Endophytic Bacillus spp. from medicinal plants inhibit mycelial growth of Sclerotinia sclerotiorum and promote plant growth

Abstract Plant growth-promoting bacteria that are also capable of suppressing plant pathogenic fungi play an important role in sustainable agriculture. There is a critical need for conducting research to discover, characterize and evaluate the efficacy of new strains of such bacteria in controlling highly aggressive plant pathogens. In this study, we isolated endophytic bacteria from medicinal plants of Bangladesh and evaluated their antagonistic capacity against an important phytopathogenic fungus Sclerotinia sclerotiorum. Growth-promoting effects of those isolates on cucumber and rice seedlings were also assessed. Among 16 morphologically distinct isolates, BDR-2, BRtL-2 and BCL-1 significantly inhibited the growth of S. sclerotiorum through induction of characteristic morphological alterations in hyphae and reduction of mycelial dry weight. When cucumber and rice seeds were treated with these endophytic bacteria, seven isolates (BCL-1, BDL-1, BRtL-2, BRtL-3, BDR-1, BDR-2 and BBoS-1) enhanced seed germination, seedling vigor, seedling growth and number of roots per plant at a varying level compared to untreated controls. All isolates produced high levels of indole-3-acetic acid (6 to 63 μg/mL) in vitro. Two most potential isolates, BDR-2 and BRtL-2, were identified as Bacillus amyloliquefaciens and B. subtilis, respectively, based on the 16S rRNA gene sequencing. These results suggest that endophytic Bacillus species from native medicinal plants have great potential for being used as natural plant growth promoter and biopesticides in sustainable crop production.

Among various reported plant growth-promoting and biocontrol bacteria, species of Bacillus showed the highest potential both in vitro and in vivo [9]. For example, B. subtilis produces various phytohormones such as indole-3-acetic acid (IAA), cytokinins, zeatin, gibberellic acid and abscisic acid that are transported into the shoot through the xylem, delay senescence and thus boost production of lettuce, tomato, cucumber and pepper [10,11]. Apart from the improvement of crop yield, B. subtilis also induces resistance to the fungal phytopathogens. Strains of B. amyloliquefaciens and B. subtilis are known to competitively colonize plants and can simultaneously act as biofertilizer and antagonists (biopesticides) of bacteria, fungi, peronosporomycetes and nematodes [12][13][14]. Colonization of cucumber plants by B. polymyxa increased enzymatic activities with a concurrent increase in cucumber yield up to 25% compared to non-treated control [15]. Production of growth-stimulating phytohormones, solubilization and mobilization of phosphates, production of siderophores, production of antibiotics, inhibition of plant ethylene synthesis and induction of plant systemic resistance to pathogens are considered as the mechanisms of plant growth promotion by endophytic bacteria [2,3,13,16,17].
Sclerotinia sclerotiorum is the most destructive phytopathogen with a wide host range, which has been reported to infect 64 plant families [18,19]. It causes white mold, cottony rot, watery soft rot, stem rot, drop, crown rot and twig blight and many other serious diseases in plants resulting in significant yield losses worldwide [18]. In the field condition, yield loss may exceed 50% due to Sclerotinia blight in peanut under a favorable environmental condition. Control of plant diseases caused by S. sclerotiorum by synthetic fungicides is problematic due to high cost, low efficacy and deleterious effects on the environment [19]. Biological agents can be the best alternatives for controlling plant diseases due to the reasons mentioned above [5,7,20]. Bangladesh is rich in diversity of medicinal plants from which discovery of novel endophytic bacteria to use as the biopesticides against notorious phytopathogens including S. sclerotiorum holds great potential. No systematic study has so far been conducted on isolation and identification of endophytic bacteria from medicinal plants of Bangladesh, and has evaluated their effects on growth promotion and protection of plants from phytopathogens. Therefore, the objectives of this study were to (i) isolate and identify potential endophytic bacteria from some important medicinal plants; (ii) evaluate inhibitory effects of the isolated endophytic bacteria against S. sclerotiorum and (iii) assess the effects of endophytes on seed germination, seedling vigor and growth of rice and cucumber.

General experimental procedure
All the chemicals and reagents used in this study were available at the Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh, previously procured from Merck AG, Germany. All photographs were taken by a high-resolution camera attached to a compound microscope.

