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Article

Use of Beauveria bassiana and Bacillus amyloliquefaciens Strains as Gossypium hirsutum Seed Coatings: Evaluation of the Bioinsecticidal and Biostimulant Effects in Semi-Field Conditions

by
Vasileios Papantzikos
1,
Spiridon Mantzoukas
1,*,
Alexandra Koutsompina
1,
Evangelia M. Karali
1,
Panagiotis A. Eliopoulos
2,
Dimitrios Servis
3,
Stergios Bitivanos
3 and
George Patakioutas
1
1
Department of Agriculture, Arta Campus, University of Ioannina, 45100 Ioannina, Greece
2
Laboratory of Plant Health Management, Department of Agrotechnology, University of Thessaly, Geopolis, 41500 Larissa, Greece
3
BASF Hellas S.A., 15125 Marousi, Greece
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(10), 2335; https://doi.org/10.3390/agronomy14102335
Submission received: 28 August 2024 / Revised: 6 October 2024 / Accepted: 9 October 2024 / Published: 10 October 2024
(This article belongs to the Special Issue Pests, Pesticides, Pollinators and Sustainable Farming)
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Abstract

:
There are many challenges in cotton cultivation, which are mainly linked to management practices and market demands. The textile commerce requirements are increasing but the effects of climate change on cotton cultivation are becoming an issue, as its commercial development depends significantly on the availability of favorable climatic parameters and the absence of insect pests. In this research, it was studied whether the use of two commercial strains as cotton seed coatings could effectively contribute to the previous obstacles. The experiment was carried out in semi-field conditions at the University of Ioannina. It used a completely randomized design and lasted for 150 days. The following treatments were tested: (a) coated seeds with a commercial strain of Beauveria bassiana (Velifer®); (b) coated seeds with a combination of Velifer® and a commercial strain of Beauveria bassiana (Selifer®); and (c) uncoated cotton seeds (control). The biostimulant effect of the two seed coatings was assessed against the growth characteristics of cotton, and the total chlorophyll and proline content. The bioinsecticidal effect was evaluated by measuring the population of Aphis gossypii on the cotton leaves. The proline effect increased by 15% in the treated plants, whereas the total chlorophyll was higher in the use of both Velifer® and Velifer®–Selifer® treatments by 32% and 19%, respectively. Aphid populations also decreased in the treated plants compared to the control plants (29.9% in Velifer® and 22.4% in Velifer®–Selifer®). Based on an assessment of the above parameters, it follows that the two seed coatings can significantly enhance the growth performance of cotton and reduce the abundance of A. gossypii.

