rus
Issue #8/2024
M. A. Keldysh, O. N. Chervyakova, O. V. Shelepova, I. V. Mitrofanova, I. V. Petrunya, K. A. Sudarikov, A. A. Gulevich, E. N. Baranova
Comparative Analysis of Optical Methods for Detection and Identification of Viral Infection During Monitoring of Vegetati
Comparative Analysis of Optical Methods for Detection and Identification of Viral Infection During Monitoring of Vegetati
DOI: 10.22184/1993-7296.FRos.2024.18.8.660.669
The article discusses the issues of assessing the prospects for detecting latent viral infection for monitoring viral pathogens using digital processing of images obtained by an optical digital camera and hyperspectral images. Information on 13 types of viruses that have been registered in some regions where Syringa L. grows is provided. Data on the species composition of Syringa viruses in the ecosystems of the Main Botanical Garden and the Moscow region and their symptoms are described. Based on the virological examination, specialized pathogens Lilac ring mottle ilarvirus (LRMV), Lilac leaf chlorosis ilarvirus (LLCV), as well as Carnation mottle carmovirus, Cucumber mosaic cucumovirus, Alfalfa mosaic alfamovirus and Potato Y potyvirus, which are not typical for lilacs, were diagnosed on lilac. As a result of system monitoring, the frequency of occurrence of seven viruses was determined.
The article discusses the issues of assessing the prospects for detecting latent viral infection for monitoring viral pathogens using digital processing of images obtained by an optical digital camera and hyperspectral images. Information on 13 types of viruses that have been registered in some regions where Syringa L. grows is provided. Data on the species composition of Syringa viruses in the ecosystems of the Main Botanical Garden and the Moscow region and their symptoms are described. Based on the virological examination, specialized pathogens Lilac ring mottle ilarvirus (LRMV), Lilac leaf chlorosis ilarvirus (LLCV), as well as Carnation mottle carmovirus, Cucumber mosaic cucumovirus, Alfalfa mosaic alfamovirus and Potato Y potyvirus, which are not typical for lilacs, were diagnosed on lilac. As a result of system monitoring, the frequency of occurrence of seven viruses was determined.
Теги: adaptivity monitoring ndvi phytoviruses pri rgb syringa l. адаптивность видовой состав мониторинг фитовирусы
Comparative Analysis of Optical Methods for Detection And Identification of Viral Infection During Monitoring of Vegetatively Propagated Lilac Cultivar
M. A. Keldysh 1, O. N. Chervyakova 1, O. V. Shelepova 1, I. V. Mitrofanova 1, I. V. Petrunya 2, K. A. Sudarikov 2, A. A. Gulevich 2, E. N. Baranova 1, 2
N. V. Tsitsin Main Botanical Garden RAS, Moscow, Russia
All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia
The article discusses the issues of assessing the prospects for detecting latent viral infection for monitoring viral pathogens using digital processing of images obtained by an optical digital camera and hyperspectral images. Information on 13 types of viruses that have been registered in some regions where Syringa L. grows is provided. Data on the species composition of Syringa viruses in the ecosystems of the Main Botanical Garden and the Moscow region and their symptoms are described. Based on the virological examination, specialized pathogens Lilac ring mottle ilarvirus (LRMV), Lilac leaf chlorosis ilarvirus (LLCV), as well as Carnation mottle carmovirus, Cucumber mosaic cucumovirus, Alfalfa mosaic alfamovirus and Potato Y potyvirus, which are not typical for lilacs, were diagnosed on lilac. As a result of system monitoring, the frequency of occurrence of seven viruses was determined.
Key words: Syringa L., RGB, NDVI, PRI, phytoviruses, monitoring, adaptivity
Article received: 13.10.2024
Article accepted: 11.11.2024
Introduction
The most widespread for urban landscaping is the highly decorative common lilac Syrínga vulgáris L., which blooms abundantly for 3–4 weeks depending on the variety and climatic conditions. To preserve valuable genotypes, dendrological collection sites, in vitro collections and cryopreservation techniques are used. When grown outdoors, lilac plants are exposed to a wide range of pathogenic organisms. It is especially difficult to cope with the consequences of infection by phytoplasmas, viruses and viroids. Qualitative methods of individual diagnostics using enzyme immunoassay, PCR methods and even sequencing are quite expensive and do not always provide an adequate answer about what kind of virus the plant is infected with. The diversity of viruses that damage plants is quite large. Meanwhile, the study of the pathogenesis of viruses shows that they cause changes in the composition of chlorophylls, carotenoids and anthocyanins.
