scholarly journals High-Throughput Phenotyping of Leaf Discs Infected with Grapevine Downy Mildew Using Shallow Convolutional Neural Networks

Agronomy ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1768
Author(s):  
Daniel Zendler ◽  
Nagarjun Malagol ◽  
Anna Schwandner ◽  
Reinhard Töpfer ◽  
Ludger Hausmann ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Disease Severity ◽  
High Throughput ◽  
Imaging System ◽  
Leaf Disc ◽  
Ground Truth Data ◽  
Breeding Lines ◽  
Grapevine Downy Mildew ◽  
Leaf Discs ◽  
Efficient Detection

Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola ((Berk. and Curt.) Berl. and de Toni) causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to considerably reduce the amount of time during leaf disc phenotyping. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.

scholarly journals High-throughput phenotyping of leaf discs infected with grapevine downy mildew using shallow convolutional neural networks

2021 ◽  
Author(s):  
Daniel Zendler ◽  
Nagarjun Malagol ◽  
Anna Schwandner ◽  
Reinhard Töpfer ◽  
Ludger Hausmann ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Disease Severity ◽  
High Throughput ◽  
Imaging System ◽  
Leaf Disc ◽  
Ground Truth Data ◽  
Breeding Lines ◽  
Grapevine Downy Mildew ◽  
Leaf Discs ◽  
Efficient Detection

Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to reduce the amount of time during leaf disc phenotyping considerably. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.

scholarly journals Effect of Detergent on the Quantification of Grapevine Downy Mildew Sporangia from Leaf Discs

HortScience ◽  
2015 ◽  
Vol 50 (5) ◽  
pp. 656-660 ◽  
Author(s):  
Atsushi Kono ◽  
Akihiko Sato ◽  
Bruce Reisch ◽  
Lance Cadle-Davidson
Keyword(s):  
Downy Mildew ◽  
Leaf Disc ◽  
Plasmopara Viticola ◽  
Inhibitory Effect ◽  
Tween 20 ◽  
Infected Leaf ◽  
Grapevine Downy Mildew ◽  
Visual Rating ◽  
Leaf Discs ◽  
Highly Correlated

Grapevine downy mildew (DM), caused by the oomycete Plasmopara viticola (Berk. & Curt.) Berlese & de Toni, is a major disease, especially in humid viticultural areas. Development of resistant cultivars is an important objective for grapevine breeding. To establish a reliable and inexpensive quantitative method to aid in breeding for DM resistance, we improved the method for counting the number of sporangia on leaf discs, and developed a method for counting the number of sporangia in solution. To prevent the loss of DM sporangia from adhesion onto plastic ware, we found as little as 0.01% Tween 20 was effective. On the other hand, this detergent was shown to have a severe inhibitory effect upon DM infection of leaves. We developed a sporangia counting method using dried droplets of DM suspensions, and the method was highly correlated with counting by hemacytometer (R2 > 0.96). The nonparametric Spearman’s rank correlations between visual rating and the number of the sporangia were as high as ρ = 0.82 to 0.91, suggesting that evaluation by the visual rating could provide a good estimate of the sporangia numbers on leaf discs. We established a high-throughput and inexpensive method with acceptable accuracy for DM resistance evaluation based on a leaf disc assay, and our results suggested that visual ratings of infected leaf discs provide a good estimate of sporangia numbers.

scholarly journals Distribution of Baseline Sensitivities to Azoxystrobin Among Isolates of Plasmopara viticola

Plant Disease ◽  
2000 ◽  
Vol 84 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Francis P. Wong ◽  
Wayne F. Wilcox
Keyword(s):  
New York ◽  
Disease Severity ◽  
Disease Incidence ◽  
Leaf Disc ◽  
Plasmopara Viticola ◽  
Grapevine Downy Mildew ◽  
Coefficients Of Variation ◽  
Control Versus ◽  
Strobilurin Fungicide ◽  
Effective Doses

An excised leaf disc assay was developed for determining the sensitivity of isolates of Plasmopara viticola (causal agent of grapevine downy mildew) to the strobilurin fungicide azoxystrobin. Five repeated assays with the same five single-sporangiophore isolates showed that the technique yielded reproducible results; that is, coefficients of variation ranged from 4.0 to 20% (mean 12%) for effective doses for 50% control (ED50 values) based on disease incidence and from 4.4 to 14% (mean 8.1%) for ED50 values based on disease severity. Then, the assay was applied to 81 single-sporangiophore isolates of P. viticola collected from 10 geographically distinct vineyards in western New York, providing a baseline distribution of sensitivities within this population. For disease incidence, individual ED50 values ranged from 0.05 to 0.94 μg/ml (mean 0.40 μg/ml), whereas for disease severity they ranged from 0.04 to 0.78 μg/ml (mean 0.24 μg/ml). When 61 of these isolates were similarly tested at a single discriminatory dose of 0.50 μg/ml, azoxystrobin provided 61% control of disease incidence versus 41 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively; for disease severity (colony diameter), azoxystrobin provided 80% control versus 57 and 1.1% control for trifloxystrobin and kresoximmethyl, respectively. These results provide information that can be utilized in future monitoring of P. viticola resistance to azoxystrobin and indicate differences in the intrinsic activities of the three strobilurin fungicides against this pathogen.

