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Article

Endophytic Strain Bacillus subtilis 26DCryChS Producing Cry1Ia Toxin from Bacillus thuringiensis Promotes Multifaceted Potato Defense against Phytophthora infestans (Mont.) de Bary and Pest Leptinotarsa decemlineata Say

by
Antonina Sorokan
,
Galina Benkovskaya
,
Guzel Burkhanova
,
Darya Blagova
and
Igor Maksimov
*
Institute of Biochemistry and genetics of the Ufa Federal Research Centre of the Russian Academy of Sciences, 450054 Ufa, Russia
*
Author to whom correspondence should be addressed.
Plants 2020, 9(9), 1115; https://doi.org/10.3390/plants9091115
Submission received: 7 July 2020 / Revised: 7 August 2020 / Accepted: 26 August 2020 / Published: 28 August 2020
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Abstract

:
Novel properties of a previously obtained Bacillus subtilis 26DCryChS strain are described. The B. subtilis 26DCryChS strain is able to produce Cry1Ia δ-endotoxin from B. thuringiensis B-5351 and to exist in internal plant tissues of potato plants in the same manner as the endophytic B. subtilis 26D source strain (487 ± 53 and 420 ± 63 CFU*103/g, respectively). B. subtilis 26DCryChS, as much as the original B. subtilis 26D strain, inhibited mycelium growth of oomycete Phytophthora infestans (Mont.) de Bary and reduced late blight symptoms development on plants by 35% compared with non-treated ones, as well as showed insecticidal activity against Leptinotarsa decemlineata. Production of the fluorescent GFP protein in the B. subtilis 26D genome allowed visualizing the endophytes around damaged sites on beetle intestines. Bacillus strains under investigation induced systemic resistance to P. infestans and L. decemlineata through the activation of the transcription of PR genes in potato plants. Thus, the B. subtilis 26DCryChS strain was able to induce transcription of jasmonate-dependent genes and acquired the ability to promote transcription of a salicylate-dependent gene (PR1) in plants infected with the late blight agent and damaged by Colorado potato beetle larvae. The B. subtilis 26DCryChS strain could be put forward as a modern approach for biocontrol agents design.