In this work, we hypothesized that viral infection should be manifested by a change in color, during instrumental analysis of photographic images and images obtained by a hyperspectral camera. We demonstrate differences in pigment composition in leaves with clearly identified viral damage and report for the first time that differences in the green part of such leaves may represent significant potential for the development of optical instrumental monitoring methods.
Materials and methods
The study was conducted in the quarantine area of the Plant Protection Laboratory of the N. V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences (MBG RAS), Moscow. Testing was performed by enzyme-linked immunosorbent assay (ELISA based on standard methodology) using the Kit Neogen Europe Ltd. The optical density of the oxidation product was determined using the AIER‑01 UNIPLAN TM enzyme immunoassay analyzer.
For RT-PCR of lilac leaf chlorosis virus (LLCV), RNA isolated from lilac leaves using the PhytoSorb magnetic particle kit (Synthol) was used. Primers to conservative regions of the Ilarvirus (Lilac Leaf Chlorosis Virus – LLCV) transport protein were used. IlaCPF: 5’-gcaatcgaacggagctagtg‑3’ IlaCPF: 5’-cacaaagctgacagaaggca‑3’. The reaction products were separated by electrophoresis in 1% agarose gel.
HSI (hyperspectral imaging) data were obtained using the hyperspectral camera of the Synergotron M.Gk (hyperspectral research module). Image analysis of LLCV-damaged leaves was carried out at subsequent sampling points for spectrophotometric analysis. To process the images the photochemical reflectance index (PRI) [1] and the normalized vegetation index (NDVI) [2] were used. The general formula for both indices is:
Index = (ρλ1 – ρλ2) / (ρλ1 + ρλ2)
PRI = (ρ531 – ρ570) / (ρ531 + ρ570)
NDVI = (ρ800 – ρ680) / (ρ800 + ρ680)
Where λ denotes specific wavelengths, ρ denotes the reflectance, and the numbers 1 and 2 denote the different wavelengths. The values of both indices vary from –1 to 1. The range of PRI index values is from –0.2 to 0.2, the range of NDVI index values is from 0.5 to 0.95.
NDVI helps to better separate the background and reveal pronounced foci of infection. NDVI is primarily sensitive to chlorophyll content [3]. The PRI index allows earlier detection of leaf damages, is sensitive to changes in carotenoid pigments, and is useful for assessing photosynthetic efficiency and stress in plants.
The images were taken with an RGB camera with an Imx 415 Sony 8.0 MP matrix. The HSV (Hue, Saturation, Brightness) color model was used, where all colors of the visible spectrum can be described by the hue value within the range of 0–360°, to construct a correlation of the color index with the chlorophyll value [4].
Results
To date, 13 types of viruses are known that affect lilacs, belonging to six families [5]. The following pathogens have been identified in lilac: elm mottle virus (EMoV), arabis mosaic virus (ArMV), cherry leafroll virus (CLRV), lilac ringspot virus (LRMV), tomato black ring virus (ToBRV), tomato bushy stun virus (ToBSV), tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), ligustrum virus A (LVA), lilac leaf chlorosis virus (LLCV). For lilac mottle virus (LMoV), lilac ringspot virus (LRSV) and lilac chlorotic leaf spot virus (LCLV), data are limited [5; 6].
As a result of systematic monitoring of lilac plantings in the Main Botanical Garden and Moscow Region, pathogens of viral etiology that are not typical for lilac culture have become widespread [7]. In the tested Syringa populations, we recorded a variety of symptoms characteristic of the phenotypic manifestation of viral diseases: chlorosis, ring spotting, linear pattern, mosaic, lightening of veins, necrosis, mottling, and various types of deformation. Symptoms appear in early spring and are extremely varied and change during the growing season. The most common and characteristic signs of the viral manifestation on lilacs are various types of mosaic, which are characterized by a change in the color of the leaves (flowers). For example, these may be small yellow spots that gradually merge. The mosaic can be limited to individual areas or cover the entire leaf, reminiscent of marbling. The white pattern is often accompanied by severe chlorosis. Leaves may become almost white with occasional green areas.
