True Leaf Stage Research Articles

Molecular identification and phylogenetic analysis of different races of Fusarium oxysporum f.sp. Melonis in muskmelon using RAPD markers under Punjab conditions

Muskmelon wilt caused by Fusarium oxysporum f.sp. melonis causes serious damage in muskmelon growing districts of Punjab. Keeping in view the importance of disease, the present study was conducted with an objective to identify the different races of muskmelon wilt using molecular techniques. Twenty isolates of F. oxysporum f.sp. melonis were collected in 2018 and 2019 from different locations of muskmelon growing areas of Punjab viz. Jalandhar (J) Kapurthala (K), Sangrur (S) and Patiala (P). Pathogenicity for the evaluation of prevalent races of F. oxysporum f.sp. melonis under Punjab conditions was performed on a set of five differentials viz. Charentais T, Charentais FOM-1, Charentais FOM-2, Margot and Isabelle under pot house conditions. After 2–3 true leaf stage, these differentials were artificially inoculated with spore suspension @ 1x106/ml of twenty isolates of F. oxysporum f.sp. melonis. Findings indicated that out of total 20 isolates, 8 isolates belonged to race 1, 8 isolates belonged to race 1.2, 3 isolates belonged to race 2 and remaining 1 belonged to race 0. Therefore, race 1 and 1.2 were prevalent in Punjab. Genetic diversity among the twenty isolates of F. oxysporum f.sp. melonis was analyzed with the Random Amplified Polymorphic DNA (RAPD) markers. Based on cluster analysis, J2 and J4 isolates were outliers and they were genetically different from other isolates. It was concluded that Fusarium wilt of muskmelon caused by FOM was prevalent in all muskmelon growing districts of Punjab. These findings are considerably important and could be used for selection and integration of muskmelon wilt resistance gene into muskmelon breeding programmes for Fusarium wilt management under Punjab conditions.

Effect of vapor pressure deficit on the photosynthesis, growth, and nutrient absorption of tomato seedlings

Vapor pressure deficit (VPD) is the main driving force of plant water transport and affects the nutrient absorption of plants. Exploring the nutrient absorption, growth, and photosynthetic characteristics of different tomato cultivars under different VPDs is of great importance for the cultivation of tomato. This experiment featured two levels of VPD (high 2.2/low 0.9 kPa) and two tomato cultivars (Solanumlycopersicum L., cv. Jinpeng and cv. Zhongza) for a total of four treatments in two controlled environment growth chambers. After about 4 weeks, the seedlings (at the three true-leaf stage) were transplanted into pots flled with inorganic medium and watered with nutrient solution. The stomatal characteristics, leaf moisture status, net photosynthetic rate (An), and plant growth of Jinpeng were not significantly different under the two VPD treatments, but high VPD resulted in a significantly increased transpiration rate (E) and whole plant hydraulic conductance (Kplant), as well as significantly increased absorption of potassium (K) and calcium (Ca) in various organs. In contrast, Zhongza had lower biomass, smaller stem height, and lower total leaf area under high vs. low VPD conditions. At high VPD, the reduction of photosynthesis in Zhongza was mainly caused by anatomical modifications of the stomata; significant reductions in stomatal aperture and length restricted photosynthesis and biomass accumulation. Moreover, the E and Kplant of Zhongza were reduced by stomatal state changes that prevent excessive transpiration. High VPD limited plant transpiration and mass flow of water, which reduced the absorption and accumulation of nutrients. Overall, these results suggest tomato cultivars differ in their stomatal sensitivity. Specifically, increasing VPD resulted in increased levels of some nutrients in certain organs in the cultivar with lower stomatal sensitivity; however, the cultivar with higher stomatal sensitivity demonstrated opposite trends.

Phenylalanine Ammonia-Lyase Enzyme Activity in the Symbiotic System Glycine max – Bradyrhizobium japonicum by Seed Inoculation Different in Activity and Virulation Strain and Treatment with Fungicides

