Effective Control of Sclerotinia Stem Rot in Canola Plants Through Application of Exogenous Hairpin RNA of Multiple Sclerotinia sclerotiorum Genes.

Sclerotinia stem rot is a globally destructive plant disease caused by Sclerotinia sclerotiorum. Current management of Sclerotinia stem rot primarily relies on chemical fungicides and crop rotation, raising environmental concerns. In this study, we developed an eco-friendly RNA bio-fungicide targeting S. sclerotiorum. Six S. sclerotiorum genes were selected for double-stranded RNA (dsRNA) synthesis. Four genes, a chitin-binding domain, mitogen-activated protein kinase, oxaloacetate acetylhydrolase, and abhydrolase-3, were combined to express hairpin RNA in Escherichia coli HT115. The effect of application of total RNA extracted from E. coli HT115 expressing hairpin RNA on disease progressive and necrosis lesions was evaluated. Gene expression analysis using real-time PCR showed silencing of the target genes using 5 ng/µl of dsRNA in a fungal liquid culture. A detached leaf assay and greenhouse application of dsRNA on canola stem and leaves showed variation in the reduction of necrosis symptoms by dsRNA of different genes, with abhydrolase-3 being the most effective. The dsRNA from a combination of four genes reduced disease severity significantly (P = 0.01). Plants sprayed with hairpin RNA from four genes had lesions that were almost 30% smaller than those of plants treated with abhydrolase-3 alone, in lab and greenhouse assays. The results of this study highlight the potential of RNA interference to manage diseases caused by S. sclerotiorum; however, additional research is necessary to optimize its efficacy.

First Report of Verticillium stripe of Canola Caused by Verticillium longisporum in North Dakota.

North Dakota leads the U.S.A. in canola (Brassica napus L.) production (5) and approximately one-third of the acreage is located in the northeastern portion of the state. A field survey conducted at the end of the 2021 growing season in northeastern North Dakota revealed the presence of a single field with approximately 15% plants exhibiting whitish discoloration symptoms in the lower third of the stems. The epidermis on the discolored areas was peeling, and the exposed tissues were dark grey to dark-brown. Stem samples taken to the laboratory were surface disinfested with a 5% aqueous solution of NaOCl for 60 sec and rinsed thrice with sterile water. Under aseptic conditions, small stem pieces were plated on V-8 medium amended with 150 mg each of penicillin and streptomycin per liter of medium and incubated at 22 oC under 16 h light daily. Under the microscope, conidiophores were verticillate, hyaline, and had three branches. Conidia were single-celled, hyaline, and measured on average 9.2 + 1.8 µm. Microsclerotia were irregularly shaped. These features match the description of Verticillium longisporum (Stark) Karapapa Bainbr. & Heale, (4). Genomic DNA was extracted from a single-spore culture of an isolate as described by Azizi et al. (1). PCR assays were conducted twice on two independent DNA samples extracted from the same isolate using V. longisporum species-specific primer set VlspF1 and VlspR4 (2) with denaturation at 95 oC for 3 min, followed by 35 cycles of amplification at 94 oC for 1 min then 56 oC for 30 sec and 72 oC for 1 min, followed by a final period at 72 oC for 5 min. The sequenced PCR product, which had 100% homology with GenBank V. longisporum reference samples KY704097 and HE972063, was assigned GenBank accession number OR088215. Pathogenicity tests were conducted in greenhouse. Briefly, twenty seeds of the canola cv. Westar were incubated on a sterilized wet paper towel for five days at 22 oC. The seedlings were carefully lifted, and their root tips cut with scissors. Ten wounded seedlings were immersed in a V. longisporum spore suspension with 2.07 x 106 spores per ml for 30 minutes and the other ten in distilled water (controls). The plants were transplanted into pots (10x10x13 cm) containing Sunshine Mix # 1 potting mix (Fison Horticulture, Vancouver, B. C.). The study was conducted twice, with individual plants as replications. Three weeks later, 20% of inoculated plants had died and at physiological maturity, the rest of them had stunted growth and blackened internal stem tissues while external stem symptoms resembled those found in the field. All control plants reached maturity without symptoms. The pathogen re-isolated from inoculated plants were morphologically identical to the one retrieved from the field. These results confirmed the isolate as Verticillium longisporum. This is the first report of Verticillium stripe on canola in the US. In North America, the disease was first reported in Manitoba, Canada, in 2014 but subsequent surveys showed it is widespread in Canada (3). Identification of genetic resistance against this disease is required to ameliorate the threat this disease represents to US canola production.