Mosaic can also appear on young leaves as light, blurred spots between the veins. Mottling on lilacs is quite common, but symptoms are usually vague. They appear in the spring and are visible for two to three weeks. A characteristic manifestation of some viruses is the formation of rings, semi-rings and spots, which can subsequently become necrotic. Usually a complex infection occurs, as a result of which the external signs are modified. The manifestation of the same viruses on different varieties and species of lilac may vary, while viruses of different species may cause similar phenotypic traits or their elements. For example, in addition to ArMV, CLRV, EMoV (Figure 1), the specialized viruses LRMoV and LLCV on lilac were diagnosed. And also, in addition to the already known ones, new and unusual viruses: carnation mottle virus (CarMoV), cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV) and potato virus “Y” (PVY) were detected. LLCV was also first registered in the Moscow region using the PCR method. The virus has become widespread in the city’s young plantings. The virus was not detected in the lilac collection or in other GBS ecosystems.
To evaluate the sampled leaves the spectral analysis methods were applied. In this case, areas with a green color (point 1), areas with signs of chlorosis (point 2) and areas with less pronounced or borderline damage (point 3) were analyzed for the quantitative content of pigments (chlorophylls a, b and carotenoids). At the sampling sites, an analysis of the color characteristics of the red, green and blue (RGB) spectrum indicators was carried out, as well as the calculation of H °, after which a correlation analysis of the entire volume of data was carried out and selectively for the data obtained in zones without damage, with chlorosis and intermediate (see Table).
The revealed correlation is valid for values greater than |0.7|. The obtained data demonstrate the possibility of analyzing damage to photosynthesis in infected plants specifically by the undamaged part of the leaves, where a correlation between the expression of the RGB color parameters and chlorophyll a was found. A correlation was also found between carotenoid content and blue range digital values.
For hyperspectral analysis, samples were taken from lilac bushes that had characteristic manifestations of chlorosis in varying degrees of severity and which were probably infected with several viruses. In further work, the samples that had confirmed Lilac Leaf Chlorosis Virus (LLCV) infection were used (Fig. 2). Thus, six samples were selected for further analysis.
The obtained data indicate the possibility of using the Synergotron M.Gk hyperspectral camera for monitoring and analysis of viral pathogens. A comparative analysis should be carried out at 3–4 points that will characterize the development of infection (Fig. 3).
We consider the use of the PRI index to be more promising, since the leaf damage zones were not obvious when using NDVI.
The results confirm that the use of hyperspectral images can significantly facilitate the work of plant pathologists and be used both for monitoring the condition of plantings and for culling planting material in propagation nurseries, as well as preventing the placement of infected material in collection banks for in vitro clonal micropropagation.
Discussion
The genus Syringa L. (Oleaceae) includes many intraspecific and interspecific varieties, the number of which reaches 2300. Lilac is one of the most common shrubs for landscaping in the world, including both northern and southern regions. This ornamental plant is also a source of many natural phytochemical and pharmacological compounds [8]. Lilac is a vegetatively propagated crop, so it is necessary to cull infected accessions in arboretums and create stable collections in vitro cultures [9; 10].
Viral pathogens demonstrate a systemic nature of infection and the infected plant remains sick throughout its life. Changes in the species composition of viruses, the ratio and structure of their populations, the spread of new infections that are not characteristic of a particular plant are shown [11].
Monitoring of viral pathogens in Syringa populations in the MBG revealed that the most noticeable trend is the predominance of viruses with a wide host range (ToMV, CMV, ArMV) and specific to other crops (AMV, CarMV, PVY, EMoV). Eight atypical and two specialized viral pathogens were also found on lilac [6]. The highest frequency rates within 55–70% of accessions were noted for CMV, followed by ArMV, TMV, EMoV, PVY, CarMV (45%, 43%, 37%, 28%, 13%). The presence of LRMoV was detected in only 7% of accessions. It was found that complex diseases caused by several viruses are predominantly common in Syringa populations.
