Green Spots Research Articles

First Report of Pestalotiopsis clavispora causing Leaf Spot on Pandanus amaryllifolius in China.

Pandanus amaryllifolius, also known as pandan, is a perennial herb, growing in Indonesia, China and the Maluku Islands (Wakte et al. 2009). It is the only plant with aromatic leaves in the Pandanaceae. It is widely used in food, medicine, cosmetics and other industries, and is also known as "Oriental Vanilla." Pandan is planted in Hainan province over 1,300 ha and is the main plant intercropped among the forest trees. From 2020, the leaf spot was surveyed for three years. Diseased leaves occurred on 30 to 80% of the surveyed plants, with an incidence of 70% and yield losses of 40%. The disease occured from mid-November to April and was most severe at low temperatures and humidity. Initial symptoms were pale green spots, that formed dark brown, nearly circular lesions. As the lesions expanded, their centers became greyish white, with yellow halos at the junction of the diseased and healthy tissue. When the humidity was high, there were small black spots scattered in the center of the lesion. Symptomatic leaf samples were collected from four different sites. The leaf surface was disinfested with 75% ethyl alcohol for 30 s and washed with sterile distilled water three times. Samples from the junction of diseased and healthy tissue (0.5 × 0.5 cm) were removed and placed on potato dextrose agar (PDA) medium containing 100 µg/mL of cefotaxime sodium and cultivated in a dark incubator at 28°C. After two days, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Following Koch's postulates, colonies from strains were used as inoculum in pathogenicity tests. Colonies with 5 mm diameter were inoculated upside onto fresh and healthy pandan leaves via wounding method (pinpricked by sterilized needles) and non-wounding method. Sterilized PDA was used as control. All plants were setted three replicates and were incubated at 28℃ for 3 to 5 days. When symptoms on leaves similar to those in the field appeared, the fungus were reisolated The colonies formed on PDA were also consistent with the original isolate (Scandiani et al, 2003). After seven days, the colony covered the whole petri dish with white, petal-shaped growth with a slight concentric, annular bulge in the center, irregular edges, with black acervuli emerging at a later stage of colony growth. Conidia were fusiform, 18.1±1.6 × 6.4±0.3 μm, showing four septations and five cells, the middle three cells were brownish black to olivaceous, and the apical cell colorless with two to three filaments, 21.8±3.5 μm long. The caudate cell was colorless with one stalk 5.9±1.8 μm long (Zhang et al. 2021; Shu et al. 2020). According to the colony and conidia characteristics, the pathogen was initially identified as Pestalotiopsis spp. (Benjamin et al. 1961). To confirm the pathogen identity, we used the universal primers ITS1/ITS4, targeting primers EF1-728F/EF1-986R and Bt2a/Bt2b sequences (Tian et al. 2018). The sequences of the PCR products were deposited in NCBI GenBank with accession numbers OQ165166 (ITS), OQ352149 (TEF1-α) and OQ352150 (TUB2). BLAST results showed that the sequences of the ITS, TEF1-α and TUB2 genes shared 100% homology with the sequences of Pestalotiopsis clavispora. The maximum likelihood method was used in the phylogenetic analysis. The result showed that LSS112 was clustered with Pestalotiopsis clavispora with a support rate of 99%. Based on morphological and molecular characteristics, the pathogen was confirmed as Pestalotiopsis clavispora. To our knowledge, this is the first report of leaf spot of pandan caused by Pestalotiopsis clavispora in China. This research will be immediately helpful for the diagnosis and control the disease on pandan.

First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare, Colombia.