Critical points for the design and application of RNA silencing constructs for plant virus resistance.

Control of plant virus diseases is a big challenge in agriculture as is resistance in plant lines to infection by viruses. Recent progress using advanced technologies has provided fast and durable alternatives. One of the most promising techniques against plant viruses that is cost-effective and environmentally safe is RNA silencing or RNA interference (RNAi), a technology that could be used alone or along with other control methods. To achieve the goals of fast and durable resistance, the expressed and target RNAs have been examined in many studies, with regard to the variability in silencing efficiency, which is regulated by various factors such as target sequences, target accessibility, RNA secondary structures, sequence variation in matching positions, and other intrinsic characteristics of various small RNAs. Developing a comprehensive and applicable toolbox for the prediction and construction of RNAi helps researchers to achieve the acceptable performance level of silencing elements. Although the attainment of complete prediction of RNAi robustness is not possible, as it also depends on the cellular genetic background and the nature of the target sequences, some important critical points have been discerned. Thus, the efficiency and robustness of RNA silencing against viruses can be improved by considering the various parameters of the target sequence and the construct design. In this review, we provide a comprehensive treatise regarding past, present and future prospective developments toward designing and applying RNAi constructs for resistance to plant viruses.

First Report of Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 µm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and ß-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (ß-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (ß-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

Identification and functional characterization of a γ-terpinene synthase in Nigella sativa L (black cumin).

Nigella sativa (Black cumin) has many applications in food and pharmaceutical industries. Thymoquinone has been considered as a main effective compound in N. sativa seeds and attracted researchers' attention mainly due to its medicinal potential. In this study, the essential oil components of leaves, flowers and seed developmental stages including half black seeds, soft black seeds and hard black seeds were analyzed in N. sativa. Whereas no terpenes were detected in flowers and leaves, seeds showed an essential oil composition that increased in its thymoquinone content during seed maturation. To study the proposed first step of thymoquinone biosynthesis, the formation of γ-terpinene from geranyl diphosphate (GDP), we identified and functionally characterized a γ-terpinene synthase (NsTPS1) in N. sativa. This monoterpene synthase was identified in RNA sequence data derived from seeds. After heterologous expression in Escherichia coli, partially purified NsTPS1 converted GDP to γ-terpinene. NsTPS1 is the first functionally characterized terpene synthase from N. sativa and displays a higher similarity to other terpene synthases from Ranunculaceae than known γ-terpinene synthases from more distant plant species. Characterization of NsTPS1 elucidates the first dedicated step in the biosynthesis of thymoquinone in N. sativa and paves the way towards metabolic engineering for high-level thymoquinone production.

Induction of resistance to Myzus persicae-nicotianae in Cucumber mosaic virus infected tobacco plants using silencing of CMV-2b gene.