Phenylalanine ammonia-lyase (PAL) is a key enzyme of the phenylpropanoid pathway and provides precursors for the synthesis of many secondary metabolites, which are necessary for the development and protection of plants from external factors of various natures, in particular plays an important role in the formation and development of their symbiosis with microorganisms. Aim. To study the activity of PAL in soybean plants in the early stages of legume-rhizobial symbiosis under the influence of seed inoculation with Bradyrhizobium japonicum strains with different symbiotic properties on the background of fungicide treatment. Methods. Microbiology (bacterial culture growing, seeds inoculation), physiological (pot experiment), biochemical (determining the PAL enzyme activity). Results. Inoculation of soybean seeds with active virulent rhizobia induces a significant decrease in PAL activity in the roots at the primordial leaf stage and a significant increase in its activity level at the first true leaf stage, compared to inactive symbiosis. At the stage of third true leaf, the activity of PAL increased more significantly in soybean root nodules formed by inactive rhizobia, compared to active symbiosis. However, at the stage of third true leaves, the activity of PAL in soybean root nodules formed by inactive rhizobia increased significantly compared to active symbiosis. The use of fungicides for pre-sowing treatment of soybean seeds induces changes in the level of PAL activity in roots and nodules, which do not affect the overall dynamics of enzyme activity in different effective symbiotic systems Glycine max - Bradyrhizobium japonicum. Conclusions. The activity of PAL in the roots and especially in the root nodules of soybeans in the early stage of plant development in the case of fungicides using and bacterization is primarily due to the action of the inoculation factor, and is determined by the symbiotic properties of rhizobia strains, in particular, their virulence and nitrogen fixation activity.

Abundance and Species of Thrips1 in Cotton on the South Plains of Texas

Thrips (Thysanoptera: Thripidae) are the major insect pest of seedling cotton, Gossypium hirsutum L., on the Texas South Plains. The western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), has historically been the most abundant thrips species in cotton in the region. A field survey during the 2017 and 2018 growing seasons collected thrips from small-plot insecticide seed treatment research experiments at multiple locations across the Texas South Plains. Species composition of adult thrips from cotyledon to four true-leaf stages cotton was determined by morphological identification with the aid of a microscope. Of the species collected during 2 years of study, 64% were onion thrips, Thrips tabaci Lindeman, followed by F. occidentalis (23%), Frankliniella exigua Hood (10%), and tobacco thrips, Frankliniella fusca Hinds (1%). Relative abundance of species varied significantly across growing seasons. In 2017, T. tabaci was the most abundant species in cotyledon to four true-leaf stages of cotton. In 2018, F. occidentalis was more prevalent than other thrips species. The percentage of adults was significantly greater than that of immatures at the cotyledon stage while immature thrips were significantly more abundant during one to four true-leaf stages of cotton. The study indicated T. tabaci and F. occidentalis were the most prevalent thrips species on seedling cotton on the South Plains of Texas.

Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens

Downy mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant–pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA-Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Effects of Seedling Types and Their Age on Phenological and Vegetative Growth Parameters of Maize (Zea Mays L.) in Burie District, West Gojjam Zone, North Western Ethiopia

The field experiment was conducted during the rainy season of 2018 in Burie District to study effects of seedling types and their growth stages on phonological and vegetative growth parameters of transplanted maize (Zea mays L.). Two types of seedlings (bare rooted and poly bagged); five levels of seedling’s growth stage (1, 2, 3, 4 and 5 true leaf) and one control (direct seeded) were laid down in RCBD design with three replications. Phenological and vegetative growth parameters were collected following standard procedures. All data were subjected to analysis of variance and mean separation for significant treatments were done by LSD. Both the main effect affected days to 50% siliking very highly significantly. Types of seedlings affected days to 50% tasseling significantly and days to 90% physiological maturity and plant height highly significantly. Days to 50% tasseling and 90% physiological maturity were affected very highly significantly by seedlings growth stages. Field survival rate and number of leaves were not significantly affected by both the main effects and the interaction. The higher plant height and earliest days to 50% tasseling, siliking and 90% physiological maturity were noticed from transplantation of poly bagged and five true leaf stage seedlings, while delayed phenological events were recorded from the control. Transplanting of poly bagged seedlings at 5 true leave stage was caused to escape the crop from terminal moisture stress. Therefore, transplanting of poly bagged seedlings at 5 true leaf stage is economically feasible and can be recommended tentatively for Burie District, Ethiopia and moisture stressed area. However; it’s also advised to repeat the study in areas having terminal moisture stress for maize production. Keywords: Bare root; Poly bagged; Seedling; Transplanting; Maize; Ethiopia DOI: 10.7176/PPAR/11-9-06 Publication date: May 31 st 2021

Alternaria alternata causing Alternaria Leaf Spot of Cucumis melo (Muskmelon) in Pakistan.