Source and preservation of Sclerotinia sclerotiorum strain
The pathogen S. sclerotiorum was collected from the stock culture of the Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh, which was previously isolated from hyacinth bean [21]. This organism was regularly cultured on the Potato Dextrose Agar (PDA) medium and preserved in a PDA slant.

Isolation of endophytic bacteria
Sixteen endophytic bacteria were isolated from different well-known medicinal plant parts such as root and leaf of Duranta plumeri, leaf of Ocimum gratissimum L., seed of Terminalia bohera and leaf of Manihot esculenta from Dhaka, Gazipur and Rajshahi districts of Bangladesh. Five-gram fresh plant sample from each of different organs were washed under running tap water and surface sterilized with 70% ethanol for 10 min followed by 1% NaOCl for 1 min and finally washed with sterile distilled water (SDW) three times to remove excess NaOCl [2]. Tissue pieces were then smashed in a sterilized mortar and pestle. Smashed plant materials were serially diluted up to 1 × 10 −6 in SDW, from which 100 μL was spread evenly on petri dishes containing yeast extract glucose agar (YGA) medium and incubated at 25 °C for 24 h. Bacterial colonies were isolated based on the colony color, shape and another unique identification criterion, and purified by repeated streak plate culture. The purified single colony isolates were preserved in 20% glycerol solution at -20 °C for subsequent experiments.

Morphological and biochemical characterization of endophytic bacterial isolates
Morphological characteristics of colonies such as size, shape, color and growth pattern were recorded under a light microscope after 24 h of growth on the YGA medium at 25 ± 2 °C as described earlier [22]. A series of biochemical tests were performed to characterize the isolated endophytic bacteria using the protocols described in Bergey's Manual of Systematic Bacteriology [23]. For the KOH solubility test, endophytic bacteria were mixed aseptically with 3% KOH solution on a clean slide with an inoculating wire loop for 1 min and observed for the formation of a thread-like mass. Catalase and oxidase tests were performed as described earlier by Hayward [24] and Rajat et al. [25], respectively.

Molecular identification of isolated endophytic bacteria
Polymerase chain reaction (PCR) amplification of the 16S rRNA gene was performed followed by sequencing to identify the active bacterial strains [7]. Initially, cells from the single bacterial colony were harvested from agar broth and re-suspended in 100 μL SDW by vortexing for 10 s. For the determination of 16S rRNA sequences of active strains, chromosomal DNA extraction was done using a commercial DNA extraction kit (ATP Biotech Inc., Taiwan) and quantified comparing with the lambda DNA marker after agarose gel electrophoresis. The 16S rRNA region was amplified by PCR using a universal primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTAC-CTTGTTACGACTT-3′

Determination of S. sclerotiorum growth suppression by endophytes
To assess antagonistic activities, seven bacterial strains were tested against S. sclerotiorum using a dual culture assay. A 6-mm-diameter mycelial plug of the pathogen was cut from an actively growing colony with a sterile cork borer and placed 3 cm apart from each endophyte colony on the PDA plate [26]. The inhibition zone was observed after incubation at 25 °C for 72 h. The radial growth of the pathogen and percentage inhibition was calculated after 10 days as described by Zohara et al. [7].
where X is the mycelial growth of the pathogen in the absence of the antagonist, and Y is the = mycelial growth of the pathogen in the presence of the antagonist. Morphological characters of approaching hyphae were observed under a light microscope and images were recorded with a digital camera attached with the microscope. At least five samples were examined for each combination of the pathogen and antagonistic endophyte.

Effect of endophytic bacterial strains on mycelial dry weight of S. sclerotiorum
Quantitative antagonistic effect of isolated endophytic bacterial strains was measured. Briefly, a block of S. sclerotiorum was grown in a conical flask containing 200 mL potato dextrose broth (PDB) for 3 days. After 3 days, 1 mL of 1 × 10 5 -, 1 × 10 6 -, 1 × 10 7 -and 1 × 10 8 -fold diluted bacterial cells were added to the broth and incubated at 25 °C for 10 days. Before inoculation of bacterial strains, mycelial dry weight of control flasks was measured to determine whether bacterial strains can decompose and decrease mycelial growth of S. sclerotiorum. After 10 days of pathogen mycelia and bacteria interaction, mycelia from each flask were placed on separate filter paper and allowed to dry for 3 h, after which time mycelial dry weight was recorded individually to determine the effect of each antagonistic bacterial strain.