Aphids such as tobacco aphid Myzus persicae-nicotianae, are among the most important plant viral vectors and plant viruses encode genes to interact with their vectors. Cucumber mosaic virus (CMV) encodes 2b protein as a suppressor of plant immune and it plays a vital role in CMV accumulation and susceptibility to aphid vectors. In this study, the resistance of tobacco plants (Nicotiana tabacum) to M. p. nicotianae was evaluated by silencing of 2b in CMV-infected plants. However, the pFGC-C.h silencing gene construct was transiently expressed using Agrobacterium tumefacience, LBA 4404 in tobacco leaves, and four days later, the plants were mechanically inoculated by CMV (Kurdistan isolate), and then, 15 days post-inoculation 1 nonviruliferous aphid was placed on each leaf for evaluation of resistance to M. p. nicotianae. To evaluate the tobacco plants resistance and susceptibility to M. p. nicotianae, the number of aphids existent per tobacco leaf, life table and, demographic parameters were recorded and used as a comparison indicator. The obtained results were analyzed using the age-stage, two-sex life table. The highest number of aphids was recorded on the control CMV-infected plants, while the lowest number on CMV infected leaves expressing CMV-2b silencing construct (pFGC-C.h). The obtained data revealed the lowest rate for all of intrinsic rate of natural increase (rm) (0.246/day), the rate of reproduction (r0) (17.04 females/generation), and finite rate of increase (λ) (1.279/day), on the pFGC-C.h treatment. The maximum generation time (T) (11.834 days) was observed on (V) treatment. However, the collected data revealed induction of resistance to tobacco aphids by silencing of CMV-2b in CMV infected plants.

Characterization of Arsenic-Resistant Endophytic Bacteria From Alfalfa and Chickpea Plants.

The present investigation was carried out to isolate arsenic (As)-resistant endophytic bacteria from the roots of alfalfa and chickpea plants grown in arsenic-contamination soil, characterize their As tolerance ability, plant growth-promoting characteristics, and their role to induce As resistance by the plant. A total of four root endophytic bacteria were isolated from plants grown in As-contaminated soil (160-260-mg As kg-1 of soil). These isolates were studied for plant growth-promoting (PGP) characteristics through siderophore, phosphate solubilization, nitrogen fixation, protease, and lipase production, and the presence of the arsenate reductase (arsC) gene. Based on 16S rDNA sequence analysis, these isolates belong to the genera Acinetobacter, Pseudomonas, and Rahnella. All isolates were found As tolerant, of which one isolate, Pseudomonas sp. QNC1, showed the highest tolerance up to 350-mM concentration in the LB medium. All isolates exhibited phosphate solubilization activity. Siderophore production activity was shown by only Pseudomonas sp. QNC1, while nitrogen fixation activity was shown by only Rahnella sp. QNC2 isolate. Acinetobacter sp. QNA1, QNA2, and Rahnella sp. QNC2 exhibited lipase production, while only Pseudomonas sp. QNC1 was able to produce protease. The presence of the arsC gene was detected in all isolates. The effect of endophytic bacteria on biomass production of alfalfa and chickpea in five levels of arsenic concentrations (0-, 10-, 50-, 75-, and 100-mg kg-1 soil) was evaluated. The fresh and dry weights of roots of alfalfa and chickpea plants were decreased as the arsenic concentration of the soil was increased. Results indicate that the fresh and dry root weights of alfalfa and chickpea plants were significantly higher in endophytic bacteria-treated plants compared with non-treated plants. Inoculation of chickpea plants with Pseudomonas sp. QNC1 and Rahnella sp. QNC2 induced lower NPR3 gene expression in chickpea roots grown in soil with the final concentration of 100-mg kg-1 sodium arsenate compared with the non-endophyte-treated control. The same results were obtained in Acinetobacter sp. QNA2-treated alfalfa plants grown in the soil plus 50-mg kg-1 sodium arsenate. These results demonstrated that arsenic-resistant endophytic bacteria are potential candidates to enhance plant-growth promotion in As contamination soils. Characterization of bacterial endophytes with plant growth potential can help us apply them to improve plant yield under stress conditions.

Development of an efficient Tef-1α RNA hairpin structure to efficient management of Lasiodiplodia theobromae and Neofusicoccum parvum.