1. Introduction

Climate change is significantly pressuring cotton cultivation [1], affecting various essential factors for the growth and production of the cotton plant [2,3]. There is a plethora of issues arising due to climate change in cotton cultivation, which are linked to the need for a continuous supply of cotton to the market [4]. One of these factors is the increase in average temperatures, which affects the range of the cotton growing seasons [4], causing problems such as heat stress [5]. Climate change is often associated with changes in the distribution of rain [6], but high water requirements are crucial for cotton as extended periods of drought can affect cotton growth and quality [7]. This can lead to a significant reduction in cotton production [8]. In addition, rising temperatures and changes in the environment may affect the type and life cycle of pathogens and pests [9], thereby affecting the need for pesticides.
The cotton aphid Aphis gossypii (Hemiptera: Aphididae) is an insect that affects cotton production in several ways. It is a serious pest and can reduce cotton production [10] by sucking plant juices and consuming important cotton nutrients [11], which can cause severe damage, especially under drought stress [12]. This can lead to nutrient losses and can affect cotton growth and production [13]. It can also reduce the cotton’s chlorophyll content [14], affecting photosynthesis and normal growth [15], and causing stress due to juice removal [16]. In addition, they can act as disease vectors [17]: while feeding, insects may sometimes transmit viruses and other pathogens to the plants [18], exacerbating cotton health problems [19]. Aphis gossypii secretions can cause the growth of the multi-phytopathogenic fungi, Ascomycetes (Sooty mold), which coats the leaves and blocks sunlight, resulting in low photosynthetic quality [20,21]. The aphid may have developed resistance to some pesticides [20,22,23], making its control an issue for cotton. To deal with the cotton aphid, farmers use a variety of control techniques, including the use of pesticides [24] and the use of natural enemies, such as some Coccinellidae species [25,26]. Also, genetic improvement may play a role in creating cotton varieties that are resistant to the aphid [27].
The above biotic and abiotic issues are directly related to the quality of cotton production and are directly linked to the constant market pressure for high-quality cotton [11,28,29,30]. Research on microorganisms that have a biostimulant [31] and/or a bioinsecticidal effect [32] enhancing cotton growth could be a sustainable way to address these issues [33].
The term beneficial microorganisms describes those that can live part of their life with certain plant species in a non-parasitic association [34], without adverse effects on plant growth. This type of symbiotic relationship confers many advantages on both sides and greatly benefits the plant’s metabolism [35]. Some microorganisms present biostimulant prospects, as they have been reported to enhance the growth of cotton plants [31]. Microorganisms such as Metarhizium ssp., transport nutrients and enhance the metabolism of cotton plants [36]. Also, some may reveal bioinsecticidal ability, while simultaneously strengthening the induced systemic resistance (ISR) of plants to pathogenic microorganisms [37].
Seed coatings are an eco-friendly alternative to the adverse effects that cotton cultivation suffers from. In recent years, several organic acid mixtures have been cited as seed coatings, such as proline, glutamate and citric acid [38]. Coating seeds with microorganisms that may have biostimulant and bioinsecticidal properties is an upcoming research field in dealing with biotic and abiotic factors that place stress on cotton cultivation [37,39]. Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) is an endophytic entomopathogenic fungus (EPF) [40,41,42], which is widely used in integrated pest management (IPM) programs. Its biostimulant effect on cotton cultivation when used as a seed inoculant has been reported [43], as have its improvements in total plant length and biomass [44] in several plant species. At the same time, it exhibits significant entomopathogenic activity against pests such as aphids [45]. The plant-growth-promoting rhizobacterium (PGPR), Bacillus amyloliquefaciens (Bacillales: Bacillaceae), is an equally interesting beneficial microorganism in the biological control of many pests for a plethora of crops, including cotton [33,46]. The biostimulant aptitude of B. amyloliquefaciens derives from its use as a growth enhancer [47]. Moreover, it has been observed to induce lignin synthesis in cotton seeds [48].
In the present study, we evaluated the biostimulant and bioinsecticidal potential of two seed coatings–one with B. bassiana and one with B. amyloliquefaciens + B. bassiana–on cotton growth characteristics and the biological control of A. gossypii nymphs.

2. Materials and Methods

2.1. Experimental Design

The experiment was performed in semi-field conditions, in a field at the agriculture faculty of the University of Ioannina, in Kostakioi Arta. The biostimulant and the bioinsecticidal effect of two commercial formulations were tested when used as cotton seed coatings in the experiment: B. bassiana, strain PPRI 5339 Velifer® OD (8 × 109 CFU/mL, 92% Excipients), and B. amyloliquefaciens, strain MBI600 Serifel® WP (5.5 × 1010 CFU/g, 95% Excipients) (BASF SE, Florham Park, NJ, USA). Two seed coating treatments were applied in the experiment: a treatment with Velifer formulation (V) and a treatment with Velifer and Serifel combined (VS). Conventional uncoated cottonseed (C) was used as a control.

2.2. Protocol for Coating Seeds with B. bassiana and B. amyloliquefaciens

Using dispensers and pipettes, the treatment solutions were prepared as the desired combined treatments. The VS treatment was prepared by adding 125 mL of formulated B. bassiana/100 Kg cottonseed and 160 mL of formulated B. amyloliquefaciens/100 Kg. The strains B. bassiana PPRI 5339 Velifer® OD and the B. amyloliquefaciens strain MBI600 Serifel® WP (BASF SE, Florham Park, NJ, USA) were added by pipette. The V treatment was prepared using 125 mL of formulated B. bassiana/100 Kg cottonseed. The required amount of water was added through the dispenser. Next, the separation of the seed samples was carried out using a precision balance (KERN PES 6200-2M), and, through the use of Wintersteiger Hege 11, the final application of the coating to the seeds was made. The seed sample was then placed in a stainless steel bucket, where stirring began. Finally, using the Eppendorf pipette, an appropriate amount of seed treatment solution was applied. The application time was ~1 min/treatment/sample. In order for the treated seeds to dry completely, they were emptied into a special sample bag, which was left open. The equipment used to apply the treatment solutions and the application bucket were carefully cleaned with ddH2O when rotating the treatments. The fungal spore concentration in the control treatment was zero.

