Bacillus subtilis 26D Triggers Induced Systemic Resistance against Rhopalosiphum padi L. by Regulating the Expression of Genes AGO, DCL and microRNA in Bread Spring Wheat.

Bacillus subtilis 26D is a plant growth-promoting endophytic bacteria capable of inducing systemic resistance through the priming mechanism, which includes plant genome reprogramming and the phenomenon of RNA interference (RNAi) and microRNA (miRNAs). The phloem-feeding insect bird cherry-oat aphid Rhopalosiphum padi L. is a serious pest that causes significant damage to crops throughout the world. However, the function of plant miRNAs in the response to aphid infestation remains unclear. The results of this work showed that B. subtilis 26D stimulated aphid resistance in wheat plants, inducing the expression of genes of hormonal signaling pathways ICS, WRKY13, PR1, ACS, EIN3, PR3, and ABI5. In addition, B. subtilis 26D activated the RNAi mechanism and regulated the expression of nine conserved miRNAs through activation of the ethylene, salicylic acid (SA), and abscisic acid (ABA) signaling pathways, which was demonstrated by using treatments with phytohormones. Treatment of plants with SA, ethylene, and ABA acted in a similar manner to B. subtilis 26D on induction of the expression of the AGO4, AGO5 and DCL2, DCL4 genes, as well as the expression of nine conserved miRNAs. Different patterns of miRNA expression were found in aphid-infested plants and in plants treated with B. subtilis 26D or SA, ethylene, and ABA and infested by aphids, suggesting that miRNAs play multiple roles in the plant response to phloem-feeding insects, associated with effects on hormonal signaling pathways, redox metabolism, and the synthesis of secondary metabolites. Our study provides new data to further elucidate the fine mechanisms of bacterial-induced priming. However, further extensive work is needed to fully unravel these mechanisms.

Additive Effect of the Composition of Endophytic Bacteria Bacillus subtilis on Systemic Resistance of Wheat against Greenbug Aphid Schizaphis graminum Due to Lipopeptides.

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides - surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future.

By Modulating the Hormonal Balance and Ribonuclease Activity of Tomato Plants Bacillus subtilis Induces Defense Response against Potato Virus X and Potato Virus Y.

Endophytic plant-growth-promoting microorganisms can protect plants against pathogens, but they have rarely been investigated as potential biocontrol agents and triggers of induced systemic resistance (ISR), regulated by phytohormones, against viruses. We studied the role of endophytic strains Bacillus subtilis 26D and B. subtilis Ttl2, which secrete ribonucleases and phytohormones, in the induction of tomato plant resistance against potato virus X and potato virus Y in a greenhouse condition. The endophytes reduced the accumulation of viruses in plants, increased the activity of plant ribonucleases and recovered the fruit yield of infected tomato plants. Both the 26D and Ttl2 strains induced ISR by activating the transcription of genes related to salicylate- and jasmonate-dependent responses. The 26D and Ttl2 strains increased the content of cytokinins and decreased the level of indolacetic acid in plants infected with PVX or PVY. PVY led to an increase of the abscisic acid (ABA) content in tomato plants, and PVX had the opposite effect. Both strains reduced the ABA content in plants infected with PVY and induced ABA accumulation in plants infected with PVX, which led to an increase in the resistance of plants. This is the first report of the protection of tomato plants against viral diseases by foliar application of endophytes.

The Role of the GSTF11 Gene in Resistance to Powdery Mildew Infection and Cold Stress.

Oilseed rape (Brassica napus) is an economically important crop. In a temperate climate, powdery mildew Erysiphe crucifertaum can drastically reduce its yield. Nevertheless, cultivars resistant to this fungal disease have not yet been selected. Glutathione S-transferase GSTF11 is involved in glucosinolate (GSL) biosynthesis and response to stress, including fungal deceases. However, the impact of exogenous GSTF11 gene expression on resistance to powdery mildew has not yet been confirmed and requires further investigation. Transgenic B. napus was generated for this purpose. It demonstrated increased GST activity and a higher GSH:GSSG ratio under normal conditions. Powdery mildew Erysiphe crucifertaum caused 50% mortality in wild type (WT) plants. In most of transgenic plants, mycelium growth was inhibited. The infection contributed to higher GSTF11 expression and increased levels of glutathione (GSH) and oxidized glutathione (GSSG) in both transgenic and WT plants. In contrast, GSTF11 mRNA content, GST activity and GSSG level were lower only in WT plants. In transgenic plants, increased resistance to powdery mildew correlated with a lower GSH:GSSG ratio, indicating a higher content of neutralized toxic molecules. GSTF11 expression was also affected by cold stress, but not drought. At -1 °C, the expression level increased only in transgenic plants. Therefore, GSTF11 appears to be nonspecific and is able to protect plants under several types of stress. This gene could be used as a target in the production of stress tolerant cultivars.

Endophytic Bacillus spp. as a Prospective Biological Tool for Control of Viral Diseases and Non-vector Leptinotarsa decemlineata Say. in Solanum tuberosum L.

Viral diseases and their damage causing significant loss to economically important crops have increased by several folds during the last decade. All the conventional approaches are not able to eradicate the viral infection. Therefore, there is a need to look for efficient and eco-friendly viral disease-preventive measures. The genomic material of the majority of deleterious viruses of higher plants is RNA. One of the possible measures to control viruses is the use of ribonucleases (RNases), which can cleave RNA in the viral genome. Based on this, we investigated the RNase activity of endophytic Bacillus spp., which can enrich in 103-105 colony-forming units per gram of wet mass of aboveground part of potato plants. A high level of RNase activity was observed in the culture medium of Bacillus thuringiensis B-6066, Bacillus sp. STL-7, Bacillus sp. TS2, and Bacillus subtilis 26D. B. thuringiensis B-5351 had low RNase activity but high ability to colonize internal plant tissues, Bacillus sp. STL-7 with high RNase activity have relatively low number of cells in internal tissues of plants. B. thuringiensis B-6066, B. subtilis 26D, and Bacillus sp. TS stimulate RNase activity in potato plants for a long time after application. Strains with high ability to colonize internal plant tissues combined with high RNase activity reduced severity of viral diseases symptoms on plants and reduced the incidence of potato viruses M, S, and Y. It is worth noting that Bacillus spp. under investigation reduced the number of Leptinotarsa decemlineata Say. egg clusters and larvae on treated plants and showed antifeedant activity. This results in increase of potato productivity mainly in the fraction of major tubers. B. subtilis 26D and Bacillus sp. TS2 combining endophytic lifestyle, RNase, and antifeedant activity may become the basis for the development of biocontrol agents for plant protection.

Mechanisms of Plant Tolerance to RNA Viruses Induced by Plant-Growth-Promoting Microorganisms.

Plant viruses are globally responsible for the significant crop losses of economically important plants. All common approaches are not able to eradicate viral infection. Many non-conventional strategies are currently used to control viral infection, but unfortunately, they are not always effective. Therefore, it is necessary to search for efficient and eco-friendly measures to prevent viral diseases. Since the genomic material of 90% higher plant viruses consists of single-stranded RNA, the best way to target the viral genome is to use ribonucleases (RNase), which can be effective against any viral disease of plants. Here, we show the importance of the search for endophytes with protease and RNase activity combined with the capacity to prime antiviral plant defense responses for their protection against viruses. This review discusses the possible mechanisms used to suppress a viral attack as well as the use of local endophytic bacteria for antiviral control in crops.