Lasiodiplodia theobromae and Neofusicoccum parvum are serious worldwide-distributed plant pathogenic fungi with a wide host range in tropical and temperate climates. They cause fruit rot, canker, and dieback of twigs in various woody plants. Protection of pruning wounds using fungicides is the prevalent strategy for the management of the diseases caused by these fungi. Chemical control of plant diseases is not environmentally safe and the residues of fungicides are a threat to nature. Furthermore, genetic resources of resistance to plant diseases in woody plants are limited. The aim of this study was to investigate the efficiency of RNA silencing using an efficient hairpin structure based on Tef-1α gene for the management of L. theobromae and N. parvum. Hairpin structure of Tef-1α was cloned in pFGC5941 binary vector and the recombinant construct was named pFGC-TEF-d. Transient expression of pFGC-TEF-d using Agrobacterium LBA4404 in grapevine (Bidaneh Sefid cv.) and strawberry cultivars (Camarosa and Ventana) led to a reduction in disease progress of L. theobromae. The disease reduction in grapevine was estimated by 55% and in strawberries cultivars Camarosa and Ventana by 58% and 93%, respectively. Further analysis of transient expression of pFGC-TEF-d in strawberry (Camarosa) shown disease reduction using Neofusicoccum parvum. Here we introduce RNAi silencing using pFGC-TEF-d construct as an efficient strategy to the management of L. theobromae and N. parvum for the first time.

Efficient silencing gene construct for resistance to multiple common bean (Phaseolus vulgaris L.) viruses.

One promising strategy to engineer plants that are resistant to plant pathogens involves transforming plants with RNA silencing constructs for resistance to multiple pathogens. Garden bean is significantly damaged by bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV) and cucumber mosaic virus (CMV). In this study, we prepared constructs producing sense, antisense and hairpin RNA (hpRNA) structures to target single as well as multiple viruses. Silencing efficiency of these constructions was analyzed using Agrobacterium (GV3101) transient expression in Nicothinia bethamiana and Phaseolus vulgaris plants. The results showed significantly reduced disease symptoms and virus accumulation in N. bethamiana plants. Generally, the efficiency of the prepared constructs was hairpin, antisense and sense, respectively, and also, there was a significant difference between mono-gene and multiple-gene constructs for reducng virus accumulation and the multiple-gene constructs showed higher effectiveness. Experiments in this study showed that using Agrobacterium harboring binary constructs containing a Caenorhabditis elegans gene, Ced-9, or a plant gene, AtBag-4, anti-apoptosis gene as a mix suspension with an Agrobacterium containing pFGC-BNC.h, a plasmid containing multiple gene fragments consisting of BCMV-CP, BCMNV-HC-Pro and CMV-2b, improved the efficiency of pFGC-BNC.h transformation. We showed reduced virus accumulation in these transgenic bean plans.

Complete Genome Sequence of Zucchini Yellow Mosaic Virus Strain Kurdistan, Iran.

The complete genome sequence of Zucchini yellow mosaic virus strain Kurdistan (ZYMV-Kurdistan) infecting squash from Iran was determined from 13 overlapping fragments. Excluding the poly (A) tail, ZYMV-Kurdistan genome consisted of 9593 nucleotides (nt), with 138 and 211 nt at the 5' and 3' non-translated regions, respectively. It contained two open-reading frames (ORFs), the large ORF encoding a polyprotein of 3080 amino acids (aa) and the small overlapping ORF encoding a P3N-PIPO protein of 74 aa. This isolate had six unique aa differences compared to other ZYMV isolates and shared 79.6-98.8% identities with other ZYMV genome sequences at the nt level and 90.1-99% identities at the aa level. A phylogenetic tree of ZYMV complete genomic sequences showed that Iranian and Central European isolates are closely related and form a phylogenetically homogenous group. All values in the ratio of substitution rates at non-synonymous and synonymous sites (dN/dS) were below 1, suggestive of strong negative selection forces during ZYMV protein history. This is the first report of complete genome sequence information of the most prevalent virus in the west of Iran. This study helps our understanding of the genetic diversity of ZYMV isolates infecting cucurbit plants in Iran, virus evolution and epidemiology and can assist in designing better diagnostic tools.