Monoinfection was detected in only 40% of the accessions tested. The species composition was constantly transforming. When two or more viruses interact in one host, the suppressive activity of one of the viruses can have a complementary effect on the viruses included in the complex and lead to infection of the plant by their entire conglomerate [12].
The main objective of our research was to study the spectrum of viral pathogens of Syringa L. Comparing the obtained images with the superimposed heat maps of the above indices, we can conclude that NDVI helps to better separate the background (non-living objects, on a painted field or on a sheet of paper), but also helps with the definition of pronounced foci of plant disease. NDVI is primarily sensitive to chlorophyll content and is widely used to estimate biomass, leaf area index and total vegetation canopy [3]. At the same time, the PRI index makes it possible to identify diseases or leaf damage at early stages; it is sensitive to changes in carotenoid content and responsive to changes in photosynthesis and stress in plants.
Acknowledgment
Study was carried out within the framework of the state assignment of the N. V. Tsitsin MBG of the RAS (No. 124030100058-4) and the ARRIAB (No. 0431 2022-0003). The results of the work were obtained using the Synergotron M.Gk Modules (ANO Institute for Development Strategy, Russia).
M. A. Keldysh 1, O. N. Chervyakova 1, O. V. Shelepova 1, I. V. Mitrofanova 1, I. V. Petrunya 2, K. A. Sudarikov 2, A. A. Gulevich 2, E. N. Baranova 1, 2
N. V. Tsitsin Main Botanical Garden RAS, Moscow, Russia
All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia
The article discusses the issues of assessing the prospects for detecting latent viral infection for monitoring viral pathogens using digital processing of images obtained by an optical digital camera and hyperspectral images. Information on 13 types of viruses that have been registered in some regions where Syringa L. grows is provided. Data on the species composition of Syringa viruses in the ecosystems of the Main Botanical Garden and the Moscow region and their symptoms are described. Based on the virological examination, specialized pathogens Lilac ring mottle ilarvirus (LRMV), Lilac leaf chlorosis ilarvirus (LLCV), as well as Carnation mottle carmovirus, Cucumber mosaic cucumovirus, Alfalfa mosaic alfamovirus and Potato Y potyvirus, which are not typical for lilacs, were diagnosed on lilac. As a result of system monitoring, the frequency of occurrence of seven viruses was determined.
Key words: Syringa L., RGB, NDVI, PRI, phytoviruses, monitoring, adaptivity
Article received: 13.10.2024
Article accepted: 11.11.2024
Introduction
The most widespread for urban landscaping is the highly decorative common lilac Syrínga vulgáris L., which blooms abundantly for 3–4 weeks depending on the variety and climatic conditions. To preserve valuable genotypes, dendrological collection sites, in vitro collections and cryopreservation techniques are used. When grown outdoors, lilac plants are exposed to a wide range of pathogenic organisms. It is especially difficult to cope with the consequences of infection by phytoplasmas, viruses and viroids. Qualitative methods of individual diagnostics using enzyme immunoassay, PCR methods and even sequencing are quite expensive and do not always provide an adequate answer about what kind of virus the plant is infected with. The diversity of viruses that damage plants is quite large. Meanwhile, the study of the pathogenesis of viruses shows that they cause changes in the composition of chlorophylls, carotenoids and anthocyanins.
In this work, we hypothesized that viral infection should be manifested by a change in color, during instrumental analysis of photographic images and images obtained by a hyperspectral camera. We demonstrate differences in pigment composition in leaves with clearly identified viral damage and report for the first time that differences in the green part of such leaves may represent significant potential for the development of optical instrumental monitoring methods.
Materials and methods
The study was conducted in the quarantine area of the Plant Protection Laboratory of the N. V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences (MBG RAS), Moscow. Testing was performed by enzyme-linked immunosorbent assay (ELISA based on standard methodology) using the Kit Neogen Europe Ltd. The optical density of the oxidation product was determined using the AIER‑01 UNIPLAN TM enzyme immunoassay analyzer.