Passiflora edulis, commonly known as passion fruit, is a vine species of passionflower native to South America. In Colombia, yellow passion fruit (P. edulis f. flavicarpa) is the most important species in terms of net production and local consumption. Recently two brevipalpus transmitted cileviruses, (i) passion fruit green spot virus (PfGSV) and (ii) hibiscus strain of citrus leprosis virus C2 (CiLV-C2H) were detected in passion fruit in Brazil and Hawaii, respectively (Ramos-González et al., 2020, Olmedo-Velarde et al., 2022). CiLV-C2H infects both citrus and hibiscus in Colombia (Roy et al., 2015, 2018) but there was no report of PfGSV elsewhere apart from Brazil and Paraguay (Costa-Rodrigues et al., 2022). Apart from emerging begomovirus diseases, five major viruses are known to infect passion fruit in Colombia: soybean mosaic virus (SMV), cowpea aphid-borne mosaic virus, passion fruit yellow mosaic virus, cucumber mosaic virus, and a tentative Gulupa bacilliform badnavirus A (Cardona et al., 2022). Current findings of CiLV-C2H in passion fruit and PfGSV in hibiscus motivated us to investigate the possibilities of cilevirus infection in passion fruit in Colombia. During surveys, along with healthy yellow passion fruit leaves, five symptomatic plant samples from Meta and three from Casanare were collected before sent to the Molecular Plant Pathology Laboratory at Beltsville, MD under APHIS permit. Passion fruit samples from Meta showed leaf mottling, rugose mosaic, and leaf distortion, whereas leaf variegation, chlorotic spots, yellowing, green spots in senescent leaves and green vein banding were observed in the Casanare samples (Supp. Fig. 1). Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, USA). To know the potential cilevirus infection in these samples, three PfGSV specific (Ramos-González et al. 2020) and a CiLV-C2 generic primer pairs (Olmedo-Velarde et al. 2021) were used in the RT-PCR assays. All five passion fruit samples from Meta failed to produce either CiLV-C2 or CiLV-C2H or PfGSV amplicon whereas all three Casanare samples successfully amplified 321, 244 and 299 nts of PfGSV-RNA1 and -RNA2 amplicons using C13F/C13R, C6F/C6R and C8F/C8R primers, respectively. Bi-directional amplicon sequencing followed by BlastN analysis revealed ≥99% nt identity with the PfGSV-RNA1 (MK804173) and -RNA2 (MK804174) genome sequences. An optimized ribo-depleted library preparation protocol was utilized to prepare two cDNA libraries using the RNA extracts of a PfGSV suspected positive (Casanare) and a negative (Meta) samples (Chellappan et al., 2022). HTS libraries of Casanare and Meta samples resulted in 22.7 to 29.5 million raw reads, respectively. After adapter trimming and filtering, clean reads were mapped to the Arabidopsis thaliana reference genome and unmapped reads were de novo assembled (Chellappan et al., 2022). BlastN analysis from the assembled contigs identified 1-3 contigs corresponding to PfGSV-RNA1 and -RNA2, respectively, from Casanare sample whereas 3 contigs of SMV were identified in Meta passion fruit sample. No other virus sequence was obtained from either of the libraries. Assembled contigs covered 99.33% of the RNA1 and 94.42% of the RNA2 genome, with read depths of 64,474 and 119,549, respectively. Meta sample contigs (OP564897) covered >99% of the SMV genome, which shared >99% nt identity with the Colombian SMV isolates (KY249378, MW655827). Both RNA-1 (OP564895) and -2 (OP564896) segments of the Casanare isolate shared 99% nt identity with PfGSV isolate (MK804173-74). Our discovery identified PfGSV in Colombia, for the first-time outside Brazil and Paraguay. The findings of PfGSV in yellow passion fruit increases the potential threat and possibility of PfGSV movement via Brevipalpus sp. from passion fruit to other hosts.

Una nueva especie de Chrysobothris (s. str.) Eschscholtz, 1829 (Coleoptera: Buprestidae) de la región Lambayeque, Perú

Chrysobothris requensis sp. nov. is described and illustrated from specimens collected in a seasonally dry forest locality of the Lambayeque region in northwestern Peru. The new species is similar to several Chrysobothris species with green spots on the elytra from which it can be distinguished by the color of the body, shape, and arrangement of green fasciae on the elytra and the male genitalia. Comments on its ecology and distribution in the country are provided.

Metabolic fingerprint of ‘WA 38’ green spot symptoms reveals increased production of epicuticular metabolites by parenchyma

Green spot is a pre-harvest physiological disorder of ‘WA 38’ apple fruit that can culminate in corking immediately underneath the peel surface. Unlike bitter pit and many other corking disorders, superficial symptoms emerge in the middle of the growing season as areas of peel discoloration, with the most severe symptoms appearing as parenchyma necrosis or corking that worsens until harvest. Our previous work demonstrated the mitigative effect of early season fruit bagging on green spot appearance, but the mechanism of its onset and the biochemical signatures associated with it remained unknown. We used metabolic profiling to, first, identify metabolites associated with green spot symptoms in the peel and cortex of ‘WA 38’ apples and, then, track changes before and after disorder onset until harvest from apples that were bagged, under a shade net, or uncovered (control). We linked metabolites from multiple metabolic pathways, including carotenoids, phenolics, triglycerides, and triterpenes, with peel and cortex green spot symptoms. Many pigment and triterpene compounds associated with cortex symptoms are typically far less abundant in cortex tissue than in peel. Light-altering treatments also impacted levels of several of these compounds during the growing season; however, the inability to predict which pre-symptomatic tissue would later develop symptoms compromised the establishment of disorder genesis and latent development using this technique. Despite these challenges, we linked novel metabolites and pathways with the disorder, including those that may indicate strengthening and isolation of symptomatic tissue, possibly to provide protections similar to the fruit surface.

