Assessing the effects of a novel biostimulant to enhance leafminer resistance and plant growth on common bean

Chemical composition of the stimulator compound

The GC–MS data (Figure S1) shows the presence of bioactive constituents and indicate that BSTC contains potential agents that have essential roles in stimulating immunity, defense signaling and enhance resistance against many pathogens and insects such as Pyridine 1,2,3,6 tetrahydro-1,2-dimethyl-(CAS), Adipic acid, 2-decyl ethyl ester, Squalene, L- Glutamic acid 5-ethyl ester and Oleic acid. While L- Glutamic acid 5-ethyl ester and citric acid enhances plant Growth, tissue repair and photosynthesis. Also, cis-9-Hexadecenoic acid, dodecanoic acid, 3-hydroxy have essential role in cell signaling, membrane stabilizer and energy source.

Analytical analysis of the five nucleobases (adenine, thymine, uracil, guanine and cytosine) in nucleic acids detected using HPLC–DAD, showed that formulated BSTC contains high concentration of nucleobases with 56 fold of the yeast extract as the main source of nucleic acids (Figure S2).

Effects of BSTC on plant growth

The foliar application with BSTC on the two P. vulgaris cvs (AlHama and Moraleda) during the two consecutive seasons recorded significantly increase in several morphological characters of vegetative growth and yield, compared to the yeast extract application (C2) used positive control and the water sprayed cvs (C1) as negative control (Fig. 1). Significant differences were observed in growth parameters after BSTC treatments, especially plant height and yield, of both AlHama and Moraleda cvs during 2019 and 2020 seasons are presented in Table 1. Nevertheless, BSTC treated AlHama cv recorded maximum increases in vegetative growth. Compared to the negative control (C1), total yield increased 150% and 96%, in addition to significant increases in plant height increase and number of pods/plants for AlHama and Moraleda, respectively. While comparing to C2 (+ ve control sprayed with YE), total yield increased 70% and 11% for AlHama and Moraleda, respectively (Table S2). The differences between P. vulgaris cvs in vegetative growth parameters could be attributed to their variations in the genetic habitation and responses to the environmental conditions. In the case of C2 treatment, although it increase the growth parameters for the two cultivars but the increases were not significant compared to the control. Results revealed that the enhancement effect of the BSTC could be attributed to the satisfactory impact of them on the metabolism and biological activity and their unexpected effect on photosynthetic pigments and the enzyme activity, which could encourage the vegetative growth of P. vulgaris.

Evaluation of foliar activators on Liriomza trifolii infestation

During the two cultivation seasons of the P. vulgaris cvs, the BSTC treated plants revealed significant decrease in the number of mines (> 62%) and mine length (60%) in the case of AlHama cv compared to the control, while the C2 treatment caused only 25% and < 4% decrease for the number of mines and mine length, respectively (Table 2). Moraleda cv, treated with BSTC caused less effect than AlHama (< 50% miner number, < 56% mine length), while C2 treated plants did not differ much than the C2 treated AlHama plants. Negative control as well as the C2 treated plants clearly showed that the ends of population tunnels are opened, the number of the mine/ leave were 6 and 8, respectively, the length of the mine was > 2 cm and the insects were able to complete the life cycle and reached the adult stage. On the other hand, BSTC treated plants caused decrease in length of mine (~ 1 cm), the number of mines/leaf (2–4) and the ends of population champers were closed that cause blocking the breathing holes of insects causing suffocation and death. BSTC treated plants showed dead larvae inside the tunnel (Fig. 2).