Graphical Abstract

1. Introduction

It is estimated that 35% of agricultural plants’ yield is lost due to biotic damage (insects, weeds, and pathogens) in the field around the world. Moreover, postharvest losses may constitute up to 15% of the total yields [1]. This has resulted in the complete dependence of crops on the use of chemical pesticides to reduce losses. Unfortunately, target organisms often develop resistance to globally used chemicals. At the moment, the most limiting productive capacity factor for potato is late blight caused by Phytophthora infestans (Mont.) de Bary infection. If left uncontrolled, late blight causes massive yield losses annually, especially under favorable conditions for P. infestans dispersion [2]. Since the last quarter of the century, different fungicides have been developed for the management of potato late blight disease, but the number of P. infestans isolates resistant to fungicides continuously increases. It is currently reported that most of the present-day P. infestans populations are fully resistant to the pesticide metalaxyl [3].
Capacity to develop insecticide resistance is the legendary feature of Leptinotarsa decemlineata Say [4]. The Colorado potato beetle is the most harmful pest of potato and its populations develop resistance to all major classes of insecticides, including δ-endotoxins produced by the most commonly used biocontrol agent Bacillus thuringiensis [5,6]. Multifaceted influences of management means on pathogens or pests are considered to slow down the development of their resistance to pesticides or biocontrol agents.
Plants are exposed to various harmful organisms simultaneously. Plant protection, in turn, demands an elevating amount of chemical pesticides upload that multiplies their negative influence on ecosystems and accelerates the development of pest resistance to chemicals. Thus, this suggests the necessity for the development of environmentally safe biocontrol agents that reveal combined biocidal impact on abundant pathogens and pests, and which will be capable to give rise to systemic resistance in host plants. Consequently, a beneficial and heterogeneous group of plant growth-promoting bacteria (PGPB), inhabiting the rhyzosphere, phyllosphere, and internal tissues of plants, is of great interest [7,8,9].
Endophytic PGPB have an advantage over rhizospheric and phyllospheric strains since inhabiting inside a plant’s tissues gives an ability to contact with the plant’s cells continually and to directly influence the plant host’s metabolism and, furthermore, penetrative pathogen or pest. This “hidden existence”, in addition, allows reducing the impact of the ambient environment on microorganisms. It has been revealed that certain PGPB strains not only stimulate both plant growth and resistance against pathogens and pests [10,11] but also show fungicidal [12], aphicidal [8,13], and insecticidal activities [14] as a consequence of synthesizing antibiotics and biosurfactants [15]. Among them, Bacillus strains are important resources for biocontrol agents development. Bacillus thuringiensis (Berliner) is a ubiquitous spore-forming bacterium which inhabits diverse dwelling places and has been considered as the most income-generating bioinsecticide during the last century [16]. The practical application of B. thuringiensis has been promoted as a result of pests’ resistance to chemical insecticides [16,17]. B. thuringiensis strains are vulnerable to UV light and flushing by rain precipitation [18,19]. Currently, the search for endophytic microorganisms producing insectotoxins, which inhabit the internal tissues of plants and are less influenced by environmental factors and more integrated in plant metabolism than rhyzo- and fillospheric PGPB, is of great interest.
At the moment, researchers are accepting that various pathogens and non-pathogenic microorganisms could serve as triggers for systemic acquired resistance (SAR) and induced systemic resistance (ISR). SAR is commonly challenged by biotrophic pathogens attack and involves salicylic acid (SA). It is generally accepted that non-pathogenic rhizospheric fungi or bacteria cause ISR when the plant interacts with them, and this process is carried out with the participation of jasmonic acid (JA)/ethylene (ET) and activates plants resistance against necrotrophic pathogens and leaf-chewing pests, such as L. decemlineata [11,20]. A lot of strains of Bacillus sp. are responsible for ISR development which does not require the SA-sensitive pathway but is dependent on JA, ethylene, and the regulatory gene NPR1. This mechanism is in accordance with the model for ISR elicited by Pseudomonas sp. At the same time, some Bacillus sp. can activate ISR through mechanisms independent of JA and NPR1 and dependent on SA [21]. Furthermore, ISR triggered by Pseudomonas sp. does not result in accumulation of transcripts of the defense gene PR1 in plants, whereas Bacillus sp.-elicited ISR can induce it [22]. Thus, the mechanisms of PGPB participation in ISR are not understood, in particular, ISR against oomycete P. infestans.
A number of bacterial determinants have recently been shown to be involved in ISR triggering. Thus, Bacillus sp. strains produce lipopeptides from different families: bacillomycin, surfactin, iturin, and fengycin [23]. It has been shown that some surfactin-producing Bacillus strains can elicit ISR to Botrytis cinerea in grape, strawberry, and tomato plants [24] and Rhizoctonia solani in lettuce [25]. Recently, aphicidal and insecticidal activity of surfactin produced by B. subtilis and surfactin-producing strain B. subtilis 26D has been shown [13,14,26].
Consequently, obtaining genetically engineered lines with an enhanced ensemble of beneficial properties is of essential interest due to their prospective value in pathogen and pest management [16]. Currently, some strains of B. megaterium, P. fluorescens, E. coli, and B. subtilis [27] have been genetically modified via insertion of Cry toxin genes. Importantly, insertion of the Btcry218 gene of Bacillus thuringiensis into the genome of the Burkholderia pyrrocinia JK-SH007 strain promotes 80% mortality of silkworms Bombyx mori larvae 24 h after eating mulberry leaves immersed in B. pyrrocinia JK-SH007 suspension [28].
Previously, we obtained the novel recombinant B. subtilis 26DCryChS (RCAM04928, ARRIAM collection) strain that expresses the gene Btcry1Ia [29] encoding Cry1Ia δ-endotoxin originated from the B. thuringiensis B-5351 strain. It was reported that B. subtilis 26DCryChS can exist endophytically in wheat plants and synthesize lipopeptide surfactin as well as the source strain B. subtilis 26D and is capable of producing Cry1Ia δ-endotoxin from the donor of the Btcry1Ia gene B. thuringiensis B-5351 strain [13]. The B. subtilis 26DCryChS strain shows aphicidal activity against Schizaphis graminum as compared with both B. thuringiensis B-5351 and B. subtilis 26D strains and inhibited the growth of the wheat pathogen Stagonospora nodorum Berk. that causes Septoria blotch [13].
The aim of this work was to investigate the capacity of the recombinant B. subtilis 26DCryChS strain expressing the gene Btcry1Ia of Cry1Ia δ-endotoxin to protect potato plants (Solanum tuberosum L.) from oomycete Phytophthora infestans (Mont.) de Bary and pest Leptinotarsa decemlineata Say.

2. Results

2.1. Assay of Endophytic Properties of Bacillus Strains

The number of B. subtilis 26DCryChS colony forming units (CFU) in interior tissues of potato plants after surface sterilization amounts to approximately 500,000 per g of plant wet mass, no less than the amount of B. subtilis 26D alive cells (Table 1). The content of B. thuringiensis B-5351 cells in potato plants was substantially lower than the B. subtilis strains under investigation.

2.2. Capacity of Bacillus Strains to Protect Potato Plants against Oomycete P. infestans

The area of late blight-damaged sites on leaves of potato plants containing B. subtilis 26D and B. subtilis 26DCryChS endophytes reduced in the same manner, whereas the effect of B. thuringiensis B-5351 was not substantial (Figure 1). Both B. subtilis 26D and B. subtilis 26DCryChS strains had antagonistic activity against P. infestans and inhibited mycelium growth (Figure 2). The donor of the gene Btcry1Ia B. thuringiensis B-5351 did not alter the growth of the oomycete mycelium.

