Aphelenchoides besseyi parasitizing cowpea (Vigna unguiculata) in Brazil

Several species of nematodes are known to cause losses to cowpea (Vigna unguiculata) throughout the world. In Brazil, Aphelenchoides besseyi was recently described causing damages on soybean, cotton, and common bean, but no report was found about the parasitism of this nematode in cowpea. The present study aimed to verify the host reaction of cowpea cultivars to A. besseyi. The experiment was conducted under greenhouse conditions, using as inoculum two A. besseyi populations, obtained from symptomatic soybean and cotton plants collected in naturally infested fields. Cultivars ‘Imponente’, ‘Aracê’, ‘Guariba’, ‘Tumucumaque’, ‘Nova Era’, and ‘Tracuateua’ were inoculated with 500 A. besseyi of each population, separately, into soil and after 30 days from the inoculation nematodes were extracted from shoot systems. Both populations were able to parasitize all the cowpea cultivars. Independently of the cultivar, cowpea plants exhibited symptoms of leaf deformation similar to those described for soybean, cotton, and common bean and, in addition, severe brooming was observed and the interior of the stems was porous and necrotic. To our knowledge, this is the first report of parasitism by A. besseyi of cowpea in Brazil, under greenhouse conditions, increasing the list of hosts of this nematode.

Natural Infection of Cichorium intybus (Asteraceae) by Groundnut Ringspot Virus (Genus Orthotospovirus) Isolates in Brazil

Leaf chicory (Cichorium intybus L.) is a nutritionally rich vegetable used in regional cuisine in Brazil. Plants of C. intybus displaying symptoms (viz. chlorotic and necrotic ringspots, mosaic, and leaf deformation) similar to that induced by orthotospoviruses (genus Orthotospovirus, family Tospoviridae) were observed in three fields (≈ 0.2 ha each) in Gama County, in the Federal District, Brazil, from September 2016 to January 2020 in plants of the cultivars ‘Folha-Larga’ and ‘Spadona’ (Fig. 1). Incidence of symptomatic plants was nearly 10% in each field. Transmission electron microscopic examination of thin sections from symptomatic leaf samples showed typical membrane-bounded orthotospovirus particles within cisternae of spongy parenchymal cells (Fig 2). Two individual leaf samples per field were collected and submitted to dot enzyme-linked immunosorbent assay with polyclonal antisera against N protein of tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV). Symptomatic samples strongly reacted only against GRSV antibodies. Total RNA was extracted (Trizol®, Sigma) from all six samples and used as template in RT-PCR assays. The primer J13 (5’-CCCGGATCCAGAGCAAT-3’) was employed for cDNA synthesis using M-MLV reverse transcriptase. PCR assays were done with the primer pair BR60/BR65 (Eiras et al., 2001) to obtain ≈ 500 bp fragment of untranslated region and partial N gene in the S RNA segment from each sample. Purified RT-PCR products of two randomly selected individual samples were directly sequenced (GenBank MW467981 and MZ126602) and their BLASTn analyses displayed 99 to 100% nucleotide identity to GRSV isolates previously reported infecting C. endivia L. in Brazil (Jorge et al., 2021). Our analyses combining N protein serology and N-gene sequencing (both directed to the S RNA segment) allowed us to confirm the GRSV infection of C. intybus, but the potential reassortant nature of these isolates (Webster et al., 2015; Silva et al., 2019) are unknown since their M RNA segments were not characterized. Individual leaf extracts (in phosphate buffer, pH 7.0) of the sequenced isolates were mechanically inoculated onto ten seedlings of two C. intybus cultivars (‘Folha Larga’ and ‘Pão-de-Açúcar’) and three plants each of the indicator hosts Capsicum chinense PI 159236, Nicandra physalodes; Nicotiana rustica; Datura stramonium; and tomato cv. Santa Clara. Systemic chlorotic and necrotic ringspots, mosaic, and leaf deformation developed in the indicator hosts and infection by GRSV was confirmed via serological assays 20 days after inoculation. However, no symptoms and no serological reaction to GRSV antibodies were observed on the C. intybus cultivars even after two successive mechanical inoculations. This transmission failure might be due to factors such as the requirement of the thrips vector(s), physicochemical barriers in the foliage or the presence of non-mechanically transmissible helper agent(s) necessary to ensure GRSV infection of C. intybus. The natural infection of C. intybus by a not fully characterized orthotospovirus (mostly likely TSWV) has been observed since 1938 in Brazil (Kitajima, 2020). Our report of GRSV infecting C. intybus is thus confirming previous speculations that similar symptoms in this vegetable crop were induced by orthotospovirus infection in Brazil. References: Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Jorge, T. S. et al. 2021. Plant Dis. 105: 714. Kitajima, E.W. 2020. Biota Neotrop. 20: e2019932. Silva, J. M. F. et al. 2019. Viruses 11: 187. Webster, C.G. et al. 2015. Phytopathology 105: 388.

