Detection of Fusarium oxysporum f.sp. lactucae race 1 and 4 via race-specific real-time PCR and target enrichment

Fusarium oxysporum f.sp. lactucae (Fol) causes a vascular disease in lettuce that results in significant yield losses. Race-specific and sensitive real-time PCR assays were developed for Fol races 1 and 4, which are prevalent in Europe. Using genotyping-by-sequencing, unique DNA loci specific to each race were identified and subsequently used for the design of primers and hydrolysis probes. Two assays per race were developed to ensure specificity. The two assays of each race could be run in duplex format, while still giving a sensitivity of 100 fg genomic DNA for all assays. Sample preparation methods were developed for plant tissue, soil, and surfaces, with an extra enrichment step when additional sensitivity was required. By controlling the incubation conditions during the enrichment step, the real-time PCR signal could be matched to the number of spore equivalents in the original sample. When enriching naturally infested soil, down to six conidiospore equivalents L-1 soil could be detected. As enrichment ensures sensitive detection and focuses on living Fol propagules, it facilitates the evaluation of control measures. The developed detection methods for soil and surfaces were applied to samples from commercial lettuce farms and confirmed the prevalence of Fol race 4 in Belgium. Monitoring of soil disinfestation events revealed that despite a dramatic decrease in quantity, the pathogen could still be detected either immediately after sheet steaming or after harvesting the first new crop. The detection method for plant tissue was successfully used to quantify Fol in lettuce inoculated with race 1, race 4 or a combination of both. Under the temperature conditions used, race 4 was more aggressive than race 1, as reflected in larger amounts of DNA of race 4 detected in the roots. These newly developed assays are a promising tool for epidemiological research as well as for the evaluation of control measures

Glasshouse-specific occurrence of basal rot pathogens and the seasonal shift of Rhizoctonia solani anastomosis groups in lettuce

Basal rot is a common disease in Belgian lettuce, which is mainly controlled by fungicides and chemical soil disinfestation. A seasonal appearance of the basal rot pathogens: Rhizoctonia solani, Sclerotinia spp., Botrytis cinerea and Pythium spp. has been reported, but lettuce growers use standard spraying schemes, irrespective of the occurrence of the pathogen. Due to stricter regulations and environmental concerns the superfluous use of fungicides should be omitted. We investigated if the use of fungicides could be reduced by only controlling the active pathogens. Therefore, lettuce was continuously grown in three glasshouses without any fungal disease control and the active pathogens causing basal rot were identified. The occurrence of basal rot pathogens appeared to be glasshouse specific and the different basal rot pathogens were active throughout the year. However, a seasonal appearance of R. solani anastomosis groups and Pythium spp. was observed with AG4-HGI and Pythium ultimum active at higher temperatures and AG2-1, AG-BI, AG1-IB and Pythium sylvaticum at lower temperatures. We report for the first time the isolation of AG-BI from infected plants. Each R. solani anastomosis group had its own optimal growth rate in vitro. Differences in pathogenicity between R. solani anastomosis groups were observed on detached leaves. AG1-IB and AG4-HGI were most pathogenic, followed by AG2-1 and AG-BI. These results show that the fungicide spraying scheme should be adapted to the occurring pathogens in the glasshouse. This information is of high importance in developing a sustainable control strategy for basal rot pathogens

Predicting Stored Period and Shelf Life Potential of Lamb’s Lettuce Using Vis/NIR Reflectance Spectroscopy

Lamb’s lettuce (Valerianella locusta L.) delivered to the vegetable auctions is not always fresh. The product can be stored for a certain time depending on the time of year. Stored and freshly harvested material is visually not distinguishable, but the shelf life potential of the first is limited. This leads to losses in distribution and a lower quality for consumers. The aim of this work was to develop a fast nondestructive methodology that uses visible/near infrared (Vis/NIR) reflectance spectroscopy that can determine whether lamb’s lettuce has been in storage and give an idea about the remaining shelf life potential. The Vis/NIR reflectance spectra were linked to the time in storage and to the quality of the samples at the end of a shelf life holding period by partial least squares regression (PLS). The prediction of the remaining shelf life potential was not successful based on colorimetric quality attributes. There was a better performance when the quality scores given by a panel of experts was used, but for the moment this prediction model lacks accuracy. The detection and quantification of a prior storage period was validated with an external test set and the root mean square error of prediction (RMSEP) was 2.5 days. Vis/NIR reflectance spectroscopy was a valid, fast and non-destructive method for determining and quantifying a storage period of lamb’s lettuce.status: publishe