2.3. Insecticidal Activity of Bacillus Strains against L. decemlineata Larvae

The B. subtilis 26D strain exhibited a moderate insecticidal influence on L. decemlineata larvae (18.1%, Table 2), whereas the eating of B. thuringiensis B-5351-immersed leaves resulted in about 77% mortality of larvae. Insecticidal activity of the B. subtilis 26DCryChS strain was less than the B. thuringiensis B-5351 strain when larvae ate surface-contaminated plants (Table 2).
The strains of Bacillus under investigation are able to colonize plant tissues. Consequently, L. decemlineata larvae swallowing potato leaves with endophytes in internal tissues become affected both by Bacillus-derived insecticides and plant defense mechanisms, primed by endophytes. Thus, feeding of plants containing B. subtilis 26D resulted in the death of 38.1% larvae. It is worth noting that the mutualistic relationship between plants and B. subtilis 26DCryChS contributed to 80.7% of larval death, and the insecticidal activity of B. thuringiensis B-5351 in this case was decreased compared to the case of surface-contaminated leaves. On the fifth day after feeding, larvae showed bacteriosis symptoms (Figure 3II).
Line B. subtilis 26DGFP expressing fluorescent protein was obtained to confirm the pathological impact of B. subtilis 26D and to visualize its cells in the digestive system of L. decemlineata. Beetles were fed with suspensions of the bacteria under investigation (applied to the surface of leaves) to observe the influence of bacterial strains in the same concentration since plants contain different numbers of endophytic cells of these strains. It was found that B. subtilis 26D violated the development of the peritrophic membrane and promoted the occurrence of darkened parts on the midgut surface. Fluorescent cells of B. subtilis 26DGFP grouped around these sites, as shown in Figure 3. Crystals of Cry-protein were observed in mesenterons of B. thuringiensis B-5351- and B. subtilis 26DCryChS-fed beetles.

2.4. Bacillus Strains Triggered Systemic Resistance in Potato Plants to P. infestans and Beetle L. decemlineata

The amount of the transcripts of potato genes StPR6, StPR1, StAOS, and StOPAR, encoding trypsin inhibitor PR6 (marker of JA-dependent pathway), basic antimicrobial protein PR1 (marker of salicylate-dependent pathway), allene oxide synthase, and 12-12-oxophytodienic acid reductase (enzymes of JA-biosynthesis, sensitive to JA), respectively, in intact and Bacillus-containing cells of plants in response to oomycete P. infestans or beetle L. decemlineata attacks, was investigated. The transcript level of JA-sensitive genes StPR6, StAOS, and StOPAR in non-affected plants containing endophytic Bacillus strains in potato plants was not altered (Figure 4), but both B. subtilis strains under study decreased the transcript level of StPR1. It is important that B. thuringiensis B-5351 increased this parameter.
P. infestans attack on water-treated potato plants did not lead to significant alterations in StOPAR and StPR1 RNA content and stimulated StPR6 transcription (Figure 4). All tested Bacillus strains positively regulated the transcription activity of JA-sensitive genes StOPAR, StAOS, and StPR6 in plants which were infected with the late blight causal agent. Infection of B. thuringiensis B-5351-containing plants increased StOPAR and StPR1 transcription but did not influence StAOS and StPR6 genes. It is important that endophytic B. subtilis 26DCryChS cells increased levels of both SA- and JA- sensitive genes under study in plants which were attacked by P. infestans.
It was found that lignin autofluorescence in cell walls around P. infestans-penetrated sites on leaves of B. subtilis 26D-treated plants was significantly increased relative to water-treated or B. thuringiensis B-5351-treated plants (Figure 5). In B. subtilis 26DCryChS-treated plants, lignin accumulated in apoplastic spaces and cell walls of the mesophyll and epidermis around infected sites, like in plants under the influence of the original strain.
The transcript level of StAOS and StPR1 genes—unlike StOPAR and StPR6—was down-regulated by L. decemlineata attacks. Treatment of plants with cells of Bacillus strains, especially B. subtilis 26DCryChS (2-fold), promoted transcription of the StPR6 gene in infected plants. Presence of endophytic B. subtilis 26D strain cells in potato plants enhanced the level of JA-biosynthesis genes and did not have any effect on the RNA content of the StPR1 gene in plant leaves damaged by larvae of L. decemlineata (Figure 6). Conversely, the pest attack on B. thuringiensis B-5351-treated plants resulted in the rise of the transcription activity of StPR1 and did not alter the StPR6 RNA level. The B. subtilis 26DCryChS strain supported the accumulation of both JA- (StAOS, StOPAR, and StPR6) and SA- (StPR1) sensitive genes in plants under the pest attack (Figure 6).