scholarly journals Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes

2017 ◽  
Vol 107 (12) ◽  
pp. 1549-1555 ◽  
Author(s):  
Konstantin Divilov ◽  
Tyr Wiesner-Hanks ◽  
Paola Barba ◽  
Lance Cadle-Davidson ◽  
Bruce I. Reisch
Keyword(s):  
Computer Vision ◽  
Downy Mildew ◽  
Vision System ◽  
Leaf Disc ◽  
Computer Vision System ◽  
Grapevine Downy Mildew ◽  
Leaf Trichomes ◽  
Cost Efficient ◽  
Percent Area ◽  
Leaf Trichome

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × ‘Horizon’ [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

Fungicide Sensitivity Survey of Plasmopara viticola Populations in Georgia Vineyards

2020 ◽  
Vol 21 (4) ◽  
pp. 256-261
Author(s):  
Sarah E. Campbell ◽  
Phillip M. Brannen ◽  
Harald Scherm ◽  
Marin T. Brewer
Keyword(s):  
Downy Mildew ◽  
Acid Amide ◽  
Leaf Disc ◽  
Plasmopara Viticola ◽  
Survey Period ◽  
Resistance Development ◽  
Grapevine Downy Mildew ◽  
Quinone Outside Inhibitor ◽  
History Of ◽  
Qoi Resistance

Grapevine downy mildew, caused by Plasmopara viticola, is among the most damaging diseases of grapes globally and in the viticultural regions of Georgia (U.S.A.). Although management of this disease typically involves fungicide applications, resistance development in P. viticola can render chemical management ineffective. The objective of this study was to survey fungicide sensitivities of P. viticola populations in vineyards across three regions in Georgia. Samples were collected from eight vineyards in 2017 and four in 2018 and tested phenotypically for sensitivity to quinone outside inhibitor (QoI), carboxylic acid amide (CAA), and phenylamide (PA) fungicides using leaf disc bioassays. In addition, DNA was extracted from P. viticola samples collected from 2015 through 2018 in these 12 vineyards and six others for a total of 18 vineyards sampled. All available DNA samples were tested for the presence of the known resistance-causing mutations G143A (QoI) and G1105S (CAA) using polymerase chain reaction (PCR). This study documented widespread occurrence of QoI resistance in P. viticola in Georgia, with 83.0% of isolates collected in 2017 and 2018 testing positive for QoI resistance based on bioassay and 82.9% of isolates collected from 2015 to 2018 testing positive for QoI resistance through PCR testing. In total, 94.4% (17/18) of surveyed vineyards had confirmed QoI resistance by the conclusion of the survey period. No reduced sensitivity to CAA or PA fungicides was identified. Due to widespread QoI resistance, these fungicides should not be relied upon for downy mildew management in Georgia vineyards with a long history of their use.

scholarly journals First Report of Pseudoperonospora cubensis Causing Downy Mildew of Trichosanthes cucumerina in Malaysia

Plant Disease ◽  
2010 ◽  
Vol 94 (5) ◽  
pp. 642-642 ◽  
Author(s):  
M. Salati ◽  
M. Y. Wong ◽  
M. Sariah ◽  
H. Nik Masdek
Keyword(s):  
Downy Mildew ◽  
Average Length ◽  
Leaf Disc ◽  
Its Region ◽  
Pseudoperonospora Cubensis ◽  
Abaxial Surface ◽  
First Report ◽  
Leaf Discs ◽  
Morphological Criteria ◽  
Field Samples

In December 2008, infected leaves of Trichosanthes cucumerina were observed on commercial cucurbit farms located in Pontian, Johor (south of West Malaysia). Bright yellow and small necrotic lesions were observed on the adaxial surface of the leaves, whereas sporangiophores were observed on pale yellowish brown-to-brown lesions on the abaxial surface. The length and width of the sporangia ranged from 19 to 36 μm (28.6) and 11 to 23 μm (17.6), respectively. The length of the sporangiophores ranged from 310 to 450 μm, with an average length of 380 μm. The pathogen was identified as Pseudoperonospora cubensis on the basis of the morphological criteria described by Palti and Cohen (2). To confirm the morphological findings, DNA was extracted from symptomatic tissue and the internal transcribed spacer (ITS) region was PCR amplified using primers ITS5-P2 and ITS4 (3). The appropriate-sized amplicon was gel excised and column purified and then submitted for direct sequencing. The resulting 802 bp amplified ITS region was 100% identical to published P. cubensis sequences (GenBank Accession Nos. EU876603, EU876584, and AY198306). This sequence was deposited with NCBI GenBank under the Accession No. GU233293. In this study, pathogenicity tests were conducted using detached leaf disc assays (1) and a P. cubensis isolate obtained from T. cucumerina. For this purpose, leaf discs were excised from 6- to 8-week-old leaves of T. cucumerina using a 20-mm cork borer. Five leaf discs were placed with their abaxial surface facing upward on moist filter paper in petri dishes. Each of four leaf discs was inoculated with four 10-μl droplets of a 1 × 105 per ml sporangial suspension, whereas the fifth disc was inoculated with water droplets and served as a control. Three replications were completed. The leaf discs were placed in darkness at 14 ± 2°C for 24 h and subsequently incubated with a 12-h photoperiod. After 10 days, sporulation was observed on the sporangia-inoculated leaf discs with similar morphological features to the initial field samples. To our knowledge, this is the first report of P. cubensis causing downy mildew of T. cucumerina in Malaysia. References: (1) A. Lebeda and M. P. Widrlechner. J. Plant Dis. Prot. 110:337, 2003. (2) J. Palti and Y. Cohen. Phytoparasitica 8:109, 1980. (3) H. Voglmayr and O. Constantinescu. Mycol. Res. 112:487, 2008.