Plant growth promoting activity of the endophytic bacteria 2.8.1 Indole-3-acetic acid production
For determination and quantification of IAA production, colonies of bacterial strains were inoculated into Jensen's broth (Sucrose 20 g/L, K 2 HPO 4 1 g/L, MgSO 4 · 7H 2 O 0.5 g/L, NaCl 0.5 g/L, FeSO 4 0.1 g/L 1 , NaMoO 4 0.005 g/L, CaCO 3 2 g/L) [27] containing 2 mg/mL l-tryptophan and incubated at 28 ± 2 °C with continuous shaking at 125 rpm for 48 h on a benchtop shaker. Two milliliters of culture solution was centrifuged at 12,000 g for 1 min, and 1 mL aliquot of the supernatant was mixed with 2 mL of Salkowski's reagent (150 mL conc. H 2 SO 4 , 250 mL distilled water, 7.5 mL 0.5 M FeCl 3 · 6H 2 O) and incubated for 20 min in darkness at room temperature, as described by Gordon and Weber [28]. IAA production was indicated by the development of a pink-red color, and the absorbance was measured at 530 nm using a spectrophotometer (PD-303U, APEL, Japan). The concentration of IAA was determined using a standard curve prepared from pure IAA solutions (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 and 65 μg/mL). Supernatants from un-inoculated test tubes were used as control.

Preparation of bacterial inocula for seed treatment
Bacterial strains that were isolated from medicinal plants were cultured separately in conical flasks containing 200 mL yeast peptone broth and incubated in an orbital shaker at 120 rpm for 72 h at 25 °C for preparation of inocula. Bacterial cells were collected via centrifugation at 15,000 rpm for 1 min at 4 °C. After washing with SDW, bacterial pellets were suspended in 0.6 mL SDW, vortexed and used for seed treatment. One gram of cucumber and rice seeds were surface sterilized with 70% ethanol for 10 min followed by washing with SDW. Seeds were then soaked in bacterial suspension in a petri dish (for how long?) and dried overnight at room temperature to ensure better coating with bacteria. The number of bacterial cells per seed was ca. 1 × 10 8 CFU, counted by using the serial dilution technique. Inoculated seeds were planted in pots containing soil mix. After germination, seedlings were watered on alternate days.

Effect of bacterial seed treatment on germination and vigor index of cucumber and rice
For determination of percent germination and seedling vigor, 100 seeds inoculated with each strain were incubated at 25 °C in petri dishes (9 cm) in three replications on a layer of moistened filter paper. Seeds soaked in water instead of bacterial suspensions were used as control.
Water was added to the petri dishes to maintain sufficient moisture. The germination percentage was recorded every 24 h for 7 days. Root and shoot lengths were measured after the seventh day. The germination percentage and vigor index were calculated using the following formula: Fisher's protected LSD test was used to determine the levels of significant differences among the mean values at p ≤ 0.05. The experimental design was completely randomized, consisting of at least three replications for each treatment and were repeated at least twice.

Biochemical characterization of endophytic bacteria
Based on in vitro antagonism against S. sclerotiorum and ability to produce IAA, seven isolates, namely, BDR-1 (root of D. plumeri), BDR-2 (root of D. plumeri), BDL-1 (leaf of D. plumeri), BRtL-2 (leaf of O. gratissimum L.), BRtL-3 (leaf of O. gratissimum L.), BBoS-1 (seed of T. bohera) and BCL-1 (leaf of M. esculenta), were selected for further morphological and biochemical study. All isolates were brown to whitish in color, fast-growing on various culture media and produced colonies of round to irregular shape with smooth surfaces. All seven isolates reacted positively to the catalase test and Gram-staining test except BRtL-3.