3. Discussion

The damage to plants caused by pathogens and insects substantially decreases their productivity and, in some cases, can totally devastate a yield. Approaches based on the use of chemical pesticides have demonstrated enormous gains in controlling populations of some harmful pests, in particular, fungal pathogens and insects. This war is an everlasting competition, since genetic changes in pests often provide restoring their virulence over plant resistance or leave the pest resistant to novel pesticides. The dependence of agriculture on chemical pesticides, the increasing frequency of pest resistance to pesticides, and environmental pollution caused by chemicals are of escalating concern. The search for ways to solve these concerns has resulted in a higher focus on biological control means. Now, it is clear that the development of stable agroecosystems cannot be achieved without the integration of agricultural plants with artificially composed microbiomes or strains, which combine some properties, and which allow to protect plants against combined environmental factors, and, at the same time, will be protected themselves through the development partnerships with the host plants. In consortiums, the set of relationships between microorganisms can lead to incalculable effects in plants under the influence of multiple biotic factors. Thus, we assume that recombinant endophytic B. subtilis strains are capable of protecting crops from a number of pathogens or insect pests [8,11,21,26].
Previously, we have shown that the source strain B. ѕubtilis 26D effectively protects potato plants against late blight causal agent [9]. The previously obtained B. subtilis 26DCгуCһЅ strain, which was developed on the basis of the endophytic B. subtilis 26D strain, is able to inhibit the propagation of oomycete P. infestans mycelium in vitro and decreased late blight lesion formation on potato leaves as well as the original strain B. subtilis 26D (Figure 1 and Figure 2). B. subtilis 26D showed proteolytic activity that can cause observed impact of B. subtilis 26DCгуCһЅ [13]. Thus, Bacillus amyloliquefaciens isolates which produce proteases display antagonistic activity against pathogenic fungi Fusarium oxysporum, Alternaria alternate, and Macrophomina phaseolina [30]. Chang W.T. et al. [31] showed that Bacillus cereus AU004 substantially inhibits the growth of Pythium ultimum, F. solani, and F. oxysporum mycelium due to the secretion of hydrolytic enzymes.
At the moment, the spore-forming bacterium B. thuringiensis, producing insecticidal crystal proteins, is the prevalent commercial agent for insect pest control [18,19]. Sensitivity of the majority of biopesticides based on commercial strains to temperature regime, atmospheric precipitations, and other conditions put bounds on their extensive use. Involvement of endophytic strains in farming techniques can solve the problem [7,18].
As was mentioned above, widespread application of B. thuringiensis cells and crystals to manage pest insects has increased the prerequisite for the distribution of pests’, in particular, the Colorado potato beetle, resistance to this entomopathogen [5,6]. Biocontrol agents combining diversified actions on different pest systems can make it possible to reduce the level of pest resistance development. Thus, the great capacity of Beauveria bassiana strain GHA and B. thuringiensis subsp. morrisoni strain tenebrionis to act in concert to control L. decemlineata was reported (the fast-operating B. thuringiensis δ-endotoxin protects defoliation of potato plants and the slow-operating B. bassiana increases adult mortality) [32]. The transgenic strain B. bassiana expressing Cyt2Ba had enhanced virulence against larvae and adults of Aedes aegypti and Ae. albopictus mosquitoes compared with the wild type of the pathogen. The median lethal time (LT50) for Ae. aegypti adults decreased by 33% at the concentration of 108 conidia/mL, 19% at 107 conidia/mL, and 47% at 106 conidia/mL in comparison with the wild type [33].
Besides, it was found previously that B. subtilis 26D disarranged L. decemlineata larvae microbiome development. Presumably, this effect can be an occasion for the further high mortality rate of the insects [34]. It was found that B. subtilis 26D interrupts the integrality of the peritrophic membrane and promotes appearance of dark sites on the midgut surface. Fluorescent B. subtilis 26D GFP was placed close by these sites, as shown in Figure 4. Production of Cry1Ia δ-endotoxin has enhanced the insecticidal potential of the B. subtilis 26DCryChS strain in comparison to that of the B. thuringiensis B-5351 strain. We assumed that it combines the influence of the source strain on the pest microbiome and peritrophic membrane and the influence of the Cry toxin on insect intestines. Probably, the entophytic lifestyle of B. subtilis 26DCryChS promotes this effect (Table 2) since the suspension of this strain was less effective than the eating of plants containing endophytes. It is essential to emphasize that the recombinant strain has not got any host specificity, since the increase in the mortality of Russian wheat aphid was observed when insects propagated on wheat seedlings which were grown from B. subtilis 26DCryChS strain-inoculated seeds [13].
The suppression of late blight symptoms on potato leaves and the survival rate of larvae of L. decemlineata on plants containing cells of Bacillus strains under investigation may be associated with the priming of immune reactions in potato plants and ISR [9,21]. The increased mortality of the pest which ate plants containing endophytic B. subtilis under investigation compared with ones that ate surface-contaminated leaves testifies to this fact.
Thus, priming is a common characteristic of PGPB-induced plant resistance. Priming initially induces a slight alteration of transcription of defense genes, increasing the plant’s ability to protect itself against further attacks (insect or/and pathogenic pests). In primed plants, subsequent defense responses can be activated more rapidly and effectively [35]. We demonstrated that Bacillus under investigation did not increase the number of transcripts of genes encoding PR proteins in potato plants, but P. infestans infection or L. decemlineata attacks were followed by a great increase in their transcription level (Figure 4 and Figure 6). Observed reactions keep up with the priming effect of microorganisms.
Resistance induced by PGPB is modulated by signal transduction networks in which JA/ET and SA have crucial roles [11,20]. The cross-talk between SA- and JA-sensitive signaling pathways and suppression of JA/ET signaling by SA and/or intermediates of SA-dependent pathways have been reported more than once [21,36]. These interactions permit plants to put forward defense mechanisms which are more effective against certain species with different characteristics of damage, thereby minimizing the development of ineffective and expensive defense mechanisms [20,21], but harmful pests use these arrangements for manipulating plant immunity. Although some non-pathogenic rhizosphere microbes stimulate ISR through the SA-dependent signaling pathway, most beneficial microbes activate ISR through the JA/ET signaling pathway [37]. However, in some cases, ISR can develop through both the SA and JA/ET signaling pathways simultaneously to accord resistance against necrotrophic and hemibiotrophic pathogens [38]. Thus, an increase in SA levels and AtPR-1 gene expression in plants of Arabidopsis after treatment with B. subtilis FB17 led to enhanced resistance to P. syringae pv. tomato [39]. ISR against Bemisia tabaci in tobacco induced by B. subtilis PY-79 showed a JA-independent mechanism of regulation [37].
Some B. thuringiensis strains can prime plants’ defense reactions against pests apart from the insecticidal effect of δ-endotoxin [16]. It was reported that Bt-induced resistance to R. solanacearum in tomato plants which was dependent on the SA signaling pathway and repressed JA-dependent mechanisms [40]. B. thuringiensis-induced plant defense against pathogens and pests is not clear yet [41,42].
Transcriptional activity of the SA- and JA-dependent genes encoding PR proteins was investigated to assess the capability of Bacillus strains to manipulate signaling cross-talks in ISR development against L. decemlineata and P. infestans in potato plants. The StPR1 gene, which encodes the protein PR1, was reported as the marker of SA-dependent reactions in some plant species [43]. Both StPR6 and JA-biosynthesis genes were JA-dependent [44]. Previously, it was shown that B. thuringiensis and B. subtilis strains can induce different transcription patterns in wheat plants [45].
We have previously shown the involvement of JA in potato protective response to the pathogen P. infestans [46] and to the pest L. decemlineata [47]. The results obtained suggest that the effect of B. subtilis 26D was closely linked to the triggering mechanism of ISR caused by JA. Results of Yarullina L.G. et al. [48] showed that inoculation of wheat with Stagonospora nodorum increased the level of transcripts of genes encoding the proteinase inhibitor PR6. The rate of the activity of this gene in intact plants of different cultivars correlated with their resistance to the Septoria blotch pathogen. In this work, we found that the source strain and B. subtilis 26DCryChS promoted a high level of JA-biosynthesis enzymes in plants under L. decemlineata and P. infestans attacks. We showed that B. thuringiensis B-5351 and B. subtilis 26DCryChS strains promoted accumulation of the RNA StPR1 gene in plants challenged by oomycete P. infestans and pest L. decemlineata (Figure 4 and Figure 6). Thus, under the influence of the recombinant strain, SA and JA reactions were activated simultaneously. Previously, [38] showed that B. cereus AR156 strain treatment led to a higher level of Arabidopsis Col-0 plants’ resistance to Pseudomonas syringae DC3000 than that in NahG, jar1, or etr1 plants. Thus, AR156-mediated ISR is implemented through the simultaneous activation of the JA/ET and SA-sensitive signaling pathways, which made it possible to speak of its cumulative effect on plant immunity in some cases.
It is worth remembering that activation of the SA-dependent StPR1 gene in plants influenced by B. thuringiensis B-5351 was not promoting plant resistance to stresses, but improving it, and then SA- and JA-dependent genes were activated together. Previously, we observed that the simultaneous influence of a low concentration of JA and SA increased potato resistance to P. infestans [46] and we assumed that B. subtilis 26DCryChS primed plant resistance that engages both pathways.
In our investigation, enhanced lignin deposition around P. infestans-penetrated sites on leaves of plants treated with B. subtilis strains under investigation was negatively correlated with the dimensions of the lesion areas. Lignin forms a physical barrier on the pathogens and may improve plant resistance against fungal infection [49]. Lignification of plant cell walls and development of a strength barrier to pathogen penetration is one of the most important ISR mechanisms, which could be triggered by PGPB [50]. In [10]’s study, when cucumber roots were treated with a cultural filtrate of hypovirulent Rhizoctonia sp., lignin content was increased after pathogen Colletotrichum orbiculare penetration in epidermal tissues of cucumber hypocotyls. It was shown that PGPB, in particular, Bacillus sp., are capable of stimulating lignin biosynthesis through activation of peroxidases and polyphenol oxidases in infected plants [41,45,51] or pest-damaged plants [11,42]. Genetically engineered plants of maize, expressing the Cry1Ab toxin, showed a higher ratio of lignin accumulation in vascular bundle sheaths and in the sclerenchyma cells near vascular bundles than wild type plants [52].

