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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

During growing season, 2011–2012, selected bean plant samples with symptoms of mosaic, vein clearing, leaf rolling, were collected from different part of Khorasan Razavi province of north-eastern Iran. To identify the virus, leaf samples were tested serologically by DAS-ELISA and tissue blot, using specific BCMV polyclonal antibody. Elisa positive samples were rechecked by RT-PCR and IC-RT-PCR using set of primers directed to the coat protein gene which were designed to detect and characterise the viral species. Similarly, the BCMV primers amplified product of approximately 373 bp in 1% agarose gel electrophoresis. In phylogenic analysis, A and B groups were formed for 12 Iranian isolate comparing with 18 BCMV isolates from GenBank. Iranian isolates were classified into three clusters indicating to have more homology 99.3% with Mexican Isolates. There are also indications of some diversities among Iranian BCMV isolates. [ABSTRACT FROM AUTHOR]

• Several endophytic bacteria were identified as Bacillus species using molecular analysis. • Endophytic bacteria identified have antagonistic activity against Ralstonia solanacearum. • These strains produce VOCs with antibacterial activity. • These VOCs inhibited virulence traits of R. solanacearum in vitro. • VOCs reduced potato wilt incidance caused by R. solanacearum in greenhouse. In this study, we investigated the effects of volatile organic compounds (VOCs) produced by endophytic bacteria belonging to the Bacillus genus on the virulence traits of Ralstonia solanacearum R32, the causal agent of potato bacterial wilt disease. The results of gas chromatography-mass spectrometry analysis showed that strains B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 able to produce 10, 4, 18, 6, and 12 VOCs with high quality, respectively. All endophytic bacteria produced VOCs that significantly reduced wilting symptoms in planta , inhibited the cell population of R. solanacearum R32 in vitro, and reduced the cell population both in the shoots and roots of inoculated plants at different levels. The VOCs significantly reduced the attachment of R. solanacearum R32cells to root tissues of potato plants, as well as chemotaxis motility towards root extract. Our results revealed that VOCs could reduce swarming-, swimming-, twitching motility, and biofilm formation by R. solanacearum R32 which are important for pathogenicity. Scanning electron microscopy analysis revealed various morphological abnormalities in the R. solanacearum R32 cells exposed to the VOCs produced by B. safensis Har267 and B. aerius Kh867 strains. Results presented in this study showed that the VOCs produced by endophytic bacteria investigated can reduce bacterial wilt disease. Investigation of the mechanisms of biocontrol activities increases our knowledge to design better methods to control bacterial pathogens. [ABSTRACT FROM AUTHOR]

Brown blotch disease of mushroom caused by Pseudomonas tolaasii , often results of severe damage to mushroom tissues. In this study, the biocontrol mechanisms mediated by endofungal bacteria Pseudomonas sp. De1, Bacillus sp. De3, Pseudomonas sp. Bi1 and Pantoea sp. Ma3 previously isolated from wild growing mushrooms against Pseudomonas tolaasii Pt18 were demonstrated. Our results provide evidence that all bacterial strains can colonize mycelia of Agaricus bisporus. All endogungal bacteria except Pseudomonas sp. Bi1 contained one or more genes encoding antibiotic production. The non-volatile compounds of endofungal bacteria significantly inhibited the biofilm formation by P. tolaasii Pt18. Results showed that all strains except Pantoea sp. Ma3 are capable of detoxify tolaasin, a major virulence factor produced by P. tolaasii Pt18. We also showed that all endofungal bacteria tested were able to significantly decrease the browning symptoms of pretreated-mushroom caps compared to inoculated caps with P. tolaasii alone. AbPPO3 gene expression levels in mycelia and fruiting bodies of A. bisporus significantly increased after P. tolaasii Pt18 inoculation. Our findings indicated that The relative expression level of AbPPO3 gene decreased in the mushroom mycelia and fruiting bodies pretreated with Pantoea sp. Ma3 and Pseudomonas sp. Bi1 strains, and the levels were significantly lower than mushroom tissues treated with P. tolaasii Pt18 alone. Overall, the findings presented in this study revealed the efficacy of the selected endofungal bacteria in biocontrol activity against P. tolaasii and the possible use of these strains as a safe microbial agents in mushroom brown blotch disease management. • The non-volatile compounds of endofungal bacteria significantly inhibited the biofilm formation by P. tolaasii. • Endofungal bacteria belongs to Pseudomonas, Bacillus and Pantoea genus significantly reduced mushroom brown blotch symptoms. • Bacillus and Pseudomonas strains detoxify tolaasin produced by Pseudomonas tolaasii under in vivo conditions. • The relative expression level of AbPPO3 gene decreased in the mushroom tissues pretreated with endofungal bacteria. [ABSTRACT FROM AUTHOR]

The purpose of this research was to explore the control of common bean root rot disease caused by Fusarium solani by the bacterial strain CTA8689 under in vitro and greenhouse conditions. The strain CTA8689 was isolated from common bean root and identified as Achromobacter xylosoxidans using 16S rDNA sequence, morphological and physiological characteristics. The strain CTA8689 suppressed the mycelial growth of F. solani in dual-culture (58.67%) and culture filtrates (41.87%) methods. The volatile compounds (VOCs) produced by CTA8689 inhibited mycelial growth and conidial germination of F. solani by 42.26% and 59.12%, respectively. Also, this strain produced biofilm, lytic enzymes such as chitinase, protease and pectinase, siderophore, IAA and gibberellin, and it was able to phosphate solubilization. Furthermore, the disease severity of common bean root rot was reduced using soil and seed treatment by 83.89% and 87.60%, respectively, 60 days after inoculation. Additionally, the strain improved the plant growth parameters in common bean under greenhouse conditions. The VOCs produced by the strain CTA8689 were analyzed by Gas chromatography-mass spectroscopy (GC-MS). Out of 23 identified VOCs, 1,3-Dimethylbenzene (21.613%), Benzene, 1,3-bis (1,1-dimethylethyl)- (13.823%%), Ethylbenzene (8.636%), Dodecane, 4,6-dimethyl- (8.145%) and Decane (5.905%), were the major bioactive components. The mode of action of the strain CTA8689 on the F. solani was through the production of antifungal compounds (antibiosis) and lytic enzymes, which suppressed mycelial growth and conidial germination of the pathogen in vitro and greenhouse conditions. Overall, our results showed that A. xylosoxidans strain CTA8689 could be a promising biological control agent (BCAs) for common bean root rot. • Strain CTA8689 was isolated from common bean root and against Fusarium solani investigated. • Strain CTA8689 was identified as Achromobacter xylosoxidans. • Strain CTA8689 antagonized the pathogen successfully in vitro and greenhouse conditions. • Strain CTA8689 improved the plant growth parameters in common bean plant. • Strain CTA8689 produced bioactive compounds with antifungal properties. [ABSTRACT FROM AUTHOR]

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 o C 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 o C for 3 min, followed by 35 cycles of amplification at 94 o C for 1 min then 56 o C for 30 sec and 72 o C for 1 min, followed by a final period at 72 o C 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 o C. 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 10 6 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.

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 β-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 (β-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 (β-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.

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)