Indole-3-acetic acid production by endophytic bacteria
Production of IAA by the isolates was examined as it is an important trait of plant growth promotion by the endophytic bacteria. In the presence of tryptophan, isolated endophytic bacteria produced IAA in concentrations between 6.3 and 63 μg/mL. The highest IAA was produced by isolate BRtL-3 (63 μg/mL) followed by BDL-1 (60.8 μg/mL), B. amyloliquefaciens BDR-2 (58.6 μg/mL) and BBoS-1 (52.3 μg/mL). However, the lowest IAA production (6.3 μg/mL) was found in B. subtilis BRtL-2. IAAs produced by BDR-1 and BCL-1 were 8.6 and 12.6 μg/mL, respectively. These results suggest that isolated endophytes remarkably varied in IAA production, and thus opportunity does exist to further explore for higher IAA-producing isolates. However, these results also suggest that endophytes with higher IAA production capacity may not show higher antagonistic activity against phytopathogens. Thus, screening of endophytes should consider all relevant criteria for selecting isolates.

In vitro interactions between Bacillus spp. and S. sclerotiorum
In a dual culture assay, hyphal growth of S. sclerotiorum toward the colonies of B. amyloliquifaciens BDR-2, B. subtilis BRtL-2 and BCL-1 was remarkably inhibited compared with the untreated control plate ( Figure 1A-D). Clear inhibition zones formed on the agar medium due to the interaction between antagonistic bacteria and S. sclerotiorum. The largest inhibition of radial growth was recorded in BCL-1 (69.1%) followed by B. amyloliquefaciens BDR-2 (66.7%) and B. subtilis BRtL-2 (54.7%). The percentages of hyphal growth inhibition by the bacterial endophytes were statistically different from each other as well as the untreated control plates (Table 1). No sclerotia were produced in plates treated with bacterial endophytes, whereas sclerotia were plentiful in the control plates. Distinct morphological alterations such as irregular and excessive branching, abnormal swelling of hyphal diameter, unusually long and pointed hyphal tips and hyphal lysis of S. sclerotiorum were observed microscopically in approaching hyphae toward the colonies of bacterial endophytes (BCL-1, BRtL-2 and BDR-2) ( Figure 1B-D). Normal polar growth of hyphae was observed under a microscope in the untreated control plates ( Figure 1A). Interactions with B. subtilis BRtL-2 caused disruption of normal radial growth of hyphae by inducing excessive branching, curling, swelling and pointed hyphae ( Figure 1B). Stunted mycelial growth with swelling, pointed tips and excessive branching was observed in S. sclerotiorum due to interaction with endophytic bacterial strain BCL-1 ( Figure 1C). The interaction of S. sclerotiorum with B. amyloliquifaciens BDR-2 resulted in hyper-branching, curly growth and broke down of hyphae ( Figure 1D). Although excessive branching and loss of polar growth in S. sclerotiorum were common, each strain in addition induced characteristic morphological alterations, indicating production of diverse inhibitory compounds by the bacterial antagonists.

Reduction of mycelial dry weight of S. sclerotiorum by the endophytic bacteria
Mycelial growth of S. sclerotiorum was remarkably affected by three bacterial antagonists in the PDB medium when bacterial isolates were added to the conical flasks containing 3 days' old culture of S. sclerotiorum (Table 1). After 10 days of incubation, the highest S. sclerotiorum mycelial dry weight (3.3 ± 0.01 g) was found in non-inoculated control flasks among all the treatments. The mycelial dry weight of S. sclerotiorum was remarkably decreased in the presence of Bacillus spp. The lowest mycelial dry weight (1.8 g) was found in a B. amyloliquifaciens BDR-2-treated flask followed by 1.8 and 2.4 g in B. subtilis BRtL-2-and BCL-1-treated flasks, respectively (Table 1).

Effect of endophytic bacteria on seed germination and vigor index of Cucumis sativus
All treatments improved seed germination and vigor index of cucumber seedlings compared to untreated control. Hundred percent germination of cucumber seeds was obtained in all bacterial isolates used in seed treatment except BBoS-1-treated seeds (98.3 ± 2.8%). Lowest germination percentage (96.7 ± 2.8%) was recorded in untreated control ( Table 2). Seedlings obtained from endophytetreated seeds showed an enhanced plant vigor index compared to untreated control. Treatment with BRtL-3 showed the highest vigor index of (871.6 ± 40), which was statistically similar to other treatments except BDR-1 (515.7 ± 25) but far higher than the untreated control (314.8 ± 30) ( Table 2). Mean values within the same column followed by different letters are significantly different by Duncan's Multiple Range Test (DMRT) at p = 0.05. Assessment of germination and vigor index was done at day 9 after placing treated seeds for germination inside petri dishes with moist paper towels. Data presented here are mean values ± SE of at least three replications. Mean values within the same column followed by different letters are significantly different by Duncan's Multiple Range Test (DMRT) at p = 0.05. Data presented here are mean value ± SE of at least three replications. a In vitro suppression of mycelial growth and sclerotia production of S. sclerotiorum. b Mycelial dry weight of S. sclerotiorum was measured after adding bacterial inocula in conical flasks and incubation for 10 days.