4. Materials and Methods

4.1. Plant, Microbe and Insect Material

Plants: sterile Solanum tuberosum L. plants (cv. Early Rose). Plants were obtained by microcloning technology and grown in tubes with Murashige and Skoog medium in a KS200 climatic chamber (SPU, Russia) with a 16-h light period (Osram L 36W/77 bulbs, illuminance 8–10,000 lux, Germany) at 20–22 °C for 21 days.
Bacteria:B. subtilis 26D, B. thuringiensis B-5351, and previously obtained B. subtilis 26DCryChS which contained the Btcry1Ia gene encoding Cry1Ia δ-endotoxin from B. thuringiensis B-5351 [13] strains held in the collection of the Laboratory of Biochemistry of Plant Immunity of the Institute of Biochemistry and Genetics UFRC [53] were used. Bacteria were grown on Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 20–22 °C.
System “Plant+endophyte”: 7-days-old plants were inoculated with 5 μL of Bacillus strains cell suspension on the stem zone adjacent to the zone of adventitious roots formation as it was described previously [34]. Concentration of bacterial cells was 108 cells/mL. Plants were grown in a gnotobiotic system for 14 days.
L. decemlineata: third instar larvae and adults were taken from seed potato (Birsk experimental farm, the Republic of Baskortostan, the Birsk region, South Ural, 55°25′52.6′′ N 55°35′58.3′′ E).
P. infestans: MO-8 (race 1.2) culture kindly provided by U.T. Diakov from the collection of the Biological Faculty, Moscow State University and stored in the collection of our laboratory was used.