Safety profile evaluation of different herbicides used on peach rootstock seedlings.

To screen out suitable herbicides for peach nurseries, we treated the potted seedlings of the peach rootstock 'Nemaguard' with eleven herbicides under recommended doses to investigate the changes of physiological indices and comprehensively evaluate the safety of different herbicides using principal component analysis (PCA). The results showed that soil application of quizalofop-p exhibited no detectable phytotoxicity on rootstock seedlings, while the remaining herbicides generated multiple symptoms, including green loss, wilting, spot, and withering. Starane caused rapid wilting and death, with a 100.0% phytotoxicity index (PI). Soil application of n-(phosphonomethyl)glycine, glufosinate-ammonium, acetochlor, and MCPA-Na showed a PI＞65.0%. As compared with the control, all herbicides inhibited leaf area growth to varying degrees, with a 10.0%-56.2% and 5.8%-44.4% reduction in young leaf area and mature leaf area, respectively. All herbicides, except quizalofop-p, increased the electrolyte permeability of leaf and root tip cells by 21.2%-145.0% and 36.9%-291.4%, respectively, and significantly inhibited root growth. The total root length, root surface area, root volume, and the number of root tips significantly decreased by 37.3%-75.3%, 35.7%-83.0%, 44.3%-89.9%, and 42.6%-73.7%, respectively. Although net photosynthetic rate (Pn) and transpiration rate (Tr) of leaves were not significantly affected by quizalofop-p, mesotrione-atrazine, MCPA-Na·bentazone, bensulfuron-methyl·quinclorac, and bensulfuron-methyl·acetochlor, there was significant reduction of 29.6%, 28.9%, 28.4% and 27.9% in Pn and 21.9%, 29.2%, 26.4%, and 19.7% in Tr post soil application of n-(phosphonomethyl)glycine, glufosinate-ammonium, acetochlor, and MCPA-Na. The overall safety ranking of the 11 examined herbicides is as follows: quizalofop-p>bensulfuron-methyl·acetochlor>bensulfuron-methyl·quinclorac>esotrione·atrazine> auizalofop-p·fluoroglycofen>acetochlor>MCPA-Na·bentazone>MCPA-Na>n-(phosphonomethyl)glycine>glufosinate-ammonium>sterane.

Influence of the urban environment on four mushroom species in the light of their elemental composition

The mushrooms are living organisms with excellent capability to adapt to the multiple challenges of their environment. Several species are well represented in urban green areas (parks, green spots, and recreation grounds). We investigated the influence of the urban environment over two saprotrophic (Bovista plumbea, Lycoperdon perlatum) and two mycorrhizal (Amanita rubescens, Suillus granulatus) species commonly present in urban parks of Cluj-Napoca, a major city of Romania. Three control sites close to the city were chosen. We determined 19 elements (Ag, Al, Ba, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Si, Sr, Ti, and Zn) in the fruiting bodies of mushrooms and in soils by ICP OES technique. S. granulatus was the most sensible species to the urban pollution; this species accumulated 130 and 4.40 mg kg−1 (dry weight) median concentrations of Al and Ni, respectively. The highest concentration of Ag, Cu and Fe was determined in B. plumbea (3.18, 83.7 and 141 mg kg−1, respectively) and L. perlatum (4.68, 91.0 and 125 kg−1, respectively) collected from the city. The saprotrophic species contained appreciably higher concentrations of Ag, Cu, Fe, Mg, P, and S than the mycorrhizal ones. Common feature of all four species was the higher Ag and Sr concentrations in the fruiting bodies of urban origin. Our results suggest that the unique defence mechanisms of the species might have higher impact over the elemental profile of the mushrooms than the soil properties. We propose L. perlatum and S. granulatus as suitable indicator species of the urban pollution with regard to inorganic pollutants.

Flat mites (Tenuipalpidae) from Bahia state, Northeastern Brazil - a checklist including new records and an illustrated key to species

Tenuipalpidae (Trombidiformes: Tetranychoidea) is an economically important family of plant-feeding mites. The family has approximately 1,100 described species in 41 genera with worldwide distribution. Currently, 40 species of Tenuipalpidae have been recorded from Brazil, and prior to this study only 11 were known from Bahia state. Five new species records are presented here for Bahia, making a total of 16 species now known from the state. Brevipalpus is considered the most important genus due to their association with, and ability to transmit, devastating plant viruses. Some diseases caused by viruses transmitted by Brevipalpus mites (VTBs) have already been recorded in the state of Bahia, such as the Passion fruit green spot virus and citrus leprosis. This paper presents a checklist of tenuipalpids reported from the state of Bahia in Brazil, including new records and an illustrated identification key to the species.