Effect of endophytic bacteria on shoot and root growth of C. sativus
The isolated endophytic bacteria had positive effects on growth and dry matter production of cucumber seedlings compared to untreated control ( Table 2). The highest shoot length (5.2 ± 0.2 cm) was found in seedlings obtained from seeds treated with BBoS-1, which was statistically similar to the effects of B. subtilis BRtL-2 (4.7 ± 0.2 cm). The highest root length was recorded in BCL-1-treated (5.3 ± 0.5 cm) seedlings. The lowest shoot and root lengths were found in non-inoculated control (0.5 ± 0.0 and 2.7 ± 0.8 cm, respectively) seedlings, which were statistically lower than those from all other treatments. The highest shoot fresh weight of cucumber seedlings was found in BBoS-1 (159.1 ± 1.9 mg) and the lowest was recorded in control (33.2 ± 0.6 mg), which was statistically different from the rest of the treatments ( Table 2). The highest root fresh weight was found in cucumber seedlings obtained from seeds treated with B. amyloliquifaciens BDR-2 (36.5 ± 1.7 mg). The lowest root fresh weight was found in untreated control seedlings (16.5 ± 2.5 mg), which was statistically different from all other treated seedlings. The seedlings obtained from seeds treated with BRtL-3 had the highest shoot (19.8 ± 0.7 mg) and root dry weight (0.3 ± 0.1 mg), respectively, whereas untreated control had 8.4 ± 0.5 and 0.1 ± 0.1 mg/seedling dry weight, respectively.

Effect of endophytic bacteria on germination, shoot and root growth of rice
Similar to cucumber seedlings, endophytic bacteria remarkably enhanced shoot and root growth of rice seedlings ( Figure 2). Rice seeds treated with three selected bacterial isolates, B. subtilis strain BRtL-2, BBoS-1 and BCL-1, had 100% germination. Root and shoot lengths were significantly enhanced by these three bacterial isolates compared to untreated control ( Figure 2). The seedlings obtained from rice seeds treated with B. subtilis strain BRtL-2 had the highest shoot length (4.7 ± 0.6 cm) followed by BBoS-1 (4.0 ± 0.3 cm) and BCL-1 (3.9 ± 0.3 cm) (Figure 2A). The lowest shoot length was recorded in untreated control (2.9 ± 0.2 cm). A significantly longer (5.0 ± 0.6 cm) root system was found in seedlings of B. subtilis BRtL-2-treated seeds compared to untreated control (2.8 ± 0.2 cm), which was the lowest.