In July 2019, leaf spot symptoms were observed on muskmelon (Cucumis melo L.) cv. Jackball-1 plants in an experimental field of 2.02 ha with a disease incidence of 30% (31°26'05.4"N 73°04'30.3"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms consisted of small, circular, brown, necrotic spots 1 to 2 mm in size covering 10 to 30% of the leaf blade, which gradually enlarged and developed concentric rings. To identify the causal agent of the disease, a total of 20 symptomatic leaves were collected. Small pieces removed from the margin between healthy and diseased tissues were surface disinfected in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Morphological observations were made on 7-day-old single-spore cultures. The colonies initially appeared white and then turned olive-green. All 20 fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 11.5 to 30 μm × 7.5 to 15 μm (n = 50) with longitudinal and transverse septa. Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. These morphological features concur with the description of Alternaria alternata (Fr.) Keissler (Woudenberg et al. 2013). For molecular identification, genomic DNA of four representative isolates (HMSMZA 07, 08, 09, 10) were extracted and PCR amplification of the internal transcribed spacer (ITS)-rDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor-1 alpha (TEF-1α) gene regions were performed (White et al. 1990, Berbee et al. 1999, Carbone & Kohn, 1999) respectively. The obtained sequences were deposited in GenBank with accession numbers MT253643.1-MT253646.1 (ITS-rDNA), MT318260.1-MT318263.1 (GAPDH), and MT318280.1-MT318283.1 (TEF-1α). BLASTn analysis of HMSMZA 07 sequences showed 100% identity with ITS rDNA (MN615420.1), GAPDH (MK637438.1) and TEF-1α (MN807795.1) sequences of A. alternata. To confirm pathogenicity, 5-6 weeks-old Muskmelon (Cucumis melo L.) cv. Jackball-1 plants (true leaf stage) were sprayed until runoff (1.5 to 2 ml per plant) with A. alternata conidial suspension (106 conidia/ml; obtained from 1 week-old cultures) amended with 0.1% (vol/vol) of Tween 20 using an atomizer in the green house. The experiment included four A. alternata isolates inoculated onto three muskmelon plants per each isolate, whereas control plants (n = 3) were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse and the experiment was conducted twice. After 5 to 7 days post inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated and confirmed by morphological and molecular (ITS) features. No disease was observed on control plants. Previously, A. alternata on muskmelon has been reported in Pakistan (Ahmad et al. 1997), however this study provides a detailed description of disease symptoms, morphological and molecular identity of the causal agent including completion of Koch's postulates. The disease could represent a threat for muskmelon crop in Pakistan due to its increasing cultivation and therefore warrants the need to develop disease management strategies.

Investigating glyphosate resistance in Amaranthus palmeri biotypes from Turkey

Amaranthus palmeri is a troublesome weed which is growing in importance worldwide. It causes serious competition in many crops. A. palmeri was introduced into Turkey 4–5 years ago and appears to adapt to different environmental conditions. Monoculture cropping systems and repeated use of the same herbicides have led to development of herbicide resistance in A. palmeri to several active ingredients including glyphosate, in USA and in South America. This study was conducted to investigate the evolution of glyphosate resistance in A. palmeri in Turkey. Seeds of 21 A. palmeri populations were collected in Turkish citrus fields where control problems with glyphosate were reported. A potentially glyphosate susceptible A. palmeri population was collected from a maize field (GS1 biotype). Seeds of putatively resistant and potentially susceptible (GS) biotypes were germinated and transplanted into large pots, and then allowed to grow in separate greenhouse chambers to obtain F2 generations. After carrying out a preliminary test experiment to exclude the most susceptible populations, a dose-response experiment was conducted in which glyphosate was applied at the 3–4 true leaf stage at 0, 332.5, 665, 1330, 2660, 5320, 10,640 and 21,280 g a.i. ha−1. Plants were harvested 21 days after treatment and dry weight was determined. Glyphosate applied at the recommended rate (1330 g a.i ha−1) controlled GS A. palmeri biotypes by more than 95% while controlling the GR biotypes at about 45%. Among those biotypes, GR1 and GR2 biotypes were confirmed to have an incipient resistance to glyphosate. The effect of glyphosate on shikimic acid accumulation was determined. Results showed that the GS2 biotype accumulated 3.1 and 1.56 times more shikimic acid than GR2 and GR1 biotypes which demonstrates that there is a lower accumulation of shikimic acid in the alleged resistant biotypes than in GS1. These findings demonstrate some increased tolerance of A. palmeri biotypes to glyphosate, which reinforces the need to implement integrated weed management to control this invasive plant in Turkey.