Control of the concentration of the macroelement phosphorus in the substrate when growing tomatoes in protected soil conditions

Although most tomatoes are grown in open ground, their cultivation in glass greenhouses or tunnel-typegreenhouses covered with plastic film is becoming more common to supply local markets with fresh fruit. In closed soil, mainly tall varieties of tomatoes are grown. They make it possible to use the area of greenhouses as efficiently as possible and get rich harvests. Tomatoes grown in greenhouses can be stored for 11 months, unlike those grown in open ground. Tomatoes are usually harvested unripe and ripen during transportation or on store shelves. Different types of tomatoes grown in greenhouses tend to have a longer shelf life, better taste and higher market value than those grown in the open field. Usually, plants are attached to the supports with a single wire, while the extra side shoots (stepchildren) are removed so that only one stem remains. The height of the tomato bushes can be gradually reduced for convenience immediately after the ripening of the first bunches of tomatoes. Shoots and inflorescences are removed to increase the length of the tomato bush, the number of fruits on it and their weight in order to meet the needs of the market. Taste, color, shape and texture are extremely important criteria for evaluating the quality of fresh tomatoes. Also, the fruits must be clean and contain no signs of decay or disease. Fresh tomatoes should be uniform in shape, symmetry and size. They should have a bright and uniform color, not contain green shoulders, spots and characteristic signs of immaturity. High concentrations of carbon dioxide (600–1000 ppm) are maintained in greenhouses to improve the process of photosynthesis, growth rate and yield. This practice is very common in low yield regions and intensive agricultural production systems. Although planting tomatoes in soil in tunnel-type greenhouses is still quite common, most intensive-type green-house tomato varieties are grown on substrates such as mineral wool. Fertigation provides adequate nutrient supply and eliminates most of the factors associated with heavy soil characteristics that are difficult to control. Growing tomatoes in greenhouses requires the use of a significant amount of water. To feed tomatoes, their producers also need to take into account the mass and degree of solubility of mineral fertilizers in water. Some elements or compounds present in irrigation water can affect the growth of plants, which is why there is a need to control their level. The paper investigates the influence of the concentration of the macroelement phosphorus in the mineral wool substrate on the growth and development of the Rosaletta (pink) tomato crop during the period of mass fruiting in protected soil conditions. It was established that the photoelectrocolorimetric method for determining the phosphorus content in mineral wool hoods is quite sensitive, simple and recommended for use in serial analyses. The determined average value of the phosphorus macroelement content (50.0 mg/l) turned out to be higher than that required for growing tomatoes in the period of mass fruiting. Controlling the phosphorus content allows you to significantly prevent tomato diseases and obtain high-quality crops.

Fruit Rot Caused by Fusarium sulawesiense on Chili in Guangxi, China

Chilli (Capsicum annuum) is considered as one of the most important vegetables and spice crops throughout the world which is widely cultivated in China. In October 2019, fruit rot symptoms were observed on chilli in Guilin, Guangxi, China (N24°18', E109°45'). Irregular dark green spots initially appeared on the middle or bottom of the fruit, then extended to larger grayish brown lesions and started to rot. In the late stage, the whole fruit dried up after water loss. Three disease samples were collected from three towns of different counties in Guilin where the disease incidence of chilli fruits was 15-30%. The margin of diseased fruits was cut into small pieces (3×3 mm), disinfected with 75% ethanol solution for 10 s, 2% NaOCl for 1 min, and rinsed in sterile distilled water three times. Tissue pieces were separately plated on potato dextrose agar (PDA) and incubated at 25°C for seven days. Fifty-four fungal isolates with similar morphology were consistently recovered from diseased tissues of three fruits, with 100% isolation frequency. Three representatives GC1-1, GC2-1 and PLX1-1 were selected for further analysis. The colonies produced abundant whitish to yellowish aerial mycelium on PDA after 7 days incubation at 25°C in the dark. Macroconidia cultured on carnation leaf agar (CLA) for 7 days were long, hyaline, falcate, with dorsal and ventral lines often gradually wider toward apex, curved apical cell and foot-shaped basal cell, mostly 2 to 5 septa, and ranged from 24.16 to 38.88 × 3.36 to 6.55 μm (average 31.39×4.48 μm), from 19.44 to 28.68 × 3.02 to 4.99 μm (average 23.02×3.89 μm), and from 20.96 to 35.05 × 3.30 to 6.06 μm (average 26.24×4.51 μm) for GC1-1, GC2-1, and PLX1-1, respectively. Microconidia were hyaline, fusoid or ovoid, one-septate or nonseptate, and ranged from 4.61 to 10.14 × 2.61 to 4.77 μm (average 8.13×3.58 μm), from 3.55 to 7.85 × 1.95 to 3.04 μm (average 5.79×2.39 μm), and from 6.75 to 18.48 × 3.05 to 9.07 μm (average 14.32×4.31 μm) for GC1-1, GC2-1, and PLX1-1, respectively. Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5F2/7cR, respectively (White et al. 1990; O'Donnell et al. 2000, 2010). Sequences were deposited in GenBank (ITS: OQ080044-OQ080046; TEF1: OQ101589-OQ101591; CAM: OQ101586-OQ101588; RPB2: OQ101592-OQ101594). A maximum Likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, CAM, TEF1, RPB2). According to morphology and phylogenetic analysis, the isolates were identified as Fusarium sulawesiense (Maryani et al. 2019). For pathogenicity tests, multiple punctures in a 5-mm-diameter circle were made with a sterilized toothpick on detached young healthy fruits, followed by inoculation with 10 μl of conidial suspension (106 spores/ml in 0.1% sterile Tween 20). Each isolate was inoculated onto eighteen fruits. Controls were inoculated with water containing 0.1% sterile Tween 20 under the same conditions. Symptoms were observed on the inoculated fruits 7 days after incubation at 25°C, whereas non-inoculated controls were asymptomatic. The fungus was re-isolated from inoculated chilli fruits, completing Koch's postulates. To our knowledge, this is the first report of Fusarium sulawesiense causing fruit rot on Chilli in China. These results will provide valuable information for prevention and management of fruit rot on Chilli.