4.2. Evaluation of Endophytic Properties of Bacillus Strains

On the 7th day after planting, sterile potato plants were inoculated with the suspension of Bacillus sp. cells (“Plant+endophyte”). After 14 days of co-culturing, leaves and stems of plants were immersed for 3 min in 70% EtOH; after this, for 3 min in a 0.03% solution of hydrogen peroxide, and then the samples were washed with sterile water three times. Sterilized potato leaves and stems (200 mg fresh weight) were homogenized in sterile bags using a BagMixer 400 W blender (Interscience, Saint Nom, France) with 2 mL sterile water added. Two consecutive 10-flast dilutions of the resultant homogenate were then performed. Aliquots (100 μL) were spread over the surface of potato-glucose agar with an L-shaped spatula until complete drying. After 24 h, Petri dishes were analyzed for the count of CFU. The ability of strains under investigation to colonize the internal plant tissues was expressed in CFU per gram of wet weight. In order to verify and confirm the identity of bacterium isolates, obtained from surface-sterilized leaves, and original strains, pure cultures were analyzed by RAPD-PCR.

4.3. Evaluation of the Ability of Bacillus Strains to Defend Potato Plants against P. infestans

Leaves of 21-day potato plants which were previously treated with bacteria (“Plant+endophyte”) or water were placed in Petri dishes on 0.04% benzimidazole-soaked cotton wool. Potato leaves were infected by spraying with 5 μL of P. infestans spore suspension (105 spores/mL). The development of symptoms on potato leaves was evaluated on the seventh day after infection by measurement of the lesion area on leaves and fixing images with the SP-800UZ Image camera (Olympus, Bekasi, Indonesia). Late blight signs became visible 1 day after the infection, and their extent was estimated by percent of affected area of the leaf blade on the 10th day after inoculation. To measure the area, leaves were photographed, and the images analyzed using the ImageJ program (NIH, USA) [30]. Ten plants of each variant group were selected for analysis, and three leaves from one plant were collected at the indicated time point per biological replicate.
Whole plants were infected by spraying with 25 5 μL of P. infestans spore suspension (105 spores/mL) for the investigation of local and systemic immune reactions.
To evaluate the antifungal activity of the strains, 5 mm-across slices of P. infestans mycelium were placed on 4 sides of Petri dishes with potato-glucose agar. Bacterial strains under investigation were placed individually in the center of the medium. Plates were cultured at 28 °C for 7 days. The test was performed in triplicate.

4.4. Evaluation of Bacillus Strains Insecticidal Activity against L. decemlineata

Then, the leaves of the control and endophytes-containing plants with petioles covered by paper gauze and inserted into tubes with water were placed in glass vessels and beetles were fed on potato leaves of these plants (1 beetle on 1 plant). Potato plants with surfaces contaminated by Bacillus strains were used to investigate the direct insecticidal effect of strains. Sterile potato plants were dipped into bacterial suspensions (108 cells/mL) for 1 min and were exposed to beetle attack at once as mentioned above.
Some adults were selected for gut investigations (Biozero BZ-X700 (“Keyence”, Japan)). Subsequently, beetles in all vessels ate non-infested plants to remove B. subtilis 26D-containing intestinal contents. The control groups of both variants were fed with potato leaves of plants treated with sterile water. Percentage of dead beetles was assessed.

4.5. Construction of Fluorescent B. subtilis 26DGFP

Genetically engineered construction on the basis of the pGFPAmy plasmid which contained a gene marking the green fluorescent protein GFPmut3 (excitation/emission-482/502 nm) was used for B. subtilis 26D recombination. Promotor Pveg of B. subtilis was amplificated by Pfu-polymerase using primers PvegF 5′-GGAGTTCTGAGAATTGGTATGCCTTAT-3′ and PvegR 5′- ACTACATTTATTGTACAACACGAGCCC-3′. pGFPamy was treated with endonuclease SmaI and ligated with Pveg [54]. Plasmid PGFPamyPveg was transformed into recipient strain B. subtilis 26D, giving ectopic insertion at the AmyE locus by a double cross-over recombination event (Figure 7I). Bacteria were selected on LB medium containing 100 µg/mL chloramphenicol. Fluorescence was observed using the microscope Biozero BZ-X700 (“Keyence”, Japan) with the standard filter (Figure 7II).

4.6. RNA Isolation and the Reverse Transcription Quantitative Polymerase Chain Reaction (qPCR)

Total RNA was isolated from plants+endophyte and sterile plants 24 h post-infection/pest impact with the TRI® reagent (Sigma-Aldrich, St. Louis, MO, USA). RNA concentration was measured using the Smart Spec Plus spectrophotometer (BioRad, Hercules, CA, USA). Concentrations of RNA in samples were equalized. The first cDNA strand was synthesized using oligonucleotide primers and M-MLV reverse transcriptase (Thermo Scientific, Madrid, Spain).
The obtained cDNA was diluted 5-fold and used for quantitative PCR (qPCR). Quantitative PCR was performed by polymerase chain reaction in real time using a set of predefined reagents N’,N’-dimethyl-N-(4-((E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl)-1-phenylquinolin-1-ium-2-yl)-N-propylpropane-1,3-diamine (SYBR Green I) (Synthol, Russia) and the device CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). qPCR was run according to the following program: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s, and at 60 °C for 1 min. After the final PCR cycle, a melting curve analysis was conducted to determine the specificity of the reaction (at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s). The efficiency of primer pairs was evaluated using 10-fold cDNA dilution series. The expression of each target gene is presented as fold change normalized to the reference gene StAct (Potato Actin) and relative to the untreated control sample. iCycler iQ5 Real-Time Detection System software (BioRad, USA) was used for data analysis. The primers used are given in Table 3.