In addition, the reduction rate (Table 3) of the leafminer infesting AlHama cv were significantly decreased during five weeks after the application of the BSTC treatments compared to the controls (C1 and C2) from 90 to 68% in the 1st season and from 92 to 57% in the 2nd season. In the case of Moraleda, reduction rate decreased from 79 to 25% in the 1st season and from 57 to 48% in the 2nd season. Reduction rates indicate the stability, effectiveness and persistence of the BSTC compound on the L. trifolii larvae insect for five weeks in 2019 and 2020 seasons. The results revealed that BSTC treatment increased the plant resistance to insect, but AlHama was more resistant than Moraleda. The mechanisms by which growth promoter compounds found in BSTC mediate disease resistance remained unexplored and elusive, yet its exact function has remained unclear. Therefore, gene expression of defense related genes were studied.

Expression of biotic stress-related genes in P. vulgaris during L. trifolii attacks

Plants respond to pathogen or insect attacks by activating the synthesis of a diverse number of defense proteins. Corroborating this, comparative analysis of disease related genes of the BSTC treated common bean with C1 and C2 plants under leafminer infestation were carried out as an attempt to understand the indirect effects of BSTC compounds insect infestation. The expression profile of 11 functional genes belonging to the four important groups of the identified genes related to biotic stress response were evaluated in order to clarify the plant response during the interaction with the leafminer L. trifolii: pathogenesis related (PvPR1 & PvPR3), antioxidant enzyme-related genes (PvPOD SOD & GST), defense and stress related (PvHPRP, PvDOX & Pvcallose), Phenyl propanoid pathway (PvPAL & Pv4CL) groups in addition to the light-harvesting chlorophyll type-I (Lhcll-I) were evaluated using RT-PCR two days post infestation with L. trifolii (Fig. 3). Significant fold differences for the expression of selected genes in the treated plants with BSTC after leafminer infestation were observed. The expression levels for pathogenesis related genes; PvPR1 (Pathogenesis-related) and PVPR3 (Chitinase class I) were increased two days post infestation with the leafminer in both AlHama and Moraleda cvs and were significantly increased with plants treated with BSTC. The expression of PvPR1 increased 1.5 fold following leafminer infestation in BSTC treated compared to the untreated plants. The results revealed that the transcription level of the SA-inducible genes PR1 and PR3 plays an important role in the defense-response in plants.

For the antioxidant enzyme related genes; PvPOD (peroxidase), SOD (Superoxide dismutase) and GSTT (glutathione S-transferase), leafminer infestation decreased their expression, but BSTC treatment caused significantly up-regulation of those enzymes indicating the capability of the SOD in the activation of antioxidant defense systems in plants. That indicates that BSTC may have important chemical compound that result in stimulating the PvGST and PvSOD gene expression.

Additionally, the transcript levels of the defense and stress related genes; PvDOX (α-dioxygenase), Pvcallose (Callose synthase) showed high transcript levels during the early stages of the interaction L. trifolii–P. vulgaris interaction, while PvHPRP (Hypersensitive-induced) showed decrease expression level in the later stages of infestation. In the case of AlHama cv, the down-regulation of the defense and stress related genes might explain the lower level of resistance and growth compared to AlHama. Those genes known are largely responsible for JA induction genes indicating that JA has important roles in the plant response to leafminer.

Moreover, the genes involved in the phenyl propanoid pathway; Pv4CL and PvPAL (phenylalanine ammonialyase), were upregulated in 2 days post interaction with L. trifolii. Although the expression of the Pv4CL (4-coumarate CoA-ligase) was significantly down regulated. However, the gene Lhcll-I, involved chlorophyll content pathway, showed down-regulation due to the L. trifolii infestation even after BSTC treatment. That indicates that leafminer infestation affects the chlorophyll content as well as plant growth. The expression of the Lhcll-I gene showed down regulation due to leafminer infection, although BSTC treated plants revealed a slide increase in its expression.

Effect of BSTC phytochemicals on L. trifolii vital proteins using docking analysis

Molecular docking is extensively used to model the interaction between a small molecule and a protein at the atomic level to elucidate the binding patterns of small molecules within the receptor active cite of the target protein.