First Report of Colletotrichum fructicola Causing anthracnose on macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) is an important commercial crop in the world and has the reputation of being the king of nuts (Liu et al. 2019). In August 2020, symptoms of anthracnose appeared on leaves of macadamia in Chongzuo, Guangxi Province, China, with an incidence of 15-20%. The disease developed from the edge of leaf. Initially, the disease symptoms on leaves were faded green spots, light yellow. After expanding and linking together, the leaves appeared brown or black irregular spots, and the edges of diseased leaves dried up and formed large necrosis, eventually leading to defoliation. A large number of orange-yellow spots (acervuli) developed on the diseased parts. Under high humidity conditions, the diseased part was grayish-brown or black, and a large number of yellowish-brown conidia were produced on the leaf surface (Fig.1 A-E). Ten symptomatic leaves were collected and washed with distilled water. Twelve lesion marginal tissues were sterilized with 75% ethanol (V/V) for 30 s and 1% NaOCl for 1min and rinsed with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 28°C under light. After 3 days, the incubated samples all produced similar cultural morphology. One isolate named GXMC2 as a representative was selected for following study. The colony by single-spore purification on PDA were grayish green with a white outer ring and cottony on surface, pale blackish green in reverse side (Fig.1 F). Conidia with oil droplets were solitary, cylindrical, transparent and measuring 13.78 to 19.25 μm (average 16.90 μm) × 5.14 to 7.33 μm (average 6.23 μm) (n=100) (Fig.1 G). Appressoria were brown to dark brown, with different shapes such as ovoid, elliptical or irregular, some with lobes. The average size was 7.89 to 13.25 μm (average 10.64 μm) × 5.76 to 9.02 μm (average 7.86 μm) (n=100) (Fig.1 H). No setae were found. The isolate was identified as Colletotrichum fructicola on the basis of the morphology of the colonies, conidia and appressoria (Park et al. 2018). The six target gene sequences, including internal transcribed spacer (ITS), β-tubulin (TUB), actin (ACT), histone3 (HIS3), chitin synthase A (CHS), and glyceraldehydes-3-phosphate dehydrogenase (GAPHD) (Qiu et al. 2020), were selected for PCR amplification. The resulting sequences were deposited in GenBank under accession numbers MZ821661, MZ821660, MZ821662, MZ821663, MZ821664 and MZ821665 respectively. Phylogenetic analysis of the concatenated sequences were performed with MEGA 7.0 software. The isolate was grouped in the same clade as other C. fructicola (Fig.2). In May 2022, Inoculation was conducted in the field. Four-year-old macadamia leaves were disinfected with 75% alcohol. The conidial suspension was sprayed on 5 unwounded healthy leaves, and 5 leaves sprayed with sterile distilled water served as control. The experiment was replicated 3 times, with each replicate containing 5 leaves. The average daily temperature and average daily relative humidity in the field were 30°C and 62%, respectively. After 2 days, yellow-brown spots appeared on the inoculated leaves and expanded outward. After 4 days, the diseased areas were dark brown, and the controls remained asymptomatic. The same fungal pathogen was reisolated and purified from inoculated leaves and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates (Fig.1 I-J). In China, C. fructicola has been reported on Passiflora edulis Sims, Brassica parachinensis, Illicium verum, Peucedanum praeruptorum, etc. (Li et al. 2021; Yu et al. 2022; Zhao et al. 2021; Ma et al. 2020). To our knowledge, this is the first report of anthracnose of macadamia caused by C. fructicola in China. This study provides the basis for further research on this disease.