4.7. Lignin Autofluorescence Registration

Localizations of lignin in leaves were studied 24 h after plant inoculation. Leaves were fixed with 96% ethanol at 4 °C for 4 h, and were placed in the mixture of ethanol/glycerin 1:1. Lignin autofluorescence on infected leaves was investigated with the laser scanning confocal microscope LSM-510 based on the inverted microscope Axiovert 200 M (“Carl Zeiss”, Oberkochen, Germany). For excitation of autofluorescence, an argon laser of 30 mW with a wavelength of 488 nm, dichroic 490 nm mirror, and 505 nm transmission filter was used [55].

4.8. Statistics

At least three biological replications in three technical repetitions each were examined in all. At least three biological replications in three technical repetitions each were examined in all experiments, including experiments with cDNA. Data presented are mean values with standard errors (±SE). Means were compared using analysis of variance (ANOVA) with p ≤ 0.05. Different letters on figures’ labels mean significant differences between treatments according to Student’s test at P < 0.05. The program Statistica 12.0 (Stat Soft, Moscow, Russia) was used.

5. Conclusions

Production of insectotoxins in recombinant lines which were obtained using endophytic bacterial strains seems to be one of the most reasonable techniques to create improved ecologically friendly biocontrol agents for pest management [17]. Artificial regulation of plant defense responses to herbivorous insects and pathogens, which frequently attack them simultaneously under field conditions, stands in need of continuous investigations. PGPB, including endophytic strains, could regulate SA- and JA-sensitive defense mechanisms in compliance with the type of damage or pathogen/pest attacks means [42,45]. Thus, the strain B. subtilis 26DCryChS is capable of stimulating both SA- and JA-dependent mechanisms in potato plants while host plants are under pest or pathogen attack.
Production of Cry1Ia δ-endotoxin in the endophytic source strain led to the development of a strain with integrated fungicidal (Figure 3) and insecticidal (Table 2) activities and capability to prime potato plants’ defense reactions through involvement of both SA- and JA-dependent pathways (Figure 4 and Figure 6). Endophytic B. subtilis 26DCryChS showed improved protective efficacy on potato plants against both oomycete P. infestans and L. decemlineata. The use of biocontrol agents based on B. subtilis 26DCryChS could be an alternative strategy of integrated plant protection.

Author Contributions

A.S.: Conceptualization, visualization, writing—original draft preparation, G.B. (Galina Benkovskaya): investigation, G.B. (Guzel Burkhanova): investigation, validation, D.B.: visualization, investigation, I.M.: writing—reviewing and editing, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by RFBR № 17-29-08014 and partially by Russian Federation State program AAAA-A16-116020350027-7.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

CFUcolony-forming units
ETethylene
JAjasmonic acid
PGPB plant growth-promoting bacteria
PR proteins pathogens-related proteins
SAsalicylic acid