Identification of a New Badnavirus in the Chinaberry (Melia azedarach) Tree and Establishment of a LAMP-LFD Assay for Its Rapid and Visual Detection

The Chinaberry tree, a member of the Meliaceae family, is cultivated in China for use in traditional medicines. In 2020, Chinaberry trees with leaf deformation symptoms were found in Hangzhou, Zhejiang province, China. In order to identify possible pathogenic viruses, a symptomatic sample was subjected to deep sequencing of small interfering RNAs. Assembly of the resulting sequences led to the identification of a novel badnavirus, provisionally designated Chinaberry tree badnavirus 1 (ChTBV1). With the recent development of China’s seedling industry and increasing online shopping platforms, the risk of tree virus transmission has increased substantially. Therefore, it is important to detect the occurrence of ChTBV1 to ensure the safety of the Chinaberry tree seedling industry. Here, we describe the development and validation of a sensitive and robust method relying on a loop-mediated isothermal amplification (LAMP) assay, targeting a 197 nt region, to detect ChTBV1 from Chinaberry tree leaves. The LAMP assay was also adapted for rapid visualization of results by a lateral flow dipstick chromatographic detection method.

Characterization of a Novel Emaravirus Affecting Ash Species (Fraxinus spp.) in Europe

We identified a novel virus in diseased European ash (Fraxinus excelsior) and manna ash (F. ornus) trees exhibiting chlorotic ringspots, mottle and leaf deformation such as curling and shoestring symptoms. High-throughput sequencing (HTS, Illumina RNASeq) of total RNA isolated from diseased leaf material in combination with RT-PCR-based amplification techniques and Sanger sequencing determined five complete genome segments, each encoding a single open reading frame. Sequence analyses of RNA1–RNA5 revealed a genome organization typical for emaraviruses, i.e., (i) conserved and complementary terminal 5′and 3′termini of each genome segment (ii) proteins showing significant homologies to the RNA-dependent RNA polymerase (RdRP) encoded by RNA1, the glycoprotein precursor (GPP) encoded by RNA2, the viral nucleocapsid protein (N, RNA3), the movement protein (MP, RNA4), and a protein of 26 kDA (P26, RNA5) highly similar to proteins of unknown function encoded by other emaraviruses. Furthermore, we identified spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) which are typical for emaraviruses exclusively in virus-infected leaf tissue exhibiting mottle and leaf deformation. Sequence comparison and phylogenetic analyses confirmed the identified novel virus as a new member of the genus Emaravirus. We established a species-specific RT-PCR detection protocol and could associate the observed disease symptoms with the infection of the novel emaravirus in F. excelsior and F. ornus. Therefore, we propose the name ash shoestring-associated emaravirus (ASaV). Investigation of ASaV-infected sample trees originating from different locations in Switzerland, Germany, Italy and Sweden provided a wide geographical distribution of the virus in affected ash species. To our knowledge, this is the first confirmation of an emaravirus affecting ash tree species with shoestring symptoms of leaves in Europe.

Flowering attributes of Henckelia Royal Queen influenced by pinching and Paclobutrazol application

An experiment was carried out to determine the effect of pinching practice and different concentrations of Paclobutrazol (PBZ) on the flowering of Henckelia Royal Queen plants.Completely randomized design with eight treatment combinations of pinching or non-pinching and different concentrations of Paclobutrazol (0, 15, 30and 60 ppm) soil drenchwas usedwith eight replicates for each treatment. Pinching was practiced twice at two weeks intervals. Flowering parameters viz. number of flower buds, days taken to flowering, number of flowers, pedicel length, corolla length and corolla width were measured at two weeks intervals from 9thweek after the treatment application. Results disclosed that plants showed better flowering performances (P<0.05) with increasing concentrations of PBZ, but degree of leaf deformation was increased. Therefore, the application of 15 ppm PBZ with pinching showed better to obtain dwarf Henckelia Royal Queen plants with improved flowering quality as commercial potted plants. Bangladesh J. Sci. Ind. Res.56(3), 177-184, 2021

A Taphrina strain infecting the model plant Arabidopsis thaliana.