Estimation of the prior storage period of lamb’s lettuce based on visible/near infrared reflectance spectroscopy

Lamb’s lettuce (Valerianella locusta L.) can be stored up to 28 days without being indistinguishable from fresh material by the human eye. However, due to the prior storage period the shelf life potential is limited and this leads to losses in distribution and a lower quality for the consumer. This work aims to develop a rapid and non-destructive methodology using visible/near infrared (Vis/NIR) reflectance spectroscopy to detect and quantify a prior storage period. Vis/NIR reflectance spectra were linked to the time in storage by partial least squares regression (PLS). Different variable selection techniques (interval PLS, Variable Importance in Projection scores, Genetic Algorithms PLS and Monte Carlo Uninformative Variable Elimination PLS) were combined to improve the accuracy and robustness of the prediction model, while decreasing the number of used wavelengths. The final model used only 10% of the original wavelengths, while the root mean squared error of cross validation decreased from 6.0 to 3.6 days. The final model was tested using 2 external test sets and had a maximum root mean square error of prediction of 3.7 days. Vis/NIR reflectance spectroscopy can be a valuable, rapid and non-destructive method for identifying and quantifying a prior storage period of lamb’s lettuce.publisher: Elsevier articletitle: Estimation of the prior storage period of lamb’s lettuce based on visible/near infrared reflectance spectroscopy journaltitle: Postharvest Biology and Technology articlelink: http://dx.doi.org/10.1016/j.postharvbio.2015.11.007 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

A thorough study of a Paratylenchus sp. in glasshouse-grown lettuce : characterisation, population dynamics, host plants and damage threshold as keys to its integrated management

In glasshouses practising monoculture of butterhead lettuce in Belgium, high densities of pin nematodes (Paratylenchusspp.) are frequently associated with reduced plant growth. Growers currently apply chemical soil disinfestation measures to manage this problem, although stricter phytosanitary regulations are forcing a shift towards integrated management. Efficient implementation of such management requires knowledge about the factors influencing nematode population dynamics, and the damage threshold for lettuce. The nematode populations in five Belgian glasshouses were monitored for at least 1 year by frequently soil sampling at 0-30 cm and 30-60 cm depth. An undescribed species of Paratylenchuswas identified in all glasshouses based on morphological and molecular features. High nematode densities (>20,000 (100 ml soil)(-1)) occurred in winter and spring. Chemical soil disinfestation lowered these populations greatly, although up to 14% survived in the deeper soil layer. After soil steaming under negative pressure, no pin nematodes were found. After 2 months of black fallow pin nematode densities were reduced by 50%-76%. Lamb's lettuce, parsley and wild rocket were found to be poor hosts in a pot experiment, while reproduction factors (P-f/P-i) on lettuce cultivars varied between 1 and 3. In three experiments with butterhead lettuce 'Cosmopolia' in pots with a series of 9 or 10 densities of Paratylenchussp. [up to 35,000 (100 ml soil)(-1)], no damage to lettuce heads was observed. However, root weight and root quality were reduced, and the corresponding damage thresholds were rather low [1,754 and 362Paratylenchussp. (100 ml soil)(-1), respectively]. Management strategies such as crop rotation, soil disinfestation or fallow are recommended to avoid pin nematode population build-up

Pratylenchus penetrans, a potential risk in glasshouse-grown lettuce : population dynamics and damage threshold

