españoles de girasol desde los años 80. Esta parásita es actualmente el principal limitante biótico de la producción de aceite de girasol en el mundo. Tanto H. maydis como O. cumana infectan la planta hospedante durante las primeras semanas después de la siembra, aunque los síntomas no se hacen visibles hasta la floración del cultivo o poco después de que ésta tenga lugar. También ambas enfermedades se controlan mediante la incorporación de genes de resistencia en las variedades cultivadas. El progreso y el éxito de los programas de mejora para resistencia a enfermedades dependen enormemente de un acertado y eficaz diagnóstico de la reacción de la planta hospedante al patógeno. Por otro lado, en la actualidad son frecuentes las técnicas basadas en el uso de sensores lejanos y/o de proximidad utilizadas en agronomía. Como alternativa a la inspección visual y al análisis de ADN destructivo, y debido a su sensibilidad a desórdenes fisiológicos en las plantas asociados al ataque de patógenos, estas técnicas pueden ser eficaces herramientas de detección en fitopatología. En el caso de proximidad, la monitorización indirecta de las plantas se efectúa principalmente mediante termometría, termografía,medidas de fluorescencia y técnicas espectrales. En el Capítulo 1 de esta Tesis Doctoral se desarrolla el estado del arte de la Marchitez tardía del maíz y del Jopo del girasol. También se presenta información científica actualizada sobre las técnicas de sensores lejanos y/o de proximidad utilizadas más comúnmente en fitopatología. Los objetivos se presentan en el Capítulo 2. En primer lugar se estudió la distribución de H. maydis en la Península Ibérica, se caracterizó su patogenicidad y también se determinaron otras especies de hongos presentes en maíz afectado por marchitez. Además, se evaluó el potencial de la termometría infrarroja para detectar las infecciones por H. maydis. En segundo lugar se detectó la presencia de O. cumana en girasol durante las fases de crecimiento subterráneo de la planta parásita utilizando para ello imágenes de fluorescencia multicolor (FMC). También se consideraron las posibles alteraciones fisiológicas en el girasol como consecuencia de la infección por O. cumana. En el Capítulo 3 se estudió la distribución geográfica de H. maydis en las principales zonas de cultivo de maíz en España y el sur de Portugal, prospectando 59 campos entre 2009 y 2013. La identidad de 14 de entre todos los aislados de H. maydis obtenidos se confirmó mediante amplificación ITS y estos mismos aislados se caracterizaron por su agresividad mediante inoculación y crecimiento de maíz susceptible crecido en condiciones de umbráculo durante todo el ciclo del cultivo. Uno de los aislados del hongo fue muy agresivo, causando síntomas severos en las plantas y reducciones significativas de peso de sus raíces y partes aéreas. Los aislados moderadamente agresivos causaron valores de enfermedad significativos, pero no todos ellos se asociaron a reducciones de peso de las plantas. En 2012 y 2013 se monitorizó la infección por H. maydis en maceta al aire libre y mediante medidas de temperatura de cubierta y del índice de estrés hídrico del cultivo en plantas control y en plantas inoculadas con el aislado más agresivo. Ambos índices respondieron a la infección por el hongo en los dos años, pudiendo detectarse dicha infección hasta 17 días antes de que los síntomas fueran visibles. Este estudio ha revelado la amplia distribución de H. maydis, que se localiza en los valles de todos los ríos de la Península Ibérica excepto el del Ebro y pone de relieve la importancia de la resistencia genética para controlar este patógeno en el sur de Europa. Además, la detección térmica de la infección previa al desarrollo de síntomas podría resultar en aplicaciones útiles para el diagnóstico presintomático y no destructivo de la enfermedad. En el Capítulo 4 se determinaron las especies de hongos asociadas a H. maydis como agente causal de marchitez de maíz. Para ello, se muestrearon 19 campos con síntomas de marchitez en las principales zonas de cultivo de la Península Ibérica entre 2011 y 2012. En el 47% de ellos no se identificó H. maydis sino otras especies: Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani y Trichoderma harzianum. En los campos restantes, junto a H. maydis se identificaron otros hongos de suelo en porcentajes apreciables: F. verticillioides (19%), F. proliferatum (19%), F. equiseti (9%), F. oxysporum (9%) y Pythium oligandrum (9%). El crecimiento vascular de H. maydis y de otras especies de hongos en