scholarly journals Screening of Croatian Native Grapevine Varieties for Susceptibility to Plasmopara viticola Using Leaf Disc Bioassay, Chlorophyll Fluorescence and Multispectral Imaging

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 661
Author(s):  
Petra Štambuk ◽  
Iva Šikuten ◽  
Darko Preiner ◽  
Ana Nimac ◽  
Boris Lazarević ◽  
...  
Keyword(s):  
Chlorophyll Fluorescence ◽  
Stress Response ◽  
Vitis Vinifera ◽  
Downy Mildew ◽  
Multispectral Imaging ◽  
Leaf Disc ◽  
Plasmopara Viticola ◽  
Post Inoculation ◽  
Time Points ◽  
Leaf Discs

In the era of sustainable grapevine production, there is a growing demand to define differences between Vitis vinifera varieties in susceptibility to downy mildew. Croatia, as a country with a long tradition of grapevine cultivation, preserves a large number of native grapevine varieties. A leaf disc bioassay has been conducted on 25 of them to define their response to downy mildew, according to the International Organisation of Vine and Wine (OIV) descriptor 452-1, together with the stress response of the leaf discs using chlorophyll fluorescence and multispectral imaging with 11 parameters included. Time points of measurement were as follows: before treatment (T0), one day post-inoculation (dpi) (T1), two dpi (T2), three dpi (T3), four dpi (T4), six dpi (T5), and eight dpi (T6). Visible changes in form of developed Plasmopara viticola (P. viticola) sporulation were evaluated on the seventh day upon inoculation. Results show that methods applied here distinguish varieties of different responses to downy mildew. Based on the results obtained, a phenotyping model in the absence of the pathogen is proposed, which is required to confirm by conducting more extensive research.

scholarly journals Elaboration and validation of a diagrammatic scale to assess downy mildew severity in grapevine

2014 ◽  
Vol 44 (8) ◽  
pp. 1384-1391 ◽  
Author(s):  
Cláudia Regina Scapin Buffara ◽  
Francislene Angelotti ◽  
Rafael Augusto Vieira ◽  
Amauri Bogo ◽  
Dauri José Tessmann ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Disease Severity ◽  
Economic Losses ◽  
Grapevine Downy Mildew ◽  
Stimulus Response ◽  
Black And White ◽  
Color Scale ◽  
Accuracy And Precision ◽  
Grapevine Leaves ◽  
Diagrammatic Scale

The downy mildew, caused by Plasmopora viticola, is one of the most important grapevine (Vitis vinifera) diseases in Southern Brazil, causing defoliation and economic losses. The evaluation of disease severity is an important decision for adoption of strategies and tactics for disease control. Therefore, the objective of this work was to elaborate and to validate a diagrammatic scale to assess downy mildew severity in grapevine, respecting the limitations of visual acuity. The diagrammatic scale with seven levels of disease severity of 1, 3, 6, 12, 25, 50 and 75% was developed in two versions of black-and-white and color scale. The scales were tested and validated by eight raters with and without previous experience with grapevine downy mildew. The raters estimated the severity of 30 grapevine leaves with different downy mildew severity, with and without the use of the scales. Minimum, intermediate and maximum severity levels were collected according to Weber-Fechner's stimulus-response law. The accuracy and precision were analyzed by linear regression between the actual and the estimated severity. Actual severity was assessed with ASSESS(r) Program. The scales provided good levels of accuracy (means of 88%) and excellent levels of precision (means of 95%). The raters showed great precision and accuracy when used the diagrammatic scale. The color scale provided more precise and accurate estimates than the black-and-white scale. The scale proposed in this work presented appropriate applicability for downy mildew evaluation in grapevine.

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes

2019 ◽  
Vol 11 (3) ◽  
pp. 954-969 ◽  
Author(s):  
Yann Dussert ◽  
Isabelle D Mazet ◽  
Carole Couture ◽  
Jérôme Gouzy ◽  
Marie-Christine Piron ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Genome Assembly ◽  
Population Genomics ◽  
Plasmopara Viticola ◽  
Plant Diseases ◽  
Host Specialization ◽  
Grapevine Downy Mildew ◽  
Downy Mildews ◽  
Genes Encoding ◽  
High Level

Abstract Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94 Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5 kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant–pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species.

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