Urban Environmental Acupuncture for Improving the Sustainability of Dense City Areas – Polish Experiences from the SALUTE4CE Project

Abstract The idea of urban environmental acupuncture postulates, that in areas with no space available for green spaces, sustainability and climate resistance can be improved by small green spots in key locations. The project SALUTE4CE established pilot environmental acupuncture strategies in Central-European cities. The author was responsible for elaborating an Action Plan for the functional area of Chorzów, Ruda Śląska and Świętochłowice, Poland. The project aimed at finding spots with potential outcomes in terms of sustainability, reducing urban heat islands and improving comfort and quality of spaces, with nature based solutions: from planting climate resistant vegetation and restoring biodiversity, through improving local retention, to green roofs or vertical walls. The concept assumes, that acting in many small places can bring synergic effects and contribute to urban regeneration. Four pilot spots have been constructed so far, and other interventions shall be realized in the coming years. So far the project results have been disseminated in project documents and reports. This paper aims at a summary of project experiences and perspectives in regard to participating Polish cities.

Identification of the clay minerals of some chosen soils in Aljabal Alakhdar district, Libya by Ethylene Glycol and Heat: X-ray Diffraction studies

Due to the widespread use of clay minerals and their importance in various industries and agricultural projects, sufficient knowledge of clay minerals as well as how to identify them is so important. Therefore, understanding these minerals can optimize the use and application of them. Al-Jabal Al-Akhdar, as it represents the only green spot in northeastern of Libya, the identification of clay minerals in this region is very important for its relationship to the management of the chemical and physical properties of its soil. Four samples were selected from the Al-Haniya area, and due to the shallow depths of its soil, these samples were taken from the last depth (35-60 cm). X-ray diffraction device used to obtain clear X-ray diffraction (XRD) curves for identification of clay minerals in selected samples from the study area. The results of the qualitative analysis of the minerals showed that there was a similarity in the types of pyrophyllite silicates and the illite is the most crystalline mineral indicated by the relative strength in these soils. The results also showed the presence of kaolinite minerals and the chlorite-vermiculite complex. The results did not show the presence of expansion minerals such as montomorollonite.

A Study on Pedestrians’ Satisfaction and Preferences for Green Patterns according to the Sidewalk Width Using VR: The Case of Seoul, South Korea

Pedestrian-friendly cities are gaining traction worldwide. This study focused on sidewalk width and green space designs that comprise the walking environment. This study examined uniform planting patterns made without considering the width of the sidewalk and derived an appropriate green area pattern created according to the width of the sidewalk. We conducted a survey using virtual reality, satisfaction and preference review, and multilinear regression analysis. The results showed that ensuring safety through planting on a narrow sidewalk with a width of less than 3 m and 3 m to 5 m had a higher effect on satisfaction, while places with sufficient shade environments had an effect on overall satisfaction for a sidewalk width of 5 m to 8 m or more. The green spots were not quite preferred; on the contrary, there was a popular opinion that many green spots felt stuffy. This study is meaningful in that it identified the reason for appropriate planting plans, according to the width of the sidewalk, by applying the changing traffic paradigm, and conducted the study from the perspective of pedestrians. In addition, this study demonstrated the possibility of using virtual reality in the planning of smart cities, by applying a new research methodology using VR to visualize tasks that are difficult to perform in reality.

Validation of endophytic bacteria colonisation in tissue culture-derived oil palm plantlets via green fluorescent visualization

This study aimed to validate the colonisation capability of endophytic bacteria (EB) isolates, Bacillus cereus EB2 and Pseudomonas aeruginosa EB35, that previously exhibited their potentials as biological control agents (BCAs) against the Ganoderma spp., a pathogen for Ganoderma disease in oil palm. Here, we demonstrated a rapid method to determine the colonisation capacity of the selected EB using oil palm tissue culture plantlets and a green fluorescent protein (GFP) visual marker. Wounded plantlet roots were inoculated with GFP-tagged B. cereus EB2 and P. aeruginosa EB35 while the plantlets without EB inoculation served as controls. The GFP signals appeared as bright green spots or lines in the inoculated GFP-tagged EB cells in root and leaf plantlet tissues, respectively, under the confocal laser scanning microscopy (CLSM) 5 days post-inoculation. In contrast, there was no intense GFP spots in neither the control root nor leaf tissues. The cracks in the roots by wounding facilitated the entry of the GFP-tagged EB cells into root tissues, allowing for endophytically colonisation of the root and above-ground tissues. Subsequent result of polymerase chain reaction (PCR)-GFP analysis further displayed the endophytic nature and early chronological colonisation of the tested EB. This is a preliminary report on root colonisation by a Gram-positive endophyte, B. cereus EB2 and leaf tissues colonisation by both EB isolates as internal colonisers, demonstrating their potential as BCAs to protect oil palm against Ganoderma spp. for a sustainable disease management.