The root-lesion nematode, Pratylenchus penetrans, causes growth reduction in glasshouse-grown lettuce and is mainly controlled by chemical soil disinfestation. Integrated management strategies require more knowledge about the population dynamics and damage threshold densities. We monitored the population during 2.5 years in a commercial glasshouse by sampling soil in the same four 1 m(2) spots at 0-30 cm and 30-60 cm depth. The grower grew lettuce in rotation with leek, applied 1,3-dichloropropene in summer and left the field fallow during winter. Growing leek reduced the nematode population slightly but chemical soil disinfestation lowered the numbers drastically, although 41% of the nematodes in the deeper layer survived. Black fallow resulted in a slight increase of the population, probably due to hatching. Two pot experiments with ten densities of P. penetrans were conducted to estimate the damage threshold for a summer and autumn cultivar ('Cosmopolia' and 'Brighton', respectively). The thresholds for lettuce weight were 669 and 3834 P. penetrans (100 ml soil)(-1) in summer and autumn, respectively, but with considerable variability in estimated parameters. The thresholds for root damage were much lower: 204 and 48 P. penetrans (100 ml soil)(-1). Nematode numbers did not increase on lettuce in the pot tests (maximum multiplication rate was 0.40) but increased slightly in the commercial setting. These results show that populations of P. penetrans build up slowly when butterhead lettuce is rotated with leek and fallow, but chemical soil disinfestation is required to avoid numbers resulting in root damage

Fusarium isolates from Belgium causing wilt in lettuce show genetic and pathogenic diversity

Fusarium oxysporum f. sp. lactucae race 4 causes vascular necrosis and wilting of lettuce. First observed in Belgium in 2015, the lack of disease resistance in commercial cultivars allowed this pathogen to spread to nearly the entire Belgian production area within 4 years. Different levels of disease development were observed in different commercial greenhouses. To help explain this variation, we collected 78 Fusarium isolates and characterized them both physiologically and genetically. Molecular race identification indicated that 91% of the isolates belonged to race 4, while 6% of the isolates belonged to race 1, which was not previously reported in Belgium. Pathogenicity assays using differential cultivars confirmed the molecular race assignment of selected isolates. Cultivar Patriot was identified as a suitable new differential cultivar to race 4. Race 4 isolates were more aggressive than race 1 isolates at 24 degrees C, but only when using chlamydospore inoculum instead of a root dip assay containing microconidia. Variation in pathogenicity and aggressiveness of the races may explain differences in disease development in commercial greenhouses. Based on genotyping-by-sequencing (GBS), race 1 and race 4 isolates were highly similar to reference isolates. Fusarium curvatum, F. oxysporum f. sp. tulipae and F. oxysporum f. sp. rhois were phylogenetically separated from F. oxysporum f. sp. lactucae races 1 and 4 based on the GBS data, but not when using multilocus sequence data. Within F. oxysporum f. sp. lactucae race 4, the GBS data differentiated two rather homogeneous groups, suggesting at least two introductions. However, the two groups did not differ in aggressiveness

A thorough study of a Paratylenchus

In glasshouses practising monoculture of butterhead lettuce in Belgium, high densities of pin nematodes (Paratylenchusspp.) are frequently associated with reduced plant growth. Growers currently apply chemical soil disinfestation measures to manage this problem, although stricter phytosanitary regulations are forcing a shift towards integrated management. Efficient implementation of such management requires knowledge about the factors influencing nematode population dynamics, and the damage threshold for lettuce. The nematode populations in five Belgian glasshouses were monitored for at least 1 year by frequently soil sampling at 0-30 cm and 30-60 cm depth. An undescribed species of Paratylenchuswas identified in all glasshouses based on morphological and molecular features. High nematode densities (>20,000 (100 ml soil)(-1)) occurred in winter and spring. Chemical soil disinfestation lowered these populations greatly, although up to 14% survived in the deeper soil layer. After soil steaming under negative pressure, no pin nematodes were found. After 2 months of black fallow pin nematode densities were reduced by 50%-76%. Lamb's lettuce, parsley and wild rocket were found to be poor hosts in a pot experiment, while reproduction factors (P-f/P-i) on lettuce cultivars varied between 1 and 3. In three experiments with butterhead lettuce 'Cosmopolia' in pots with a series of 9 or 10 densities of Paratylenchussp. [up to 35,000 (100 ml soil)(-1)], no damage to lettuce heads was observed. However, root weight and root quality were reduced, and the corresponding damage thresholds were rather low [1,754 and 362Paratylenchussp. (100 ml soil)(-1), respectively]. Management strategies such as crop rotation, soil disinfestation or fallow are recommended to avoid pin nematode population build-up