maíz se confirmó analizando plantas con marchitez procedentes de tres campos diferentes. Tanto H. maydis como F. graminearum, F. equiseti, F. proliferatum y T. harzianum se recuperaron de la inserción entre la raíz y tallo y a 10 cm de altura en el tallo de las plantas. El efecto de la infección por H. maydis sobre la producción de las plantas de maíz se cuantificó en macetas y condiciones seminaturales en 2011. El peso de las mazorcas de plantas inoculadas se redujo en un 54%. Estas plantas también tuvieron pesos de raíz y de parte aérea (tallo y hojas) significativamente menores que los de las plantas control. Estos resultados apuntan al gran impacto que puede tener la Marchitez tardía sobre la producción de maíz en campo. Además, y aunque la patogenicidad de los hongos de suelo identificados en maíz debería ser confirmada, los resultados de este trabajo sugieren que la Marchitez tardía del maíz puede tener una etiología compleja. En cuanto al Capítulo 5, en él se analizó por primera vez la fluorescencia emitida por la clorofila de girasol en las bandas espectrales con máximos en el rojo (F680) y en el rojo lejano (F740). Se incubaron plantas sanas de girasol en macetas y condiciones de invernadero y, entre la segunda y la quinta semana de crecimiento se compararon los patrones de emisión de fluorescencia de los cuatro primeros pares de hojas (PHs) tanto en la superficie de la hoja como entre PHs. Los PHs de plantas sanas de girasol presentaron similares patrones de fluorescencia, tanto en el rojo como en el rojo lejano, que variaron dependiendo del grado de desarrollo de la hoja. La utilidad de F680 y F740 como indicadores de la infección de girasol por O. cumana durante las fases de desarrollo subterráneo de la plana parásita se evaluó en condiciones experimentales similares. En plantas infectadas por O. cumana se detectaron aumentos tempranos de F680 y F740, así como reducciones del ratio F680/F7403. Por otro lado, la significación de las diferencias de fluorescencia emitida por plantas control y plantas inoculadas dependió del PH que se considerara en cada momento. Las medidas de contenido clorofílico y de contenido de clorofila total apoyaron los resultados de la FMC, aunque fueron menos sensibles en la discriminación de plantas control y plantas inoculadas. Al final del experimento se confirmó la infección del girasol por la presencia de nódulos en las raíces de las plantas. Este trabajo revela el potencial de la fluorescencia en las regiones del rojo y el rojo-lejano para detectar de forma temprana la infección de girasol por O. cumana, lo que podría ser especialmente interesante para llevar a cabo un fenotipado temprano de material de programas de mejora. Más aún, y hasta donde hemos podido conocer, este es el primer trabajo donde se analiza el efecto de una planta parásita sobre su hospedante utilizando imágenes de fluorescencia en el rojo y en el rojo lejano. En el Capítulo 6 se analizó la emisión de fluorescencia azul y verde (FAV) en hojas de plántulas sanas de girasol. Además, se aplicaron tanto la FAV como la técnica de termografía para detectar la infección del girasol por O. cumana durante el desarrollo subterráneo de la planta parásita. En ambos experimentos se incubaron las plantas de girasol en macetas en invernadero y las medidas se tomaron tras el traslado temporal a cámara de condiciones controladas. En el primer experimento se observó que la FAV emitida por hojas de girasol sano aumentaba a lo largo de su desarrollo. En el caso de girasol parasitado, las hojas presentaban emisiones de FAV menores, y esta diferencia respecto a las hojas de las plantas control fue consistente a lo largo de todo su desarrollo. Al final del experimento se obtuvieron menores concentraciones de pigmentos, lo que sugiere que en las hojas de girasol ocurre un descenso de metabolitos secundarios tras la infección por O. cumana. Por otro lado, a lo largo de todo el experimento se detectaron mayores temperaturas de hoja en girasol inoculado con O. cumana en comparación con la temperatura de hojas de plantas control. Esto podría indicar que el ataque de la planta parásita induce un cierre estomático y una reducción de la transpiración del girasol. En el trabajo de nuevo se ha demostrado que es posible efectuar una monitorización no destructiva de la infección de girasol por O. cumana, en este caso utilizando FAV y termografía, y que dicha monitorización podría aplicarse al fenotipado rápido de girasol. Además, ambas técnicas se han revelado como útiles aproximaciones para estudiar los procesos