Effects of E-genes and photoperiod duration on assimilation processes in isogenic lines of soybean

The aim of the research. To reveal the patterns of genetic control of assimilation processes in isogenic by E-genes lines of soybean conditions of different photoperiod duration. Materials and methods. The plant material – 5 isolines of soybean (Glycine max (L.) Merr.) Cv. Clark: short-day (SD) lines (genotypes E1E2E3, E1e2e3) and neutral-day (ND) lines (genotypes e1E2e3, e1e2E3, e1e2e3). From the third true leaf stage, one part of the plants was grown on a natural day (16 hours), and the other was exposed to a short day (9 hours). On the day of the start of the experiment, after 7 and 14 days, the dry weight of leaves and stems, the number and area of leaves were measured, based on which assimilation indices were calculated – RGR, NAR, LAR, SLA, LWR. Results. During the studied ontogenetic period (two weeks) in all isolines, regardless of the genotype by E-genes and the duration of the photoperiod, the assimilation processes increased. The RGR and NAR under the short day decreased in the first week and then increased in the second week of the experiment. The degree of change in the indices varied depending on the isoline genotype by E-genes. The LAR and LWR were lower under the short day in SD lines. These indices were the same in the ND lines under short and long day. Under the short photoperiod the SLA in SD lines was higher, and in ND lines it was practically the same for both photoperiods. Conclusions. The assimilation processes in the studied isogenic lines during the experience (two weeks) increase, but less intensively under short-day conditions. The studied lines, differed in the nature and intensity of assimilation processes, depending on the genotype by E-genes. Assimilation processes in the studied soybean lines are probably determined by the interaction of E-genes and the duration of the photoperiod, which is one of the important conditions for the adaptation of soybean to the environmental factors

Novel Transcriptome Study and Detection of Metabolic Variations in UV-B-Treated Date Palm (Phoenix dactylifera cv. Khalas).

Date palm (Phoenix dactylifera) is one of the most widespread fruit crop species and can tolerate drastic environmental conditions that may not be suitable for other fruit species. Excess UV-B stress is one of the greatest concerns for date palm trees and can cause genotoxic effects. Date palm responds to UV-B irradiation through increased DEG expression levels and elaborates upon regulatory metabolic mechanisms that assist the plants in adjusting to this exertion. Sixty-day-old Khalas date palm seedlings (first true-leaf stage) were treated with UV-B (wavelength, 253.7 nm; intensity, 75 μW cm−2 for 72 h (16 h of UV light and 8 h of darkness). Transcriptome analysis revealed 10,249 and 12,426 genes whose expressions were upregulated and downregulated, respectively, compared to the genes in the control. Furthermore, the differentially expressed genes included transcription factor-encoding genes and chloroplast- and photosystem-related genes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to detect metabolite variations. Fifty metabolites, including amino acids and flavonoids, showed changes in levels after UV-B excess. Amino acid metabolism was changed by UV-B irradiation, and some amino acids interacted with precursors of different pathways that were used to synthesize secondary metabolites, i.e., flavonoids and phenylpropanoids. The metabolite content response to UV-B irradiation according to hierarchical clustering analysis showed changes in amino acids and flavonoids compared with those of the control. Amino acids might increase the function of scavengers of reactive oxygen species by synthesizing flavonoids that increase in response to UV-B treatment. This study enriches the annotated date palm unigene sequences and enhances the understanding of the mechanisms underlying UV-B stress through genetic manipulation. Moreover, this study provides a sequence resource for genetic, genomic and metabolic studies of date palm.

First report of bacterial leaf blight caused by Xanthomonas hortorum pv. carotae on carrots in Spain.

In September 2019, symptoms resembling those of bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus Hoffm.) cv. Romance cultivated in commercial plots in Chañe (Segovia), Spain. Symptoms were observed in two plots surveyed representing three hectares, with an incidence greater than 90%, and also in some plots in other nearby municipalities sown with the same batch of seeds. The lesions observed at the ends of the leaves were initially yellow that develop dark brown to black with chlorotic halos on leaflets that turned necrotic. Yellow, Xanthomonas-like colonies were isolated onto YPGA medium (Ridé 1969) from leaf lesions. Two bacterial isolates were selected and confirmed by real-time PCR using a specific primer set for Xanthomonas hortorum pv. carotae (Temple et al. 2013). All isolates were gram-negative, aerobic rods positive for catalase, able of hydrolyzing casein and aesculin and growing at 2% NaCl, while were negative for oxidase and urease tests. Sequences of 16S rRNA gene showed 100% similarity with Xanthomonas campestris, X. arboricola, X. gardneri, X. cynarae strains (GenBank accession numbers: MW077507.1 and MW077508.1 for the isolates CRD19-206.3 and CRD19-206.4, respectively). However, the resulting phylogeny of multilocus sequence analysis (MLSA) of a concatenation of the housekeeping genes atpD, dnaK, and efp (Bui Thi Ngoc et al. 2010), by using neighbour-joining trees generated with 500 bootstrap replicates, grouped the two isolates with the X. hortorum pv. carotae M081 strain (Kimbrel et al. 2011) (GenBank accession numbers: MW161270 and MW161271 for atpD for the two isolates, respectively; MW161268 and MW161269 for dnaK; MW161272 and MW161273 for efp). A pairwise identity analysis revealed a 100% identity between all three isolates. Pathogenicity of the isolates was tested by spray inoculation (Christianson et al. 2015) with a bacterial suspension (108 CFU/ml) prepared in sterile distilled water at 3 to 4 true-leaf stage (six plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 72 h, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 20 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through PCR analysis. Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Spain, and the first molecular and pathological characterization. It is important to early detect this pathogen and take suitable measures to prevent its spread, since it could cause yield losses for a locally important crop such as carrot.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Okra in China.