The dynamic change in the degradation and in vitro digestive properties of porcine myofibrillar protein during freezing storage

The myofibrillar protein (MP) degradation and in vitro digestive properties of porcine longissimus during freezing at −8, −18, −25 and − 40 °C for 1, 3, 6, 9 and 12 months were investigated. As the freezing temperature and duration of frozen storage increased, the amino nitrogen and TCA (trichloroacetic acid)-soluble peptides of the samples were significantly increased, while the total sulfhydryl content and band intensity of myosin heavy chain, actin, troponin T, tropomyosin were significantly decreased (P < 0.05). At higher freezing storage temperatures and durations, the particle size of MP samples and the green fluorescent spots detected using a laser particle size analyzer and confocal laser scanning microscopy became large. After 12 months of freezing, the digestibility and the degree of hydrolysis of the trypsin digestion solution of the samples frozen at −8 °C were significantly decreased by 15.02 % and 14.28 %, respectively, when compared to fresh samples, whereas, the mean surface diameter (d3,2) and mean volume diameter (d4,3) were significantly increased by 14.97 % and 21.53 %, respectively. Therefore, frozen storage induced protein degradation and impaired the ability of digestion in the pork proteins. This phenomenon was more evident as the samples were frozen at high temperatures over a long storage period.

Identification of the clay minerals of some chosen soils in Aljabal Alakhdar district, Libya by Ethylene Glycol and Heat: X-ray Diffraction studies

Due to the widespread use of clay minerals and their importance in various industries and agricultural projects, sufficient knowledge of clay minerals as well as how to identify them is so important. Therefore, understanding these minerals can optimize the use and application of them. Al-Jabal Al-Akhdar, as it represents the only green spot in northeastern of Libya, the identification of clay minerals in this region is very important for its relationship to the management of the chemical and physical properties of its soil. Four samples were selected from the Al-Haniya area, and due to the shallow depths of its soil, these samples were taken from the last depth (35-60 cm). X-ray diffraction device used to obtain clear X-ray diffraction (XRD) curves for identification of clay minerals in selected samples from the study area. The results of the qualitative analysis of the minerals showed that there was a similarity in the types of pyrophyllite silicates and the illite is the most crystalline mineral indicated by the relative strength in these soils. The results also showed the presence of kaolinite minerals and the chlorite-vermiculite complex. The results did not show the presence of expansion minerals such as montomorollonite.

Green roofs and pollinators, useful green spots for some wild bee species (Hymenoptera: Anthophila), but not so much for hoverflies (Diptera: Syrphidae)

Urbanisation has become one of the major anthropogenic drivers behind insect decline in abundance, biomass and species richness over the past decades. As a result, bees and other pollinators' natural habitats are reduced and degraded. Green roofs are frequently recommended as ways to counter the negative impacts of urbanisation on nature and enhance the amount of green space in cities. In this study we evaluated the pollinator (more specifically wild bees and hoverflies) diversity, abundance and species richness on twenty green roofs in Antwerp, Belgium. We analysed the influence of roof characteristics (age, surface area, height, percent cover of green space surrounding each site) on species richness or abundance of pollinators. In total we found 40 different wild bee species on the green roofs. None of the physical roof characteristics appear to explain differences in wild bees species richness and abundance. Neither could we attribute the difference in roof vegetation cover, i.e. roofs build-up with only Sedum species and roofs with a combined cover of Sedum, herbs and grasses, to differences in diversity, abundance, or species richness. We found a positive trend, although not significant, in community weighted mean body size for wild bees with an increase in green roof surface area. Roof wild bee communities were identified as social polylectic individuals, with a preference for ground nesting. Only eleven individuals from eight different hoverfly species were found. Our results show that green roofs can be a suitable habitat for wild bee species living in urban areas regardless of the roofs’ characteristics, but hoverflies have more difficulties conquering these urban green spaces.