Discussion
The use of synthetic chemicals as fertilizers and pesticides not only increases the production cost of crop but also causes deleterious effects on the environment and health of humans and other organisms in the ecosystem. The application of novel strains of endophytic bacteria has been found as natural alternatives to synthetic agrochemicals and an essential component of sustainable agriculture [3,9]. In the present study, we isolated and characterized seven plant growth-promoting endophytic bacteria from a few traditional medicinal plants of Bangladesh. Out of total 16 isolated and screened endophytic bacteria, three, namely BCL-1, BDR-2 and BRtL-2, significantly inhibited growth and sclerotia production of the phytopathogenic fungus S. sclerotiorum (Table 1; Figure 1). Seven isolates significantly (p ≤ 0.05) enhanced germination of seeds, increased vigor index of seedling and promoted growth of cucumber and rice compared to untreated control (Table 2; Figure 2). Two potent plant growth-promoting endophytes were identified as B. subtilis BRtL-2 and B. amyloliquefaciens BDR-2. Promotion of plant growth and suppression of phytopathogens by endophytic Bacilli have previously been reported [29][30][31]. Both B. subtilis and B. amyloliquefaciens have been commercialized in many countries as biological (biocontrol and biostimulant) alternatives to synthetic pesticides [32]. To the best of our knowledge, this is the first report of isolation and characterization of plant growth-promoting and antagonistic endophytic bacteria under Bacillus genus from traditional herbal medicinal plants of Bangladesh.
One of the important findings of the current study is that the three isolates, namely BCL-1, BDR-2 and BRtL-2, significantly inhibited growth of hyphae (55%-69%) and production of sclerotia of an economically important fungal pathogen S. sclerotiorum (Table 1; Figure 1), and two out of these three isolates belonged to Bacillus spp., as revealed by 16S rRNA gene sequencing. Inhibition of hyphal growth of various fungi including S. sclerotiorum by several strains of B. subtilis and B. amyloliquifaciens has previously been reported [17,29,[33][34][35]. Isolate BRD-2 also inhibited about 68% radial growth of rice sheath blight fungus Rhizoctonia solani (Sultana et al. unpublished personal communication). The production of diverse classes of secondary metabolites and lytic enzymes is considered as a chemical weapon of Bacillus species against phytopathogens [9,13,17,34]. Another interesting finding of this study was characteristic morphological alterations in hyphae of S. sclerotiorum during interactions with B. subtilis BRtL-2, B. amyloliquifaciens BDR-2 and BCL-1, indicating production of metabolites involved with antibiosis [5,17]. Suppression of wheat blast fungus Magnaporthe oryzae Triticum through antibiosis by plant endophytic bacteria has recently been reported [14]. Our findings suggest that BDR-2 and BRtL-2 isolated from the native medicinal plants could be used as biocontrol agents against major phytopathogens in rice and cucumber. Strains of endophytic bacteria from locally adapted plant species may have higher potential to survive and multiply when used as biostimulant or biopesticide through augmentative inoculation. Further study is needed to isolate and characterize the bioactive metabolites from these Bacillus species.
A total of seven endophytic bacteria (BRtL-2, BDR-2, BCL-1, BDL-1, BDR-1, BRtL-3 and BBoS-1) isolated in this study significantly increased seed germination, and enhanced vigor and growth of cucumber seedlings compared to untreated control (Table 2). Similarly, three isolates (BCL-1, BBoS-1 and B. subtilis strain BRtL-2) also enhanced shoot and root development in rice seedlings ( Figure 2). All seven strains produced high amounts of IAA. These findings suggest that the production of IAA by these bacterial endophytes is linked with growth promotion of cucumber and rice seedlings although a quantitative relationship of IAA amount and growth promotion could not be established. Furthermore, we found that B. amyloliquefaciens strain BDR-2 remarkably promoted growth and increased grain yield of rice up to 21% in field trial under varying doses of fertilizer application to the soil (Sultana et al. unpublished personal communication). These results indicate that endophytic Bacillus spp. from medicinal plants can colonize and promote the growth of unrelated host plants. High plant colonization and promotion of growth of rice by the endophytic bacteria isolated from unrelated host willow have been reported [36]. Beneficial bacteria with potential to provide growth and yield enhancement of multiple crops and suppression of harmful microbes are major candidates for biological pesticides to be used for sustainable agriculture. The enhancement of plant growth by IAA-producing bacteria including members of Bacillus genus has previously been reported [3,37,38]. Synthesis of phytohormones triggers the activity of specific enzymes (e.g. α-amylase), which promote early germination and increase the availability of starch assimilation [39].
In this study, we isolated and partially characterized some plant growth-promoting endophytic bacteria from medicinal plants of Bangladesh. Two potential isolates were identified as B. subtilis BRtL-2 and B. amyloliquifaciens BDR-2 by 16S rRNA gene sequencing. These isolates significantly suppressed mycelial growth and sclerotia production of a phytopathogenic fungus S. sclerotiorum. In addition, cucumber and rice seed treatment with these endophytic bacteria significantly enhanced the percentage of seed germination and plant vigor likely due to the higher amount of IAA production. These findings indicate that two Bacillus spp. isolated from local medicinal plants can be used as biostimulant and biocontrol agents for sustainable production of cucumber and rice in an ecofriendly manner. Further studies are needed to determine the modes of action of these medicinal plant endophytes and also to test their efficacy as biostimulant and biocontrol agents in field conditions.