mediante los cuales O. cumana altera la fisiología de su hospedante (metabolismo secundario y fotosíntesis). Por último, la discusión general de todos los resultados obtenidos en esta Tesis Doctoral y las conclusiones derivadas se presentan en los Capítulos 7 y 8 respectivamente.Some crop diseases caused by soilborne pathogens are characterised by very late symptoms appearance. This is the case of Late wilt of maize (caused by the fungus Harpophora maydis) and that of Broomrape of sunflower (caused by the root parasitic plant Orobanche cumana). Harpophora maydis was first reported in the Iberian Peninsula in 2010, and during the last years it has become a major concern to maize growers. On the contrary, attacks of O. cumana are frequent in sunflower growing areas of Spain since the 1980’s. Currently, the parasite is the first biotic constraint to sunflower oil production worldwide. Both H. maydis and O. cumana infect the host plant during the first weeks after sowing but symptoms are not observed until flowering or shortly after it. Also, they are controlled through the incorporation of genes of resistance into the crop varieties. The advancement and success of breeding programmes is highly dependent on an accurate and fast screening of the reaction of the host plant to the pathogen. On the other hand, techniques based on the use of remote and/or proximal sensors are frequently used with agronomical purposes. As an alternative to visual inspection and to destructive analyses of DNA, and because its sensitivity to physiological disorders in plants associated with pathogen attack, these techniques can constitute efficient detection tools in phytopathology. In the case of near distance, indirect monitoring of plants is majorly conducted by means of thermometry, thermography, fluorescence measurements and spectral techniques. In Chapter 1 of this Ph.D. Thesis, the state of the art of maize late wilt and sunflower broomrape is presented, as well as updated scientific information about the remote and proximal sensing techniques that are most commonly used in plant pathology. The objectives are presented in Chapter 2. First, the distribution of H. maydis in the Iberian Peninsula and its pathogenic characterization were addressed as well as the identification of other fungal species found in symptomatic maize. Also, the utility of infrared thermometry on the detection of maize infections by H. maydis was assessed. Second, the presence of O. cumana in sunflower was analysed during underground development stages by means of multicolour fluorescence (MCF). Possible physiological disorders in sunflower as a consequence of O. cumana infection were also considered. In Chapter 3 the geographical distribution of H. maydis in the main maize growing areas in the South of Portugal and Spain was determined by prospecting 59 fields from 2009 to 2013. Fourteen out of all the isolates of H. maydis were molecularly confirmed by ITS amplification, and their aggressiveness was analysed by inoculation and growth of susceptible maize under shadehouse conditions for the whole growing season. One of the isolates was highly aggressive, causing severe symptoms as well as significant weight reductions of both aboveground parts and roots of the inoculated plants. Moderately aggressive isolates caused significantly high symptoms severity, but not all of them were related to reductions in plant weight. In 2012 and 2013, the infection by H. maydis was monitored outdoors by means of measurements of canopy temperature and crop water stress index of potted control plants and plants inoculated with the most aggressive isolate. Both indices responded to the presence of fungal infection in both years, this infection being detected up to 17 days before symptoms in the plants were visible. This study shows the distribution of H. maydis in all the river valleys of the Iberian Peninsula, except that of the Ebro River, and highlights the importance of genetic resistance for controlling the pathogen in southern Europe. In addition, the thermal detection of the infection prior to symptoms development was possible, what might be further applied to the non-destructive pre-symptomatic diagnosis of Late wilt of maize. The species of fungi that are associated to H. maydis as the causal agent of maize wilt were identified in Chapter 4. Surveys were conducted in 2011 and 2012 in 19 fields where symptomatic plants were collected. The fields were located in the main maize growing areas of the Iberian Peninsula. In 47% of them the fungus infecting