In the current study, we screened various natural compounds presented in the BSCT with 6 vital proteins that have essential roles in pathway of life cycle of the insect: acetyl cholinesterase, elongation factor, histone subunit3, arginine kinase, HSP70 and HSP90. To assess their stability and reliability, a range of methods were used to estimate the efficiency of the predictive model acetyl cholinesterase, elongation factor, histone subunit3, arginine kinase, HSP70 and HSP90 protein model. PROCHECK analysis, which quantifies the residues in the Ramachandran plot, was used for eventually validation. The ERRAT tool, which defines the protein’s overall quality factor, was used to study statistics on no-bonded interactions among different types of atoms. The molecular interactions of protein–ligand were docked by SAMSON 2020 software through predicting the potential affinity, molecular structure, geometry optimization of structure, vibration frequencies of coordinates of atoms, bond length and bond angle. The best scoring poses out of 100 poses were used to calculate the binding affinity of the ligands. Shortlist of screening exhibit best binding ligands to the selected proteins is presented in Table S3. Ligand with the highest binding score with each protein was identified. The grid was configured with the following parameters: size X, Y, Z, structural properties of each molecule and calculated binding affinities to the six model proteins for L. trifolii insect. Based on docking results, we shortlisted of three compounds, disodium 5′-inosinate binds exhibit best binding with acetyl cholinesterase and HSP90 exhibited, delphinidin 3-glucoside best binds with elongation factor and HSP90, while hyperoside best binds histone subunit 3, arginine kinase and cytochrome with binding score ranged between − 10.2 to − 5.0 (Table S3). The best-docked compounds were evaluated for their 2D and 3D structures and the important interacting residues obtained during molecular docking (Fig. 4). The predicted docking binding energy point to the possible of biological activity of the title molecule on inhibiting the protein function and may participate in insect mortality.

Discussion

Foliar application of soluble biochemical represents an additional source for delivering essential nutrients to plants biofortification²⁴. Leaves can uptake the nutrients either through penetrating the cuticle or through the stomata²⁴. A safe and environmental friendly compounds is required for cropping production systems²⁵. In this research, we highlighted the effects of applying a new, inexpensive and easy to prepare biobased stimulator compound on enhancing growth development, yield and leafminer resistance of common bean under normal agronomical practices. BSTC is prepared from compound obtained from yeast autolysate as a rich source of high quality protein, essential amino acids, essential minerals and vitamins that enhance cell division and enlargement²⁶. The prepared suspension is supplemented with citric acid and salicylic acid that enhance plant growth²⁷ and ascorbic acid which has an essential role in plant cell division, cell expansion, growth and development.

Effects of chemical composition and micronutrient chelated compounds

Micronutrient metals such as iron and zinc are essential nutrient elements required for healthy plant growth⁶. Zinc is an essential micronutrient that works as a co-factor of over 300 enzymes in plants and it plays important roles in protein synthesis, regulation of growth and development disease resistance²⁸. Therefore, the presence of zinc chloride in the formulated BSTC allow zinc chelating by amino acids to improve foliar uptake in plants (Fig. 5). The aminochelatos are used to overcome iron and zinc deficiencies correction in plants²⁷ and evolve plant defenses against pests and pathogens²⁹. Several researches reported the effect of macronutrient fertilization and damage by insect herbivory^30,31. From another perspective, high concentrations of amino acids presented in the formulated BSTC are considered as precursors for protein synthesis, indicating their importance for stimulation of cell growth and stimulate defense against pests and pathogens²⁷.

The HPLC–DAD used for nucleobases analysis presented in this work has the potential to trace the nitrogen and carbon derived from nucleic acids. HPLC results revealed that the formulated BSTC has high concentration of nucleobases, one of the key elements that enhance growth and insect resistance in treated plants as they increase the absorption of the nutrient compounds. This is the first time using nucleobase as source of nutrient in plants. The presences of high concentration of nucleobases have an essential role in growth performance, and development of immunity when combined with yeast extract. Nucleotides and nucleosides are conditionally essential nutrients in tissues requiring rapid cell replication³² and development of immunity³². They are safely added to infant formula and animal feed ingredients^33,34 to regulate growth performance and reduce the susceptibility to various stressors and diseases, which was correlated with enhanced growth.