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Figure 1. The influence of Bacillus strains on late blight disease symptoms on the potato leaves on 10th day after P. infestans inoculation. (I) Percentage of leaf area with late blight symptoms. Data represented as mean values ± standard error, values followed by the same alphabet are not significantly different from each other according to Student’s test p < 0.05. (II) Late blight lesions highlighted using ImageJ software (red areas on leaves).
Figure 1. The influence of Bacillus strains on late blight disease symptoms on the potato leaves on 10th day after P. infestans inoculation. (I) Percentage of leaf area with late blight symptoms. Data represented as mean values ± standard error, values followed by the same alphabet are not significantly different from each other according to Student’s test p < 0.05. (II) Late blight lesions highlighted using ImageJ software (red areas on leaves).
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Figure 2. Antagonistic effect of Bacillus sp. on pathogenic oomycete P. infestans growth in vitro.
Figure 2. Antagonistic effect of Bacillus sp. on pathogenic oomycete P. infestans growth in vitro.
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Figure 3. Mesenteron structure of L. decemlineata beetles 24 h after feeding of potato plants, which contained cells of Bacillus strains. Scale bars, 200 um. (I) Healthy larva; (II) bacteriosis on alive larva on the 5th day after eating of plants containing Bacillus sp.
Figure 3. Mesenteron structure of L. decemlineata beetles 24 h after feeding of potato plants, which contained cells of Bacillus strains. Scale bars, 200 um. (I) Healthy larva; (II) bacteriosis on alive larva on the 5th day after eating of plants containing Bacillus sp.
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Figure 4. The effect of Bacillus strains on transcriptional activity of StPR6, StPR1, StAOS, and StOPAR genes in potato plants after 24 h post infection with P. infestans. Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test p < 0.05.
Figure 4. The effect of Bacillus strains on transcriptional activity of StPR6, StPR1, StAOS, and StOPAR genes in potato plants after 24 h post infection with P. infestans. Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test p < 0.05.
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Figure 5. Lignin fluorescence in P. infestans-penetrated sites on leaves of water- and Bacillus sp.-treated potato plants. Scale bars 20 µm.
Figure 5. Lignin fluorescence in P. infestans-penetrated sites on leaves of water- and Bacillus sp.-treated potato plants. Scale bars 20 µm.
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Figure 6. The effect of Bacillus strains on transcriptional activity of StPR6, StPR1, StAOS, and StOPAR genes in potato plants after 24 h post damage caused by larvae of L. decemlineata. Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test P < 0.05.
Figure 6. The effect of Bacillus strains on transcriptional activity of StPR6, StPR1, StAOS, and StOPAR genes in potato plants after 24 h post damage caused by larvae of L. decemlineata. Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test P < 0.05.
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Figure 7. (I) Construction of the gene engineering plasmid pGFPamyPveg for B. subtilis 26D transformation. (II) B. subtilis 26DGFP fluorescens (Scale bar 50 nm). AmyE’ front, and amyE back—5’ and 3’ parts of B. subtilis 168 a-amylase gen; Cap encodes chloramphenicol acetyl transferase; Amp encodes beta-lactamase; SmaI and PstI—restriction sites, ColE1ori—origin of replication in E. coli.
Figure 7. (I) Construction of the gene engineering plasmid pGFPamyPveg for B. subtilis 26D transformation. (II) B. subtilis 26DGFP fluorescens (Scale bar 50 nm). AmyE’ front, and amyE back—5’ and 3’ parts of B. subtilis 168 a-amylase gen; Cap encodes chloramphenicol acetyl transferase; Amp encodes beta-lactamase; SmaI and PstI—restriction sites, ColE1ori—origin of replication in E. coli.
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Table
Table 1. Content of Bacillus sp. in plant tissues after surface sterilization, CFU*103/g of plant wet mass (14th day post inoculation).
Table 1. Content of Bacillus sp. in plant tissues after surface sterilization, CFU*103/g of plant wet mass (14th day post inoculation).
VariantCFU*103/g
water (0)-
B. subtilis 26D420 ± 63a
B. thuringiensis B-53517.5 ± 0.25b
B. subtilis 26DCryChS487 ± 53a
Note: Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test p < 0.05.
Table
Table 2. Effect of feeding on surface contaminated with Bacillus spp. or containing endophytic Bacillus sp. cells in the internal tissues of potato plants on L. decemlineata larvae mortality.
Table 2. Effect of feeding on surface contaminated with Bacillus spp. or containing endophytic Bacillus sp. cells in the internal tissues of potato plants on L. decemlineata larvae mortality.
Variant/ParameterMortality, % (7th Day after Feeding)
Surface-Contaminated PlantsPlant + Endophyte
Water4.1 ± 0.7a6.7 ± 0.5a
B. subtilis 26D18.1 ± 3.5b38.1 ± 5.2b
B. thuringiensis B-535176.7 ± 6.35c55.6 ± 5.8c
B. subtilis 26DCryChS48 ± 11.8d80.7 ± 13.3d
Note: Data presented as mean values ± standard error, values labeled by the same character are not significantly different from each other according to Student’s test P < 0.05.
Table
Table 3. Primers used for quantitative PCR.
Table 3. Primers used for quantitative PCR.
GeneGene ProductNCBI Access NumberPrimers
StPR6Trypsin inhybitor, PR-6AY089962F 5’-gctgaggattggtgagaggta-3’
R 5’-ccacatcaccataatccaact-3’
StPR1basic antimicrobial protein PR-1AY050221F 5’-tgggtggtggttcatttcttgt-3’
5’-catttaattccttacacatcataa-g-3’
StAOSallene oxide synthaseDQ174273F 5’-gcacactttccctctaccttac-3’
R 5’-ccaagtttctccgcttcatcta-3’
StOPAR12-12-oxophytodienic acid reductaseJN241968F 5’-gggatacacagattaccctttcc-3’
R 5’- tcgggcttcacaagttcttac-3’
StActactinX55749F 5’-gatggtgtcagccacac-3’
R 5’-attccagcagcttccattcc-3’

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MDPI and ACS Style

Sorokan, A.; Benkovskaya, G.; Burkhanova, G.; Blagova, D.; Maksimov, I. Endophytic Strain Bacillus subtilis 26DCryChS Producing Cry1Ia Toxin from Bacillus thuringiensis Promotes Multifaceted Potato Defense against Phytophthora infestans (Mont.) de Bary and Pest Leptinotarsa decemlineata Say. Plants 2020, 9, 1115. https://doi.org/10.3390/plants9091115

AMA Style

Sorokan A, Benkovskaya G, Burkhanova G, Blagova D, Maksimov I. Endophytic Strain Bacillus subtilis 26DCryChS Producing Cry1Ia Toxin from Bacillus thuringiensis Promotes Multifaceted Potato Defense against Phytophthora infestans (Mont.) de Bary and Pest Leptinotarsa decemlineata Say. Plants. 2020; 9(9):1115. https://doi.org/10.3390/plants9091115

Chicago/Turabian Style

Sorokan, Antonina, Galina Benkovskaya, Guzel Burkhanova, Darya Blagova, and Igor Maksimov. 2020. "Endophytic Strain Bacillus subtilis 26DCryChS Producing Cry1Ia Toxin from Bacillus thuringiensis Promotes Multifaceted Potato Defense against Phytophthora infestans (Mont.) de Bary and Pest Leptinotarsa decemlineata Say" Plants 9, no. 9: 1115. https://doi.org/10.3390/plants9091115

APA Style

Sorokan, A., Benkovskaya, G., Burkhanova, G., Blagova, D., & Maksimov, I. (2020). Endophytic Strain Bacillus subtilis 26DCryChS Producing Cry1Ia Toxin from Bacillus thuringiensis Promotes Multifaceted Potato Defense against Phytophthora infestans (Mont.) de Bary and Pest Leptinotarsa decemlineata Say. Plants, 9(9), 1115. https://doi.org/10.3390/plants9091115

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