2.3. Experimental Set-Up

A mixture of peat and perlite in 1:1 ratio (v/v) and in in 9 L pots was used as a growth substrate for the cotton plants (var. Olivia). The experiment was conducted in a completely randomized design. The beginning point (day 0) was 19 May 2023, and the last experimental day was 16 October 2023 (day 150). Each treatment consisted of 21 cotton seeds, each placed in a pot filled with growth substrate. The pots were irrigated daily through a drip system (ARGOS Electronics 2014), automatically controlled by a computer. Irrigation quantity and frequency were based on climatic data taken from temperature and humidity sensors. In addition, to further control the irrigation adequacy, frequent measurements of the relative pot’s moisture were made using a soil moisture meter (ΔΤ-SM150 Kit, Delta T Devices, Cambridge, UK). Initial fertilization was applied to each pot with an N30-P10-K10 fertilizer, which was repeated after 50 days.

2.4. Recording of A. gossypii Population and Cotton Growth Characteristics

The natural presence of A. gossypii on cotton leaves was systematically recorded on a weekly basis from the beginning of the experiment. Cotton growth characteristics such as total plant length (cm); central shoot diameter (mm); and the total number of shoots, internodes, leaves, and cotton bolls were measured. Leaf area (cm2) was calculated from an image analysis of the leaves, using the Image j protocol [49]. At the end of the experiment (day 150), the total plant biomass was measured by dividing each plant into roots, shoots, leaves and seedcotton, taking the values of the fresh weight (g) of each of the previous categories for each plant separately. Following this, dry biomass (g) was recorded after 72 h of 80 °C oven-drying.

2.5. Total Chlorophyll Content

Measurement of the total chlorophyll (TCHL) content in the cotton leaves was carried out weekly during the experiment in a non-destructive way, using the SPAD (Minolta Co., Ltd., Tokyo, Japan) instrument method. For the accuracy of the method, the linear correlation of the SPAD method with the chemical method of chlorophyll determination was carried out (R2 = 0.901) in randomly selected cotton leaf samples, according to the protocol of [50], with some modifications: 10 mL of acetone was used as the extraction solvent of 0.04 g cotton leaf tissue, which was crushed in a porcelain mortar with a pestle. Each sample was placed in a 10 mL glass tube, vortexed, and left overnight at 4 °C. The absorbance was measured in a spectrophotometer (Jasco-V630 UV-VIS, JASCO INTERNATIONAL Co., Ltd., Tokyo, Japan), using the equations described by Lichtenthaler and Buschmann [51], and the result was expressed in g of TCHL of fresh leaf per cm2 of cotton leaf area:
Ca (μg/mL) = 11.24 × A661.6 − 2.04 × A644.8
Cb (μg/mL) = 20.13 × A644.8 − 4.19 × A661.6

2.6. Proline Content

The total proline content was determined to assess the cotton plants’ stress from the environmental conditions according to [52] with some modifications: 4 mL of extractant solution containing 70% ethanol was poured into a mortar containing 0.1 g of fresh cotton leaf plant tissue and was crushed with a pestle until it became a homogeneous mix. The samples were added to glass tubes, which were centrifuged at 4000 g, for 10 min. In a new set of tubes, 1 mL of supernatant extract and 2 mL of fresh acid–ninhydrin solution were placed. Then the samples were vortexed and incubated in a dark water bath at 95 °C for 25 min and the reaction mixture was cooled directly in an ice bath. When at room temperature, the samples were centrifuged for 5 min at 4000 g. The absorbance was determined at 520 nm in a spectrophotometer (Jasco-V630 UV-VIS). The results were reported in μmol of proline, g−1 of fresh cotton leaf weight.

2.7. Statistical Analysis

One-way ANOVA was performed with Tukey’s post hoc test to compare the means of the treatments for the effect on plant growth, proline, total chlorophyll content (TCHL), and cotton fresh and dry mass. Two-way ANOVA was performed for the Insect Population with two variables: population and time. All statistical analyses were conducted using SPSS v. 25 (IBM-SPSS Statistics, Armonk, NY, USA).

3. Results

3.1. Insect Pest Population

The average population of A. gossypii nymphs per treatment in the cotton crop was recorded. More specifically, in all seven samplings, the variation in the average number of A. gossypii nymphs was statistically significant among the treatments (F = 11.881, df = 2.511, p < 0.001). Forty days after treatment, the average number of aphids was significantly higher in the control samples than in the treated samples (Figure 1). The average change in the aphid population at the maximum, 56 days, was 12.38 ± 1.35 aphids for the V plants and 13.71 ± 0.38 aphids for the VS plants. In the control plants, the aphid population was 17.68 ± 0.92 aphids (Figure 1). Fewer aphids were almost always counted on the plants treated with V than on the plants inoculated with other strains.