Yeasts are important plant-associated organisms that can modulate host immunity to either promote or prevent disease. Mechanisms of plant-yeast interactions, specifically of yeast perception by the plant innate immune system, remain unknown. Progress has been hindered by the scarcity of yeast species associated with the model plant Arabidopsis thaliana (Arabidopsis). We have previously isolated Taphrina strain M11 from wild Arabidopsis in the field. Taphrina are poorly studied dimorphic yeast-like fungi that are plant pathogens, often producing plant hormones and causing tumour-like leaf deformation symptoms on their hosts. Here we characterize the interaction of M11 with Arabidopsis. Infection of Arabidopsis with the birch pathogen T. betulina, used as a non-host control, shows early HR, enhanced ROS accumulation, and limitation of growth, demonstrating that Arabidopsis has immunity against non-adapted yeasts. M11 triggered limited cell death, an attenuated ROS response, and grew in planta, as well as subtle but clear leaf deformation symptoms, demonstrating it is pathogenic. Hormone responsive promoter-reporter analysis demonstrated activation of cytokinin signalling during infection. Mutant infection assays indicate jasmonate and ethylene were required for immunity against M11. Analysis of the Taphrina M11 genome was used to mine evidence for yeast specific PAMPs which may underlie host immune responses against yeast-like fungi.

Begomovirus Caboniensis: a New Bipartite Begomovirus Isolated From Cnidoscolus Urens in Brazil

A novel bipartite begomovirus infecting Cnidoscolus urens (Euphorbiaceae) from Pernambuco State, Brazil has been characterized. The complete DNA-A (2657 to 2692 nt) and DNA-B (2622 nt) components of the viral isolates showed a typical genome organization of New World bipartite begomoviruses. DNA-A of the isolates had the highest percentage of nucleotide identity (88.6–88.9%) with the Cnidoscolus mosaic leaf deformation virus (NC_038982). Based on the current classification criteria for the genus Begomovirus, a new member infecting C. urens was reported, and the name Begomovirus caboniensis was proposed for these viruses, adopting the standardized binomial system.

Complete Genome Sequence of Two Isolates of Spiraea Yellow Leafspot Virus (Genus: Badnavirus) From Spiraea x Bumalda ‘Anthony Waterer’

Isolates of spiraea yellow leafspot virus (SYLSV) were collected from Spiraea (Spiraea x bumalda) ‘Anthony Waterer’ plants showing virus-like symptoms including yellow spotting and leaf deformation in Minnesota and Maryland. The complete genome sequence of SYLSV-MN (Minnesota) and SYLSV-MD (Maryland) was 8,017bp in length. The sequences share 95% of identity at the nucleotide level. Both isolates have the same genome organization containing three open reading frames (ORFs), with ORF3 being the largest encoding the putative polyprotein of 232 kDa with conserved domains including Zinc finger, pepsin-like aspartate protease, reverse transcriptase (RT), and RNase H. Pairwise comparisons between members of the genus badnavirus showed that Gooseberry vein banding associated virus GB1 (HQ852248) and Rubus yellow net virus isolate Baumforth's Seedling A (KM078034) were the closest related virus sequences to SYLSV sharing 73% of identity at the nucleotide level. Bacilliform virions with dimensions of 150 nm x 30 nm were observed in virus preparations from symptomatic but not in asymptomatic plants.

Imaging analysis method to quantify leaf deformation in response to sub-lethal rates of dicamba

Dicamba is a synthetic auxin herbicide that is prone to off-target movement, including drift and volatilization. Due to the increased acreage of dicamba-resistant soybean to control glyphosate-resistant weeds, dicamba drift injury to neighboring vegetable crops is of concern. A method to quantify leaf deformation (often referred to as leaf cupping) caused by dicamba injury was developed and compared to visual rating techniques to determine its accuracy and suitability. A second objective was to determine the relative dicamba sensitivity of several economically important vegetable crops. Soybean, snap bean, tomato, and cucumber were grown in the greenhouse and exposed to dicamba at 0, 56, 112, 280, 560, 1120, and 2240 mg ae ha−1, which is respectively 0, 1/10000, 1/5000, 1/2000, 1/1000, 1/500, and 1/250 of the maximum recommended label rate for soybean application (560 g ae ha−1). Plants were evaluated visually and using an imaging analysis technique where leaf deformation index (LDI) was measured using a leaf area scanner. LDI is calculated by dividing the two-dimensional projection of the area of the leaf in its natural configuration by the area of the flattened leaf. Across all four crops, log-logistic regression analysis indicated the LDI method had lower I50 values with lower standard error, demonstrating the LDI method gives more precise estimates of sensitivity. This novel method provides an objective, quantitative method to measure dicamba drift injury and determine relative sensitivities of valuable specialty crops.