The chemical composition of BSTC revealed from GC–MS showed that it contains important chemicals that may have essential role induce immunity and resistance against many pathogens and insects, enhance plant growth, and defense signaling that may lead to enhancing plant growth and insect resistance in a direct or indirect manner. Previous documents showed that adipic acid ethyl ester and pyridine 1,2,3,6 tetrahydro-1, 2-dimethyl both induce immunity and induces resistance against many pathogens and insects^33,34,35; oleic acid that induces defense signaling, enhanced salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens in plant³⁵ and the squalene that mediates steroid synthesis and stimulates the immune system³⁶. Another chemicals found in BSTC could be involved in stimulating of gene expression of essential genes in the plant cells.

Effects on plant growth and yield

The foliar application with BSTC on the two P. vulgaris cvs (AlHama and Moraleda) during the two consecutive seasons recorded significantly increase in several morphological characters of vegetative growth and yield, compared to the C1 and C2 cvs. The foliar application with BSTC on the two P. vulgaris cvs (AlHama and Moraleda) during the two consecutive seasons recorded significantly increase in several morphological characters of vegetative growth and yield, compared to the C1 and C2 cvs. Nevertheless, BSTC treated AlHama cv recorded maximum increases in vegetative growth, as the yield increased more than double increased and plant height increased 50% compared to the Moraleda cv, that may be due to change in the genetic background between the two cvs which may act different with the foliar fertilizers. There is evidence that nutrient supply from the YE increase growth parameters in date palm³⁷, soybean³⁸, grape²⁰, sweet potato³⁹, pea⁴⁰ and snap bean⁴¹. Several bioactive compounds were used as plant growth regulators to enhance crop quality such as salicylic acid, nitric oxide, jasmonic acid, ascorbic acid and citric acid were used to improve the immunity, yield and quality of various crops⁴².

Effects on leafminer resistance

Apart from growth improvements, a new phenomenon was observed on the BSTC treated plants, the leafminer were not able to continue their life cycle causing the death of the larvae. The leafminer L. trifolii insect is an economically important insect that causes great losses in several crops^2,3. Usually, synthetic insecticides and biological control are used to control leafminers infestation^2,5. We recorded significant reduction in infestation after foliar spraying with BSTC with both P. vulgaris cvs compared to the controls. Also, treating infected plants showed close at the end of the tunnel causing larvae suffocation and death. In general, differences in leafminer infestation between the two cvs during the two tested seasons were insignificant. Results indicate that BSTC may contain stimulator to increase plant resistance to leafminer. No previous report indicated the effect of any foliar applications with natural compounds on insect infestation.

In this investigation, we developed for the first time safe and easily prepared biobased stimulator compound to control leafminer infestation.

Nevertheless, the mechanisms by which growth promoter compounds found in BSTC mediate disease resistance remained unexplored and elusive, yet its exact function has remained unclear. Therefore, gene expression of defense related genes were studied. Adding ascorbic acid, citric acid and salicylic to the formulated BSTC has positive effects on stimulating the plant growth hormones JA and SA., which play important roles in the plant metabolism in response to biotic and abiotic stresses^15,43,44. In general, JA-mediated signaling pathways are implicated in the regulation of insect defenses, while the SA pathway is associated with defense responses against pathogens¹⁵.

