First report of leaf spot disease caused by Stemphylium eturmiunum on garlic in Korea.

Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.

Physiological Responses of Ocimum basilicum, Salvia officinalis, and Mentha piperita to Leaf Wounding

The investigation about the leaf wounding effect on plant physiological procedures and on leaf pigments content will contribute to the understanding of the plants’ responses against this abiotic stress. During the experiment, some physiological parameters such as photosynthesis, transpiration and stomatal conductance as well as the chlorophyll and anthocyanin leaf contents of Ocimum basilicum, Salvia officinalis, and Mentha piperita plants were measured for about 20–40 days. All the measurements were conducted on control and wounded plants while in the latter, they were conducted on both wounded and intact leaves. A wide range of responses was observed in the wounded leaves, that is: (a) immediate decrease of the gas exchange parameters and long-term decrease of almost all the measured variables from O. basilicum, (b) immediate but only short-term decrease of the gas exchange parameters and no effect on pigments from M. piperita, and (c) no effect on the gas exchange parameters and decrease of the pigments content from S. officinalis. Regarding the intact leaves, in general, they exhibited a similar profile with the control ones for all plants. These results imply that the plant response to wounding is a complex phenomenon depending on plant species and the severity of the injury.

Micropropagation of Pelargonium odoratissimum (L.) L’Her. through petioles and leaves

Pelargonium odoratissimum (L.) L’Her is a hard rooting plant and the common methods of propagation via stem cuttings are not successful with this species. therefore, tissue culture methods have been experienced for the mass-propagation of this high-valued species. Intact leaves, leaf segments and petiole sections derived from nodal explants in vitro were employed for the optimization of P. odoratissimum micropropagation. The treatment combinations used were MS and 1/2 MS media supplemented with 6-benzylaminopurine, BAP (1, 1.5, 2 and 4.5 mg.L-1) and 1-naphthaleneacetic acid, NAA (0.1, 1 and 1.5 mg.L-1). With leaf segments, the lowest browning incidence, the greatest callogenesis and the highest number of shoots were obtained with the media containing 1.5 mg.L-1 BAP and 1 mg.L-1 NAA. Two mg.L-1 BAP + 0.1 mg.L-1 NAA kept the same results for petiole explants. Intact leaves showed the best results for the three mentioned treatments with 1 mg.L-1 BAP + 1 mg.L-1 NAA. 0.2 mg.L-1 NAA caused the highest rooting percentage and the greatest mean data for the number and length of the roots. Rooted plantlets were transferred to the pots containing 1:1 peat-moss and perlite. Acclimatization of the plantlets was followed by 90 % of survival rate in the greenhouse. The protocol employed would be a potent one to present for the extension section.

Photosynthetic linear electron flow drives CO2 assimilation in maize leaves

Photosynthetic organisms commonly develop the strategy to keep the reaction centre chlorophyll of photosystem I, P700, oxidised for preventing the generation of reactive oxygen species in excess light conditions. In photosynthesis of C4 plants, CO2 concentration is kept at higher levels around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) by the cooperation of the mesophyll and bundle sheath cells, which enables them to assimilate CO2 at higher rates and to survive under drought stress. However, the regulatory mechanism of photosynthetic electron transport for P700 oxidation is still poorly understood in C4 plants. Here we assessed gas exchange, chlorophyll fluorescence, electrochromic shift, and near infrared absorbance in the intact leaves of NADP-malic enzyme subtype of C4 plants maize in a comparison with the C3 plant field mustard. Instead of the alternative electron sink due to photorespiration, photosynthetic linear electron flow was strongly limited between photosystems I and II dependent on the proton gradient across the thylakoid membrane (ΔpH) in response to the suppression of CO2 assimilation in maize. The increase of ΔpH for P700 oxidation was caused by the regulation of proton conductance of chloroplast ATP synthase but not by promoting cyclic electron flow, which was supported by linear relationships among CO2 assimilation rate, linear electron flow, P700 oxidation, ΔpH, and the oxidation rate of ferredoxin. At the scale of intact leaves, the ratio of PSI to PSII was estimated almost 1:1 in both C3 and C4 plants. Overall, the photosynthetic electron transport was regulated for P700 oxidation in maize through the same strategies as in C3 plants only except for the capacity of photorespiration despite the structural and metabolic differences in photosynthesis between C3 and C4 plants.