Okra [Abelmoschus esculentus (L.) Moench], which belongs to the family Malvaceae, is widely grown in the tropics, sub-tropics and warmer areas of the temperate zones for its immature seed pods which are consumed as a vegetable. In China, okra pods are consumed as not only vegetables but also as a traditional medicine to cure dental diseases and gastric ulcers. During September 2018 to June 2019, extensive spots on okra leaves were observed in several commercial fields (approximately 2.0 hectares), with disease incidence of approximately 25%~50% in the Yanqing District (115°98'E, 40°46'N) of Beijing, China. Symptoms of the disease initially appeared as small pale brown spots with yellow haloes. As the disease progressed, some spots gradually coalesced, forming larger irregular dark brown lesions. The centers of the lesions became grayish white. A total of 13 small fragments (3 to 5 mm) excised from the lesion margins were sterilized in 1% sodium hypochlorite (NaClO) for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. In total, 21 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 286/span> to 711 μm (avg. = 578 μm, n = 50). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3-17-septate, and measuring 52 to 231 μm (avg. = 182 μm, n = 50). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). Pathogenicity tests were conducted on potted okra plants cv. 'Jiayuan'. Twenty four healthy okra plants at the true leaf stage were sprayed with conidial suspensions (1 × 106 conidia/mL), incubated at a glass cabinet maintained at 25°C and 90% relative humidity (RH). To each leaf approximately 10 mL of conidial suspension was applied. Plants sprayed with water were used as controls. Seven days later, dark brown spot, which were identical to those observed in the fields, were observed on inoculated leaves, whereas the control plants remained healthy. C. cf. flagellaris was reisolated from symptomatic leaves, confirming Koch's Postulates. Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). The nuclear ribosomal internal transcribed spacer region (ITS), and portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone & Kohn 1999). The resulting 542 bp ITS, 226 bp ACT, 410 bp HIS3 and 306 bp TEF1 sequences of isolate QK14091813 were deposited in GeneBank (Accession nos. MT949700, MT949701, MT949702 and MT949703, respectively). The ITS, ACT, HIS3 and TEF1 sequences shared 99.42% to 100% identities to previously published sequences of C. cf. flagellaris (Accession nos. MN633275 for ITS, MF680960 for ACT, MK991295 for HIS3, and MK991292.1 for TEF1, respectively). Multi-locus phylogenetic analyses (ITS, ACT, HIS3, and TEF1) were performed by neighbor-joining method using MEGA 7.0. The resulting trees showed that C. cf. flagellaris isolate QK14091813 (this study) nested within the clade that includes other isolates of C. cf. flagellaris with a 99% confidence level. To our knowledge, this is the first report of C. cf. flagellaris causing leaf spot on okra (Farr and Rossman 2020). The pathogen has a worldwide distribution and an unusually broad host range, which can be of great significance, and the plant protection policy of priority to prevention and synthetical prevention should be followed.

