Warburgia ugandensis Leaf and Bark Extracts: An Alternative to Copper as Fungicide against Downy Mildew in Organic Viticulture?

In organic viticulture, copper-based fungicides are commonly used to suppress Downy Mildew infection, caused by the oomycete Plasmopara viticola. However, the frequent and intensive use of such fungicides leads to accumulation of the heavy metal in soil and nearby waters with adverse effects on the ecosystem. Therefore, alternative, organic fungicides against Downy Mildew are urgently needed to reduce the copper load in vineyards. In this study, the use of Warburgia ugandensis Sprague (Family Canellacea) leaf and bark extracts as potential fungicides against Downy Mildew were evaluated. In vitro (microtiter) and in vivo (leaf discs, seedlings) tests were conducted, as well as field trials to determine the efficacy of the extracts against Downy Mildew. The results revealed an MIC100 of 500 µg/mL for the leaf extract and 5 µg/mL for the bark extract. Furthermore, experiments with leaf discs and seedlings demonstrated a strong protective effect of the extracts for up to 48 h under (semi-) controlled conditions. However, in field trials the efficacy of the extracts distinctly declined, regardless of the extracts’ origin and concentration.

The Role of Betacyanins in Plant Salt Tolerance

<p>Soil salinity is a major threat to future food stability. Almost 20% of irrigated land is currently too saline to grow traditional crops. Moreover, rising sea levels, scarcity of fresh water, and more intense and prolonged periods of drought are exacerbating the problem. Saline soils severely reduce yields of most crop plants. By contrast, halophytes, which naturally thrive on saline substrates, have a variety of mechanisms to tolerate both the osmotic and cytotoxic components of salt stress. There has been concerted scientific effort worldwide to understand these mechanisms, and to introduce genes that may increase salinity tolerance in crop plants. Many halophytes in the Caryophyllales are pigmented red owing to a tyrosine-derived alkaloid called betacyanin. Recent studies using Disphyma australe, a succulent halophyte common on coastal dunes and rocky outcrops throughout New Zealand, have indicated a role for betacyanins in salinity tolerance. This thesis focuses on how the mechanism through which betacyanins might affect salt tolerance mechanisms in D. australe and whether the putative benefits of betacyanins on salt tolerance might be transferred to naturally non-betacyanic plants. Effects of betacyanin on Na+ distribution in salt-stressed leaves of red and green morphs of D. australe were studied using fluorescence microscopy, cryo-scanning electron microscopy with energy dispersive X-ray analysis, and atomic absorption spectrometry (AAS). In betacyanic leaves Na+ accumulated in the epidermis, while in green leaves Na+ was distributed more evenly across the epidermis and mesophyll. Both leaf types had similar numbers of salt glands, but salt secretion rates were higher in red than in green leaves. Betacyanic leaves under salt stress were able to maintain relatively high K+/ Na+ ratios, essential for many metabolic processes, while the leaves of green plants were not. Leaf sections stained with fluorescein diacetate and propidium iodide showed that mesophyll viability decreased significantly in green leaves under salt stress, while there was almost no decrease in mesophyll viability in the presence of betacyanins. Thus, betacyanic leaves might protect the photosynthetically active mesophyll from cytotoxic effects of Na+ by accumulating Na+ in the epidermis instead of the mesophyll. This in turn leads to more efficient salt secretion and higher K+/ Na+ ratios in the mesophyll, resulting in increased mesophyll viability under salt stress. Effects of high apoplastic sodium concentrations on ion flux kinetics in mesophyll tissue was studied using the non-invasive microelectrode ion flux estimation technique. Mesophyll cells of both betacyanic and green leaves showed a highly unusual K+ flux response; most crop plants leak K+ out of cells upon salt stress, but D. australe and the native Australian Disphyma crassifolium both showed K+ influx upon salt stress. Actively taking up K+ from the apoplast to maintain a high cytosolic K+/ Na+ ratio during salt stress might be an entirely new mechanism to combat the cytotoxic stress component of salinity stress in these halophytes. The salt induced K+ uptake was dependent on the presence of Cl- and Cl- was also taken up into mesophyll cells upon salt stress. Taking up both cations and anions at the same time could avoid membrane depolarisation. Voltage-gated channels, which are involved in the salt induced K+ efflux in glycophytes, would not be activated and this could be a new mechanism to avoid a K+ leak during salt stress. To test whether the beneficial effect of betacyanin production on salt tolerance could be transferred to naturally non-betacyanic plants, transgenic betacyanin-over-expression (BtOE) mutants of Nicotiana tabacum were generated by our colleagues at Plant & Food Research Ltd. Betacyanins in leaf discs of N. tabacum were associated with decreased chlorophyll degradation upon high light and high salt stress. Additionally, the decline in maximum quantum efficiency of PSII after high light and salt treatment was significantly greater in green than in betacyanic leaves. Placing a polycarbonate filter with a similar absorption spectrum to betacyanin over green N. tabacum leaf discs had a similar effect to the presence of betacyanin. Thus, betacyanins probably have a photoprotective effect in N. tabacum, which is essential as both high light and salinity can impair photosynthesis. To assess if the salt tolerance enhancing effect of betacyanin production observed in the leaf discs also occurs in whole N. tabacum plants, the ability to recover from exposure to saturating light was assessed. Betacyanic plants were able to fully recover quicker after exposure to saturation light than green leaves. This research shows that the presence of betacyanins during salt stress correlates with an altered Na+ distribution in leaf tissues and a higher salt secretion rate, which contributed to higher mesophyll viability. Moreover, a completely new ion flux response to salt stress was observed in D. australe and D. crassifolium. The observed salt induced K+ uptake into the mesophyll cells during salt stress might be an entirely new mechanism, to maintain a high K+/ Na+ ratio in the cytosol and avoid the cytotoxic effects of Na+ in photosynthetically active tissue. The beneficial effects of betacyanins could also be transferred to non-betacyanic species, by introducing betacyanin production. These results strongly suggest that betacyanins play a role in salt tolerance in halophytes and might be a valuable resource in increasing the salt tolerance of naturally non-betacyanic crop plants.</p>