A modified method for enzymatic isolation of and subsequent wax extraction from Arabidopsis thaliana leaf cuticle

Background The plant cuticle represents one of the major adaptations of vascular plants to terrestrial life. Cuticular permeability and chemical composition differ among species. Arabidopsis thaliana is a widely used model for biochemical and molecular genetic studies in plants. However, attempts to isolate the intact cuticle from fresh leaves of Arabidopsis have failed so far. The goal of this study was to optimise an enzymatic method for cuticle isolation of species with a thin cuticle and to test it on several A. thaliana wild types and mutants. Results We developed a method for isolation of thin cuticles that allows reducing the isolation time, the separation of abaxial and adaxial cuticles, and avoids formation of wrinkles. Optical microscopy was used for studying cuticle intactness and scanning electron microscopy for visualisation of external and internal cuticle structures after isolation. Wax extracts were analysed by GC–MS. Isolation of intact cuticle was successful for all tested plants. The wax compositions (very-long-chained fatty acids, alcohols and alkanes) of intact leaves and isolated cuticles of wild type Col-0 were compared. Conclusions We conclude that the optimised enzymatic method is suitable for the isolation of A. thaliana adaxial and abaxial cuticles. The isolated cuticles are suitable for microscopic observation. Analysis of wax composition revealed some discrepancies between isolated cuticles and intact leaves with a higher yield of wax in isolated cuticles.

Unraveling the leaf-dropping behavior behind bat folivory: do bats use biological control against roost parasites?

Folivory in bats, the behavior of chewing bites of leaves to extract the liquid portion and discard the fiber as oral pellets, is seen as a strategy that might provide vitamins, micronutrients and proteins not always available in fruits, and even secondary metabolites (hormonal precursors) that stimulate/inhibit reproductive processes. While examining chewed leaves, we noticed an important amount of completely intact leaves below the roosts of Artibeus amplus and Artibeus lituratus, and decided to systematically quantify them during a year. We recovered 639 intact leaves from six plant species and found that both species are constantly feeding on leaves, but invariably leaving 26–78% of these intact, dropping them on the floor just below their roosting sites. These large proportions of dropped, intact leaves suggest a completely different, novel, non-nutritional use by bats. Several studies in birds have revealed that fresh leaves are effectively used in nests to control diverse organisms that include blood suckling ectoparasites, protozoans, fungus and bacteria. We report for the first time this biological control behavior performed by bats by selecting, bringing and dropping intact leaves below their roosting sites. The chemical properties of the leaves need to be investigated to reveal those active components against specific pathogens.

A commonly used photosynthetic inhibitor fails to block electron flow to photosystem I in intact systems

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions made from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INT’s chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO2 fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O2 evolution and O2 consumption in isolated thylakoids and CO2 fixation in intact leaves, using two stable isotopes of oxygen (16O2,18O2) and CO2 (12C,13C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO2 fixation, therefore its use may produce artefacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

Is nitric oxide a critical key factor in ABA-induced stomatal closure?

The role of nitric oxide (NO) in abscisic acid (ABA)-induced stomatal closure is a matter of debate. We conducted experiments in Vicia faba leaves using NO gas and sodium nitroprusside (SNP), a NO-donor compound, and compared their effects to those of ABA. In epidermal strips, stomatal closure was induced by ABA but not by NO, casting doubt on the role of NO in ABA-mediated stomatal closure. Leaf discs and intact leaves showed a dual dose response to NO: stomatal aperture widened at low dosage and narrowed at high dosage. Overcoming stomatal resistance by means of high CO2 concentration ([CO2]) restored photosynthesis in ABA-treated leaf discs but not in those exposed to NO. NO inhibited photosynthesis immediately, causing an instantaneous increase in intercellular [CO2] (Ci), followed by stomatal closure. However, lowering Ci by using low ambient [CO2] showed that it was not the main factor in NO-induced stomatal closure. In intact leaves, the rate of stomatal closure in response to NO was about one order of magnitude less than after ABA application. Because of the different kinetics of photosynthesis and stomatal closure that were observed, we conclude that NO is not likely to be the key factor in ABA-induced rapid stomatal closure, but that it fine-tunes stomatal aperture via different pathways.