Effects on expression of defense related genes

Plants respond to pathogen or insect attacks by activating the synthesis of a diverse number of defense proteins. Corroborating this, comparative analysis of disease related genes of the BSTC treated common bean with healthy plants under leafminer infestation were carried out. The expression of the pathogenesis-related genes encoding antibiotic protein; PvPR1 and PvPR3 increased upon insect attach, but the expression of PvPR1 increased 1.5 fold following leafminer infestation in BSTC treated compared to the untreated plants. In accordance with these findings, the upregulation of PvPR1 was reported during the incompatible interaction between the common bean and fungal infection⁴⁵. While, PR3 gene (chitinase) induces signaling molecules that may work as defensive mechanism elicitors^43,44.

The antioxidant enzyme-related genes; PvPOD, PvGST and PvSOD, presented a progressive increase in expression following infectious development in BSTC treated plants. That indicates that BSTC has important chemical compound that stimulate the PvGST and PvSOD gene expression. Additionally, the high expression of the defense and stress related genes; PvDOX, PvHPRP and Pvcallose, may indicate their role in insect infestation, except for the PvHPRP expression that showed down regulation.

The expression responses of defense and stress related genes observed in the present study are in agreement with the results of previous investigations. During the interaction between Rhizoctonia solani–P. vulgaris, highly expressed of PvDOX was recorded to protect plant tissues by undergoing excessive necrosis associated with oxidative stress during pathogenesis^44,45. In fact, oxidative stress improves the ROS scavenging capacity in plant by increasing the expression of related enzymatic activities, such as glutathione S-transferase⁴⁵. Also, PvHIPRP expressed under hypersensitive responses, was induced at the early stages of infection in infected tissue of P. vulgaris⁴⁶. Plants unable to undergo programmed cell death⁴⁷ or incapable of generating HR^’ are more resistant to such pathogens. On the other hand, callose induction is an effective barrier presented at early stages of pathogen invasion and is associated with incompatible responses⁴⁸. While the genes involved in the phenylpropanoid pathway, the PvPAL and Pv4CL, upregulated during infestation in AlHama cv but not with Pv4CL gene in Moraleda cv. That may explain the differences in response in the two cvs upon insect infestation.

On the other hands, the expression level of the Pv4CL gene in P. vulgaris infected with fungi was recorded as transient expression, with the greatest upregulation in the early stages of infection⁴⁶. In Arabidopsis, mutation analysis in the PAL pathway caused rapid production of SA associated with local cell death⁴⁹. Certain phenylpropanoid compounds, such as PvPAL and Pv4CL, leads to the synthesis of lignin, and many defense molecules, which are crucial for plant defense against abiotic and biotic stress factors⁵⁰.

The expression of the Lhcll-I gene showed down regulation due to leafminer infection, although BSTC treated plants revealed a slide increase in its expression. Lhcll-I encodes the chlorophyll a-b binding protein which work as receptor for light harvesting, it captures and delivers excitation energy to photosystems. Its expression is regulated by multiple environmental and developmental cues⁵¹.

Due to the overexpression of PvPOD, PvDOX, PvPAL, Pv4CL Pvcallose in BSTC treated AlHama we hypothesize that they are involved in defense mechanism against leafminer infestation.

In silico studies for molecules inhibiting the protein function

Molecular docking revealed that the main ligands supplemented by BSTC for bind the selected insect proteins are delphinidin 3-glucoside which binds with the insect HSP90 and elongation factor with high score (− 10.2 and − 9.2 kcal/mol, respectively), followed by disodium-5′-guanylate that bind with HSP70 with -9 kcal/mol while the binding energy of hyperoside binds with arginine kinase and histone subunit3 by − 8.8 and − 7 kcal/mol, respectively. The obtained results indicate that those ligands may have an essential role in insect death. In general, molecular docking revealed that binding the BSTC ligands have high docking scores with the insect essential proteins and could have negative effects in insect survival^52,53. From the obtained results, we concluded that controlling leafminer infestation with BSTC implementation has positive influence on plant growth and yield quality and quantity improvement. However, further research is needed for mode of action of the BSTC that cause insect death and whether the BSTC have positive effects on controlling other harmful insects.