The Role of Betacyanins in Plant Salt Tolerance

<p>Soil salinity is a major threat to future food stability. Almost 20% of irrigated land is currently too saline to grow traditional crops. Moreover, rising sea levels, scarcity of fresh water, and more intense and prolonged periods of drought are exacerbating the problem. Saline soils severely reduce yields of most crop plants. By contrast, halophytes, which naturally thrive on saline substrates, have a variety of mechanisms to tolerate both the osmotic and cytotoxic components of salt stress. There has been concerted scientific effort worldwide to understand these mechanisms, and to introduce genes that may increase salinity tolerance in crop plants. Many halophytes in the Caryophyllales are pigmented red owing to a tyrosine-derived alkaloid called betacyanin. Recent studies using Disphyma australe, a succulent halophyte common on coastal dunes and rocky outcrops throughout New Zealand, have indicated a role for betacyanins in salinity tolerance. This thesis focuses on how the mechanism through which betacyanins might affect salt tolerance mechanisms in D. australe and whether the putative benefits of betacyanins on salt tolerance might be transferred to naturally non-betacyanic plants. Effects of betacyanin on Na+ distribution in salt-stressed leaves of red and green morphs of D. australe were studied using fluorescence microscopy, cryo-scanning electron microscopy with energy dispersive X-ray analysis, and atomic absorption spectrometry (AAS). In betacyanic leaves Na+ accumulated in the epidermis, while in green leaves Na+ was distributed more evenly across the epidermis and mesophyll. Both leaf types had similar numbers of salt glands, but salt secretion rates were higher in red than in green leaves. Betacyanic leaves under salt stress were able to maintain relatively high K+/ Na+ ratios, essential for many metabolic processes, while the leaves of green plants were not. Leaf sections stained with fluorescein diacetate and propidium iodide showed that mesophyll viability decreased significantly in green leaves under salt stress, while there was almost no decrease in mesophyll viability in the presence of betacyanins. Thus, betacyanic leaves might protect the photosynthetically active mesophyll from cytotoxic effects of Na+ by accumulating Na+ in the epidermis instead of the mesophyll. This in turn leads to more efficient salt secretion and higher K+/ Na+ ratios in the mesophyll, resulting in increased mesophyll viability under salt stress. Effects of high apoplastic sodium concentrations on ion flux kinetics in mesophyll tissue was studied using the non-invasive microelectrode ion flux estimation technique. Mesophyll cells of both betacyanic and green leaves showed a highly unusual K+ flux response; most crop plants leak K+ out of cells upon salt stress, but D. australe and the native Australian Disphyma crassifolium both showed K+ influx upon salt stress. Actively taking up K+ from the apoplast to maintain a high cytosolic K+/ Na+ ratio during salt stress might be an entirely new mechanism to combat the cytotoxic stress component of salinity stress in these halophytes. The salt induced K+ uptake was dependent on the presence of Cl- and Cl- was also taken up into mesophyll cells upon salt stress. Taking up both cations and anions at the same time could avoid membrane depolarisation. Voltage-gated channels, which are involved in the salt induced K+ efflux in glycophytes, would not be activated and this could be a new mechanism to avoid a K+ leak during salt stress. To test whether the beneficial effect of betacyanin production on salt tolerance could be transferred to naturally non-betacyanic plants, transgenic betacyanin-over-expression (BtOE) mutants of Nicotiana tabacum were generated by our colleagues at Plant & Food Research Ltd. Betacyanins in leaf discs of N. tabacum were associated with decreased chlorophyll degradation upon high light and high salt stress. Additionally, the decline in maximum quantum efficiency of PSII after high light and salt treatment was significantly greater in green than in betacyanic leaves. Placing a polycarbonate filter with a similar absorption spectrum to betacyanin over green N. tabacum leaf discs had a similar effect to the presence of betacyanin. Thus, betacyanins probably have a photoprotective effect in N. tabacum, which is essential as both high light and salinity can impair photosynthesis. To assess if the salt tolerance enhancing effect of betacyanin production observed in the leaf discs also occurs in whole N. tabacum plants, the ability to recover from exposure to saturating light was assessed. Betacyanic plants were able to fully recover quicker after exposure to saturation light than green leaves. This research shows that the presence of betacyanins during salt stress correlates with an altered Na+ distribution in leaf tissues and a higher salt secretion rate, which contributed to higher mesophyll viability. Moreover, a completely new ion flux response to salt stress was observed in D. australe and D. crassifolium. The observed salt induced K+ uptake into the mesophyll cells during salt stress might be an entirely new mechanism, to maintain a high K+/ Na+ ratio in the cytosol and avoid the cytotoxic effects of Na+ in photosynthetically active tissue. The beneficial effects of betacyanins could also be transferred to non-betacyanic species, by introducing betacyanin production. These results strongly suggest that betacyanins play a role in salt tolerance in halophytes and might be a valuable resource in increasing the salt tolerance of naturally non-betacyanic crop plants.</p>