diseased plants was not H. maydis but Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani and/or Trichoderma harzianum. In the remaining fields H. maydis was identified together with other soilborne fungi that were also frequently isolated from diseased plants: F. verticillioides (19%), F. proliferatum (19%), F. equiseti (9%), F. oxysporum (9%) and Pythium oligandrum (9%). The vascular growth of H. maydis and other fungi into the host was confirmed by means of tissue analyses of diseased plants collected at three different locations. Harpophora maydis, as well as F. graminearum, F. equiseti, F. proliferatum and T. harzianum were recovered from the root-stem insertion, and from stem tissues up to 10 cm high. The effect of the infection by H. maydis on maize yield was assessed in inoculated potted plants that were grown in shadehouse in 2011. Cob production was reduced in 54% upon fungal infection. In addition, significantly low weights of roots and aboveground parts (stems and leaves) were obtained. These results point to the great economic impact that Late wilt can have on the yield of maize under field conditions. Likewise, this work suggests that it can be a disease of a complex etiology. Further work should address the pathogenicity of fungal species other than H. maydis on maize, so that the role they may play on disease incidence and on symptoms severity can be determined. Concerning Chapter 5, the fluorescence emitted by chlorophyll (Chl) of sunflower leaves in the spectral bands with peaks near red (F680) and far-red (F740) was analysed for the first time. Healthy sunflowers were grown in pots under greenhouse conditions. Fluorescence emission patterns across the leaf surface and throughout the plant were compared for the first four leaf pairs (LPs) and between the second and fifth weeks of growth. Similar fluorescence patterns, with a delay of three or four days between them, were obtained for LPs of healthy sunflower, showing that red and far-red fluorescence varied with the developmental stage of the leaves. The use of F680 and F740 as indicators of the infection of sunflower by O. cumana during underground development stages of the parasite was also evaluated under similar experimental conditions. Early increases in F680 and F740 as well as decreases in F680/F740 were detected upon infection, significant differences between inoculated and control plants being dependent on the LP that was considered at any time. Measurements of Chl contents and final total Chl content supported the results of MCFI, although they were less sensitive in differentiating healthy from inoculated plants. The infection of sunflowers was confirmed by the presence of broomrape nodules in the roots at the end of the experiment. This work revealed the potential of MCFI in the red and far-red regions for early detecting O. cumana in sunflower, what might be particularly interesting for early phenotyping in sunflower breeding programmes. Furthermore, and to the best of our knowledge, this is the first time that the effect of a parasitic plant in its host is analysed by means of MCFI. In Chapter 6 we analysed the blue-green fluorescence (BGF) emission of leaves of healthy sunflower plantlets, and we implemented BGF and thermal imaging in the detection of the infection by O. cumana during underground parasite development. In both experiments sunflowers were grown in pots and under greenhouse conditions and measurements were made after temporary movement to chamber of controlled conditions. Increases in BGF emission were observed in leaf pairs of healthy sunflowers during their development. Besides, lower BGF emission was consistently detected in parasitised plants throughout leaf expansion, and low pigment concentration was obtained at final time, supporting the interpretation of a decrease in secondary metabolites upon parasite infection. Also, parasite-induced stomatal closure and transpiration reduction were suggested by warmer leaves of inoculated sunflowers throughout the experiment. Techniques of BGF and thermal imaging allowed the non-destructive monitoring of sunflower broomrape, and they could be implemented for fast screening of sunflower genotypes. Additionally, these techniques were shown as valuable approaches to assess the processes by which O. cumana alters physiology (secondary metabolism and photosynthesis) of sunflower. Finally, the general discussion of all the results from the work included in this Ph.D. Thesis and the conclusions drawn from them are presented in Chapters 7 and 8 respectively