Response of Palmer amaranth (Amaranthus palmeri S. Watson) and sugarbeet to desmedipham and phenmedipham

AbstractA prepackaged mixture of desmedipham + phenmedipham was previously labeled for control of Amaranthus spp. in sugarbeet. Currently, there are no effective POST herbicide options to control glyphosate-resistant Palmer amaranth in sugarbeet. Sugarbeet growers are interested in using desmedipham + phenmedipham to control escaped Palmer amaranth. In 2019, a greenhouse experiment was initiated near Scottsbluff, NE, to determine the selectivity of desmedipham and phenmedipham between Palmer amaranth and sugarbeet. Three populations of Palmer amaranth and four sugarbeet hybrids were evaluated. Herbicide treatments consisted of desmedipham and phenmedipham applied singly or as mixtures at an equivalent rate. Herbicides were applied when Palmer amaranth and sugarbeet were at the cotyledon stage, or two true-leaf sugarbeet stage and when Palmer amaranth was 7 cm tall. The selectivity indices for desmedipham, phenmedipham, and desmedipham + phenmedipham were 1.61, 2.47, and 3.05, respectively, at the cotyledon stage. At the two true-leaf application stage, the highest rates of desmedipham and phenmedipham were associated with low mortality rates in sugarbeet, resulting in a failed response of death. The highest rates of desmedipham + phenmedipham caused a death response of sugarbeet; the selectivity index was 2.15. Desmedipham treatments resulted in lower LD50 estimates for Palmer amaranth compared to phenmedipham, indicating that desmedipham can provide greater levels of control for Palmer amaranth. However, desmedipham also caused greater injury in sugarbeet, producing lower LD50 estimates compared to phenmedipham. Desmedipham + phenmedipham provided 90% or greater control of cotyledon-size Palmer amaranth at a labeled rate but also caused high levels of sugarbeet injury. Neither desmedipham, phenmedipham, nor desmedipham + phenmedipham was able to control 7-cm tall Palmer amaranth at previously labeled rates. Results indicate that desmedipham + phenmedipham can only control Palmer amaranth if applied at the cotyledon stage and a high level of sugarbeet injury is acceptable.

Interactive effects of Andrographis paniculata extracts and cancer chemotherapeutic 5-Fluorouracil on cytochrome P450s expression in human hepatocellular carcinoma HepG2 cells

Andrographis paniculata is an annual herbaceous plant widely used in many East Asian countries to help relieve symptoms of the common cold, fever, and non-infectious diarrhea. It is often co-administered with prescribed drugs. The present work aimed to study the interactive effects of A. paniculata extracts and the cancer chemotherapeutic drug, 5-Flurouracil (5-FU), on the expression of four common drug metabolizing cytochrome P450 s (CYP450), including CYP1A2, CYP3A4, CYP2C9, and CYP2D6, in human hepatocellular carcinoma HepG2 cells. Two different types of A. paniculata extracts were tested, the ethanolic extract at the first true leaf stage (FTLEE) and the water extract at the mature leaf stage (MLWE), along with their major phytochemical compound, andrographolide (AP1). The results showed that FTLEE, MLWE, and AP1 sensitized HepG2 cells to the cytotoxicity of 5-FU. Western Blotting assay demonstrated that both extracts and AP1 single treatments decreased CYP2C9 and CYP2D6 but increased CYP3A4 levels. 5-FU single treatment reduced the level of CYP1A2 and CYP2D6. Combination of 5-FU only with FTLEE produced an increase in CYP1A2 level compared to 5-FU treatment alone. Combination of either extracts or AP1 with 5-FU produced a further reduction of CYP2D6 levels. The data suggest the influence of harvesting stage of A. paniculata on CYP1A2 induction, and the synergistic effect of A. paniculata and 5-FU on CYP2D6 reduction. The combinations of A. paniculata with anti-cancer drugs should be critically considered. However, the interactive effects of A. paniculata extracts or AP1 with 5-FU on CYP450 s need more preclinical studies.

Cucurbit leaf crumple virus (CuLCrV) associated to watermelon disease in Campeche state, Mexico.