For RT-PCR of lilac leaf chlorosis virus (LLCV), RNA isolated from lilac leaves using the PhytoSorb magnetic particle kit (Synthol) was used. Primers to conservative regions of the Ilarvirus (Lilac Leaf Chlorosis Virus – LLCV) transport protein were used. IlaCPF: 5’-gcaatcgaacggagctagtg‑3’ IlaCPF: 5’-cacaaagctgacagaaggca‑3’. The reaction products were separated by electrophoresis in 1% agarose gel.
HSI (hyperspectral imaging) data were obtained using the hyperspectral camera of the Synergotron M.Gk (hyperspectral research module). Image analysis of LLCV-damaged leaves was carried out at subsequent sampling points for spectrophotometric analysis. To process the images the photochemical reflectance index (PRI) [1] and the normalized vegetation index (NDVI) [2] were used. The general formula for both indices is:
Index = (ρλ1 – ρλ2) / (ρλ1 + ρλ2)
PRI = (ρ531 – ρ570) / (ρ531 + ρ570)
NDVI = (ρ800 – ρ680) / (ρ800 + ρ680)
Where λ denotes specific wavelengths, ρ denotes the reflectance, and the numbers 1 and 2 denote the different wavelengths. The values of both indices vary from –1 to 1. The range of PRI index values is from –0.2 to 0.2, the range of NDVI index values is from 0.5 to 0.95.
NDVI helps to better separate the background and reveal pronounced foci of infection. NDVI is primarily sensitive to chlorophyll content [3]. The PRI index allows earlier detection of leaf damages, is sensitive to changes in carotenoid pigments, and is useful for assessing photosynthetic efficiency and stress in plants.
The images were taken with an RGB camera with an Imx 415 Sony 8.0 MP matrix. The HSV (Hue, Saturation, Brightness) color model was used, where all colors of the visible spectrum can be described by the hue value within the range of 0–360°, to construct a correlation of the color index with the chlorophyll value [4].
Results
To date, 13 types of viruses are known that affect lilacs, belonging to six families [5]. The following pathogens have been identified in lilac: elm mottle virus (EMoV), arabis mosaic virus (ArMV), cherry leafroll virus (CLRV), lilac ringspot virus (LRMV), tomato black ring virus (ToBRV), tomato bushy stun virus (ToBSV), tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), ligustrum virus A (LVA), lilac leaf chlorosis virus (LLCV). For lilac mottle virus (LMoV), lilac ringspot virus (LRSV) and lilac chlorotic leaf spot virus (LCLV), data are limited [5; 6].
As a result of systematic monitoring of lilac plantings in the Main Botanical Garden and Moscow Region, pathogens of viral etiology that are not typical for lilac culture have become widespread [7]. In the tested Syringa populations, we recorded a variety of symptoms characteristic of the phenotypic manifestation of viral diseases: chlorosis, ring spotting, linear pattern, mosaic, lightening of veins, necrosis, mottling, and various types of deformation. Symptoms appear in early spring and are extremely varied and change during the growing season. The most common and characteristic signs of the viral manifestation on lilacs are various types of mosaic, which are characterized by a change in the color of the leaves (flowers). For example, these may be small yellow spots that gradually merge. The mosaic can be limited to individual areas or cover the entire leaf, reminiscent of marbling. The white pattern is often accompanied by severe chlorosis. Leaves may become almost white with occasional green areas.
Mosaic can also appear on young leaves as light, blurred spots between the veins. Mottling on lilacs is quite common, but symptoms are usually vague. They appear in the spring and are visible for two to three weeks. A characteristic manifestation of some viruses is the formation of rings, semi-rings and spots, which can subsequently become necrotic. Usually a complex infection occurs, as a result of which the external signs are modified. The manifestation of the same viruses on different varieties and species of lilac may vary, while viruses of different species may cause similar phenotypic traits or their elements. For example, in addition to ArMV, CLRV, EMoV (Figure 1), the specialized viruses LRMoV and LLCV on lilac were diagnosed. And also, in addition to the already known ones, new and unusual viruses: carnation mottle virus (CarMoV), cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV) and potato virus “Y” (PVY) were detected. LLCV was also first registered in the Moscow region using the PCR method. The virus has become widespread in the city’s young plantings. The virus was not detected in the lilac collection or in other GBS ecosystems.