Metabolomic analysis reveals key metabolites alleviating green spots under exogenous sucrose spraying in air-curing cigar tobacco leaves

Cigar variety CX-010 tobacco leaves produce localized green spots during the air-curing period, and spraying exogenous sucrose effectively alleviates the occurrence of the green spots. To investigate the alleviation effect of exogenous sucrose spraying, the total water content and the number and size of green spots on tobacco leaves were investigated during the air-curing period under four treatments; CK (pure water), T1 (0.1 M sucrose), T2 (0.2 M sucrose) and T3 (0.4 M sucrose). The results showed that the total water content of tobacco leaves showed a trend of T3 < CK < T2 < T1 in the early air-curing stage, and the number and size of green spots showed a trend of T3 < T2 < T1 < CK. All sucrose treatments alleviated the green spot phenomenon, and T3 had the fewest green spots. Thus, the tobacco leaves of the T3 and CK treatments at two air-curing stages were used to perform metabolomics analysis with nontargeted liquid chromatography‒mass spectrometry to determine the physiological mechanism. A total of 259 and 178 differentially abundant metabolites (DAMs) between T3- and CK-treated tobacco leaves were identified in the early air-curing and the end of air-curing stages, respectively. These DAMs mainly included lipid and lipid-like molecules, carbohydrates, and organic acids and their derivatives. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the T3 treatment significantly altered carbohydrate metabolism (pentose phosphate pathway, sucrose and starch metabolism and galactose metabolism) and amino acid metabolism (tyrosine metabolism and tryptophan metabolism) in air-curing tobacco leaves. Sucrose treatment alleviated green spots by altering DAMs that affected chlorophyll degradation, such as tyrosine and citric acid, to promote the normal degradation of chlorophyll.

First Report of Puccinia modiolae Causing Rust Disease on Malva verticillata in Korea.

Malva verticillata (Malvaceae), commonly called Chinese mallow or whorled mallow, is an annual herb native to East Asia and is currently distributed worldwide. In Korea, this plant is cultivated as a leafy vegetable and cooked like spinach or used in soups and also as a medicine material. In March 2022, typical symptoms of rust disease were observed on M. verticillata in a plastic house (37°22'12″ N, 127°34'30" E) in Yeoju, Korea. Yellow or light green round chlorotic spots appeared on the upper surface of infected leaves, while reddish-brown or dark brown rust pustules formed on its lower surface. Infection occurred in 10% of M. verticillata plants surveyed, and disease severity ranged between 30-90%. A representative sample was deposited in the Kunsan National University Herbarium (KSNUH1762). Telia were mostly hypophyllous, reddish-brown to dark brown, round, mostly grouped, and 0.3-0.7 mm in diameter. Teliospores were mostly two-celled, but rarely one or three-celled, yellowish to light brown, fusoid, and 42.9-101 × 10.8- to 18.8 μm (average 72.7 ± 12.3 × 14.2 ± 1.92 μm [mean ± SD]; n = 50), with a smooth, hyaline to yellowish wall of 1.0-2.5 μm thickness. The morphological characteristics were similar to those reported for Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). To confirm the morphological identification, genomic DNA was extracted from the teliospores of an infected leaf. The internal transcribed spacer (ITS) with primers ITS5-u and ITS4rust (Pfunder and Schürch 2001) and the large subunit (LSU) rDNA with primers LRust1R and LRust3 (Beenken et al. 2012) were amplified for sequencing. The resulting sequences were deposited in GenBank with accession numbers ON631218 for ITS and ON631226 for LSU. BLASTn search showed that the Korean sample was identical to the ITS sequences of P. modiolae from Modiola caroliniana (MK458693-MK458697) and the LSU sequences from M. caroliniana, Malva sylvestris, and Alcea rosea (MH742976, MH742977, and MH742978). In the phylogenetic trees of the ITS and LSU sequences, the Korean sample was grouped with the reference sequences of P. modiolae, with the maximum supporting value. For the pathogenicity test, rust-infected leaf discs were placed on the upper or lower surfaces of leaves of three healthy M. verticillata. Three non-inoculated plants served as controls. Inoculated and non-inoculated plants were maintained in a growth chamber at 22°C, a 16/8 h light cycle, and 80% humidity. After three weeks, all inoculated plants developed evident rust symptoms on the upper and lower surfaces of the leaves on which the leaf discs were placed, whereas the control plants remained symptomless. The pathogen present on the inoculated plants was confirmed to be the same pathogen as that observed in the field, fulfilling Koch's postulates. Based on the morphological investigation, sequence analysis, and pathogenicity tests, P. modiolae was identified as the causal agent of rust disease on M. verticillata. To date, this pathogen has been reported on seven Malvaceae plants, including Alcea rosea, Althaea officinalis, Lavatera arborea, Malva parviflora, Malva sylvestris, Modiola caroliniana, and Modiola sp., in North and South America (Farr and Rossman 2022). However, it has not been reported in Asia or Korea. This study is the first report of rust disease on M. verticillata worldwide. Considering its high incidence rate and severe damage, this pathogen is a potential concern for the cultivation of M. verticillata in Korea. This finding could contribute to developing phytosanitary and control treatments for this disease.

Interaction between Movement Proteins of Hibiscus green spot virus.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.