An annual recurrent disease causing yield reduction in cultivated watermelon (Citrullus lanatus) was documented by the growers in different farms of Campeche state, Mexico. In April 2019 and March 2020 open field grown watermelon plants showed symptoms such as leaf curling, crumpling, and leaf basal or apical necrosis (Figure S1), with an incidence ranging from 30 up to 80%. These plants also presented high populations of whitefly, especially in the most affected fields. In order to identify the causal agent of the disease, a total of 22 symptomatic watermelon plants were collected in four locations from Campeche state. Total nucleic acids (DNA and RNA) were extracted from these leaf samples. Initially, RT-PCR analysis was performed with specific primers (Table S1) for cucurbit-infecting Crinivirus transmitted by whitefly but the expected size PCR product for those viruses was not amplified in any of these samples. To investigate the presence of cucurbit-infecting begomoviruses, PCR was performed by using specific primers for those begomoviruses reported in Mexico and north/central America including Squash leaf curl virus (SLCV), Watermelon chlorotic stunt virus (WmCSV), Melon chlorotic leaf curl virus (MCLCuV), and Cucurbit leaf crumple virus (CuLCrV) (Table S1). Only the expected amplicon size of ~1089 bp for CuLCrV was amplified from DNA extracts from all 22 watermelon samples, suggesting a single cucurbit-associated virus. The putative complete genome of the CuLCrV Campeche isolate was amplified by circular DNA enrichment using a Rolling Circle Amplification (RCA) procedure from two representative samples, followed by enzymatic digestion using BamHI, EcoRI, KpnI, and SacI enzymes (Inoue-Nagata et al., 2004). Expected linearized full-length viral components (~2.7 kb) were obtained with EcoRI and SacI, and both products, from one selected sample, were cloned in to pGreen0029 vector and were fully sequenced. Sequence analysis of the EcoRI clone, designated as LV2019Camp_A (deposited in GenBank accession no. MW273384) revealed the highest identity of 97.52% to CuLCrV DNA-A isolate Baja California Sur isolate (GeneBank accession no. MN625831.1), whereas the KpnI clone, designated as LV2019Camp_B (deposited in GenBank accession no. MW273385), shared 94.87% identity with DNA B of CuLCrV isolate Arizona (GeneBank accession no. AF327559.1). Subsequently, CuLCrV isolate Campeche-derived agroinfectious clone, was obtained by constructing a partial dimeric tandem repeat of both DNA-A and DNA-B components (Bang et al., 2014). Twelve watermelon plants were agroinfiltrated with the infectious clone at the fourth true leaf stage, resulting in symptomatic plants (11/12) exhibiting leaf yellowing, curling, and crumpling 15 days after agroinfiltrated (Figure S1), and CuLCrV infection was confirmed by PCR specific detection using DNA extract from non-inoculated leaves. Previously CuLCrV has been detected in the USA (Arizona, Texas, California, Florida, South Carolina, and Georgia), and north Mexico (Coahuila) infecting cucurbits including squash, cucumber, cantaloupe, pumpkin, and watermelon (Brown et al., 2000., Keinath et al., 2018), in both single and mixed infection with other whitefly transmitted RNA viruses (CYSDV, genera Crinivirus), and DNA viruses (SLCV, genera Begomovirus) (Kuo et al., 2007). To our knowledge, this is the first report of CuLCrV infecting a cucurbit crop in the Campeche state from the Yucatán peninsula, in Mexico.

Role of salicylic acid in heat stress tolerance in tri-genomic Brassica napus L.

Tri-genomic Brassica napus L.wasdeveloped by the cross between Brassica napusand Brassica nigra. The crop is animportant source of vegetable seed oil in Pakistan,after cotton. The low oilseed rape yield is attributed to high temperature in the production zones. Interspecific hybridization using these two speciescan be helpful to produce heat resistant hybrids. On the other hand, it has been found that foliar application of different plant growth regulators can be used to reduce the heat stress in Brassica. The objectiveof this studywas to test the response of three different tri-genomic hybrids to high temperature stressat seedling stage. Seedlings were foliar sprayed with 0.13 mM salicylic acid (SA) prior to exposure tohigh temperatureat two true leaf stage. The plants were harvested after 30 days of sowing for growth and biochemical analysis. Plants ofV38 showed the highest values for all morphological traits and biochemical activities among the threehybrids. In general, plants exposed to the temperature stress exhibited a significant decline in growth, chlorophyll content and enzyme activity.Foliar application of SA significantly improved leaf and root biomass under heat stress.Further, antioxidativeenzyme activities significantly increased in response to SA either compared to control or to plants exposed to temperature stress.It is concluded thatapplication of salicylic acid elevated activity of antioxidative enzymes and was helpful in mitigating the detrimental effects of high temperature inoil seed rape.

Occurrence of Leaf Spot Caused by Alternaria alternata on Eggplant (Solanum melongena) in Pakistan.

Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 μm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (β-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), β-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for β-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, β-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan.

Effect of Vanadium on the Uptake and Distribution of Organic and Inorganic Forms of Iodine in Sweetcorn Plants during Early-Stage Development