To evaluate the sampled leaves the spectral analysis methods were applied. In this case, areas with a green color (point 1), areas with signs of chlorosis (point 2) and areas with less pronounced or borderline damage (point 3) were analyzed for the quantitative content of pigments (chlorophylls a, b and carotenoids). At the sampling sites, an analysis of the color characteristics of the red, green and blue (RGB) spectrum indicators was carried out, as well as the calculation of H °, after which a correlation analysis of the entire volume of data was carried out and selectively for the data obtained in zones without damage, with chlorosis and intermediate (see Table).
The revealed correlation is valid for values greater than |0.7|. The obtained data demonstrate the possibility of analyzing damage to photosynthesis in infected plants specifically by the undamaged part of the leaves, where a correlation between the expression of the RGB color parameters and chlorophyll a was found. A correlation was also found between carotenoid content and blue range digital values.
For hyperspectral analysis, samples were taken from lilac bushes that had characteristic manifestations of chlorosis in varying degrees of severity and which were probably infected with several viruses. In further work, the samples that had confirmed Lilac Leaf Chlorosis Virus (LLCV) infection were used (Fig. 2). Thus, six samples were selected for further analysis.
The obtained data indicate the possibility of using the Synergotron M.Gk hyperspectral camera for monitoring and analysis of viral pathogens. A comparative analysis should be carried out at 3–4 points that will characterize the development of infection (Fig. 3).
We consider the use of the PRI index to be more promising, since the leaf damage zones were not obvious when using NDVI.
The results confirm that the use of hyperspectral images can significantly facilitate the work of plant pathologists and be used both for monitoring the condition of plantings and for culling planting material in propagation nurseries, as well as preventing the placement of infected material in collection banks for in vitro clonal micropropagation.
Discussion
The genus Syringa L. (Oleaceae) includes many intraspecific and interspecific varieties, the number of which reaches 2300. Lilac is one of the most common shrubs for landscaping in the world, including both northern and southern regions. This ornamental plant is also a source of many natural phytochemical and pharmacological compounds [8]. Lilac is a vegetatively propagated crop, so it is necessary to cull infected accessions in arboretums and create stable collections in vitro cultures [9; 10].
Viral pathogens demonstrate a systemic nature of infection and the infected plant remains sick throughout its life. Changes in the species composition of viruses, the ratio and structure of their populations, the spread of new infections that are not characteristic of a particular plant are shown [11].
Monitoring of viral pathogens in Syringa populations in the MBG revealed that the most noticeable trend is the predominance of viruses with a wide host range (ToMV, CMV, ArMV) and specific to other crops (AMV, CarMV, PVY, EMoV). Eight atypical and two specialized viral pathogens were also found on lilac [6]. The highest frequency rates within 55–70% of accessions were noted for CMV, followed by ArMV, TMV, EMoV, PVY, CarMV (45%, 43%, 37%, 28%, 13%). The presence of LRMoV was detected in only 7% of accessions. It was found that complex diseases caused by several viruses are predominantly common in Syringa populations.
Monoinfection was detected in only 40% of the accessions tested. The species composition was constantly transforming. When two or more viruses interact in one host, the suppressive activity of one of the viruses can have a complementary effect on the viruses included in the complex and lead to infection of the plant by their entire conglomerate [12].
The main objective of our research was to study the spectrum of viral pathogens of Syringa L. Comparing the obtained images with the superimposed heat maps of the above indices, we can conclude that NDVI helps to better separate the background (non-living objects, on a painted field or on a sheet of paper), but also helps with the definition of pronounced foci of plant disease. NDVI is primarily sensitive to chlorophyll content and is widely used to estimate biomass, leaf area index and total vegetation canopy [3]. At the same time, the PRI index makes it possible to identify diseases or leaf damage at early stages; it is sensitive to changes in carotenoid content and responsive to changes in photosynthesis and stress in plants.
Acknowledgment
Study was carried out within the framework of the state assignment of the N. V. Tsitsin MBG of the RAS (No. 124030100058-4) and the ARRIAB (No. 0431 2022-0003). The results of the work were obtained using the Synergotron M.Gk Modules (ANO Institute for Development Strategy, Russia).
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