What Could Arrest an Eriophyoid Mite on a Plant? The Case of Aculops allotrichus from the Black Locust Tree

Aculops allotrichus is a vagrant eriophyoid that lives gregariously on the leaves of the black locust tree. This study demonstrated that conspecifics can have a significant impact on A. allotrichus females on unprofitable, old black locust leaves and can arrest them on those leaves. The effect was more pronounced in females that were exposed to artificially injured individuals than to intact ones. They not only prolonged their sojourn on leaf discs with pierced conspecifics, but also preferred the leaf disc halves with damaged individuals to clean ones. Aculops allotrichus is the first described herbivore in which artificially injured conspecifics, instead of causing alarm, keep the foraging individuals within a risky patch. Other objects, such as artificially injured or intact heterospecifics, pollen or sand, were irrelevant to the eriophyoid females on old leaf patches. In tests with old leaves of maple, magnolia and hard kiwi vine, the females postponed their movement from non-host leaf discs, which suggests that they may need more time to recognise and evaluate unfamiliar plants than familiar ones.

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

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.

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

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.

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

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.

A Leaf Disc Assay for Evaluating the Response of Tea (Camellia sinensis) to PEG-Induced Osmotic Stress and Protective Effects of Azoxystrobin against Drought

Tea (Camellia sinensis), a globally cultivated beverage crop, is sensitive to drought, which can have an adverse effect on the yield and quality of tea. Azoxystrobin (AZ) is one kind of fungicide considered as an agent to relieve damage caused by stress. Initially, the response of tea plant to osmotic-gradient stress was evaluated using leaf disc assays with PEG-induced osmotic stress. The decline of the maximum quantum yield of PSII (Fv/Fm), actual photosynthetic efficiency of PS II (Y(II)), total chlorophylls, carotenoids, DPPH radical scavenging capacity, reducing power, total phenols, and the increase in MDA was observed in leaf discs treated with a gradient of PEG solutions (22.8, 33.2, 41.1% PEG, and blank). These results revealed that efficiency of photosystem II (PSII), photosynthetic pigments, and antioxidant ability in leaf discs were inhibited with an aggravated lipid peroxidation under PEG-induced osmotic stress, and indicated leaf disc assay with moderate PEG iso-osmotic condition would reflect a portion of tea plant response to drought stress. Therefore, the protective effect of AZ (0.125 and 1.25 g a.i. L−1) on tea plants suffering from drought was evaluated using leaf disc assays with 22.8% PEG iso-osmotic condition. Pretreatment of AZ (0.125 a.i. g L−1) reversed Fv/Fm, Y(II), DPPH radical scavenging capacity, and reducing power with reduced MDA in PEG-treated leaf discs, but photosynthetic pigments, total phenols, and ascorbate peroxidase activity were irresponsive to AZ. An Alleviated physiological damage in tea leaf with AZ applying was preliminarily revealed in this study. A Rapid screening of agents for tea plants against drought was developed to assist in the selection of protective agents.