Iodine and vanadium are elements that are closely related to organisms in water environments. Iodine and vanadium are known as “beneficial elements” that stimulate the growth and development of higher plants. Iodine is an essential element for the synthesis of the thyroid hormones triiodothyronine and thyroxine in the human body, with vanadium also known to be involved in the synthesis of thyroid hormones. The cooperation of both elements in the human body and in algae presents a question regarding the impact of vanadium interaction on iodine uptake in higher plants. The absorption of iodine from seawater in algae is known to be more efficient in the presence of vanadium, with key role in this process played by the iodoperoxidase enzyme, with vanadium acting as a cofactor. The study of the nature of the absorption of iodine by higher plants, and in particular by crops such as corn, remains insufficiently studied. The aim of this study was to investigate the effect of vanadium on iodine uptake via vanadium-dependent iodoperoxidase (vHPO) activity in sweetcorn plants (Zea mays L. subsp. Mays Saccharata Group) “Złota Karłowa”. The experiment was carried out with organic and inorganic iodine compounds, namely potassium iodide (KI), potassium iodate (KIO3), 5-iodosalicylic acid (5-ISA), and 2-iodobenzoic acid (2-IBeA), each used in a dose of 10 μM. These compounds were applied with and without vanadium in the form of ammonium methavanadate (NH4VO3) at a dose of 0.1 μM. A double control was used, the first without iodine and vanadium and the second with vanadium but without iodine. Root length, root mass, and above-ground weight were significantly higher after iodine and vanadium compared to controls. Plants were collected at the five true leaf stage. vHPO activity level was much higher in the roots than in the leaves, but greater variation in the leaves was observed between treatments in terms of vHPO activity. Vanadium was shown to accumulate in the roots. The use of a relatively low dose of vanadium may have caused changes in the accumulation of this element in the aerial parts of the plant, leaves, and shoots. Fertilization with iodine and vanadium compounds decreased the accumulation of most minerals, macroelements, and microelements compared to controls. The obtained results of iodine accumulation in individual parts after applying iodine and vanadium fertilization testify to the stimulating effect of vanadium on iodine uptake and accumulation.

Developing Triploid Maples

Maples are common street and shade trees throughout the temperate zone. They are widely used for their wide range of ornamental traits and adaptability, particularly to urban settings. Unfortunately, some species such as Acer tataricum ssp. ginnala (Amur maple) and A. platanoides (Norway maple) have escaped cultivation to become pests or in some cases threaten native flora. However, these species remain economically important and are still asked for by name. To ameliorate potential future ecological damage from additional escapes, we have been breeding for sterile forms using ploidy manipulation and backcrossing to develop triploids. We began with a series of experiments to develop tetraploids of Amur, Norway, and trident (A. buergerianum) maples. Treatment of seedlings at the cotyledon or first true leaf stage was successful in inducing tetraploids of each species. Mortality, cytochimeras, and tetraploids varied among species. After identifying tetraploids, they were field planted alongside diploid cultivars and seedlings, which served as pollinizers in open-pollination. Seedlings derived from open-pollinated tetraploids were generally found to be a high percentage triploids. Thus far, no Norway or trident maple triploids have flowered but after three years we observed five, 22, and 22 Amur maple triploids flowering over three respective years with no seedlings recovered to date. Further evaluation is required but our findings are encouraging that the triploids we have developed thus far will be sterile and provide new cultivars for nursery growers and land managers.

Pixel-Level Weed Classification Using Evolutionary Selection of Local Binary Pattern in a Stochastic Optimised Ensemble

In the early growth stages of young crops, weeds compete for living resources while often grow close together and with overlaps. Conventional weeding techniques such as mechanical weeding and herbicide application are not precision methods. To allow for high precision target and spraying of weeds, autonomous field robots equipped with computer vision systems could be deployed to manage and control the weeds in the crop line. The robots are therefore required to classify weeds at pixel-level accuracy with high recall rates. This paper presents a pixel-level weed classification model using rotation-invariant uniform local binary pattern (LBP) features. The model design is based on an ensemble with a two-level optimisation structure. The first-level leverages on Genetic Algorithm (GA) optimisation to select the best rotation-invariant uniform LBP configurations. The second level utilises covariance matrix adaptation evolution strategy (CMA-ES) in the Neural Network (NN) ensemble to select the best combinations of voting weights of the predicted outcome for each classifier. This model design allows for different feature inputs selected by GA for each of the NN classifiers in the ensemble, unlike classical ensembles which share the same input data. For ensemble weight optimisation, we compared differential evolution, particle swarm optimisation, and CMA-ES. A crop/weed field image dataset public dataset consisting of 60 images of weeds and carrots at early true leaf stage was used for validation with existing works. The results demonstrated that our LBP-based pixel-level classifier exhibits excellent predictive capability with high recall and F1 scores when compared to the literature. It was found that CMA-ES optimised ensemble gave the best configuration and demonstrated a classification accuracy of 87.9%, which exceeded the existing statistical feature classification of 85.9%. This highlights some potentialities for chip-based application-specific integrated circuit LBP pixel-level weed classification in smart agriculture applications.