3.2. Effect on Plant Growth

The evaluation of the morphological features of the tested plants was based on a recording of the plants’ length, shoots, internodes, the number of leaves and cotton bolls, and the stem diameter. In general, V plants and VS plants resulted in better growth than the untreated plants. V plants and VS plants, after 150 days, had statistically more length compared to the control plants (F = 9.553, df = 2, p < 0.001) (Figure 2). For the shoots, all the plants, treated and untreated, had the same average growth (F = 1.523, df = 2, p = 0.951) (Figure 3).
The V plants and VS plants caused an increase in the number of internodes (F = 6.759, df = 2, p = 0.011) (Figure 4) and cotton bolls (F = 8.759, df = 2, p = 0.009) (Figure 5). The leaf areas (cm2) after 150 days were as follows: for the control, 241.15 ± 53.55; for the Velifer treatment, 372.70 ± 44.98; and for the Velifer–Serifel treatment, 397.50 ± 45.81 (F = 11.553, df = 2, p < 0.001). The differences proved to be not statistically significant for the number of leaves: F = 2.159, df = 2, p = 0.870 (Figure 6). A similar increase (not statistically significant) was recorded for the stem diameter (F = 3.111, df = 2, p = 0.811) (Figure 7).

3.3. Effect on Proline and Total Chlorophyll Content (TCHL)

The effect on the proline was found to be not statistically significant in all the plants by the end of the experiment (F = 2.197, df = 2, p = 0.790): after 150 days, the proline was the same in all the plants (Figure 8).
The chlorophyll concentration increased in the V and VS plants after 30 days and remained above that of the control plants until the end of the experiment (F = 13.220, df = 2, p < 0.001) (Figure 9). The increase in TCHL was attributed to the endophytic effect on the leaves. This effect was especially evident at 56 days due to the low infestation of aphids and high TCHL values for the V plants. The decrease in the TCHL values after 128 days was expected due to the leaf maturation.

3.4. Cotton Fresh and Dry Mass

The fresh and dry mass measured at the final harvest are summarized in Figure 10A and Figure 10B, respectively. The V treatment significantly increased the total fresh mass (F = 34.198, df = 2, p < 0.001) and dry mass (F = 25.811, df = 2, p < 0.001) of the above-ground part of the cotton plants. The V and VS plants showed significantly increased fresh mass in the roots (F = 24.308, df = 2, p < 0.001), and the V treatment significantly increased the roots’ dry mass (F = 19.398, df = 2, p < 0.001). The V and VS plants significantly increased the shoots’ fresh mass (F = 14.118, df = 2, p < 0.001) and their dry mass (F = 18.128, df = 2, p < 0.001). Also, in the case of leaf weight, the V and VS treatments increased the fresh mass (F = 17.228, df = 2, p < 0.001). The leaves’ dry mass increased only with the V treatment (F = 8.118, df = 2, p < 0.001). Finally, for seedcotton, we did not find a significant difference in the fresh (F = 2.118, df = 2, p = 0.964) or the dry mass (F = 1.918, df = 2, p = 0.916).

4. Discussion

Some EPFs and PGPRs can be used as seed dressings for a wide range of plant species [37,53,54]. Cottonseed arouses an interest in experimentation on such microorganisms as seed coatings in order to improve the plant’s metabolism, as it is a high-demand crop. Due to its increased use for commercial purposes [55], it is important to find eco-friendly solutions to combat its insect pests, such as A. gossypii. In this study, the bioinsecticidal activity of fungi and bacteria coatings was demonstrated by the significant reduction in the A. gossypii population on the cotton plants. This outcome conforms with our last work [56], where the inoculated cottonseed coatings (with the same B. bassiana strain) was effective in minimizing the A. gossypii population. The coating with B. bassiana showed a higher bioinsecticidal effect, with a longer duration than its co-inoculated application with B. amyloliquefaciens. In field conditions, a B. bassiana coating on maize Zea mays (Poales: Poaceae) seed has shown bioinsecticidal action on Spodoptera frugiperda (Lepidoptera: Noctuidae) [57], with no negative effects on beneficial insects, such as honeybees. A similar result was presented in the study by Mishra et al., 2013 [58], where a B. bassiana formulated strain was effective on seeds via encapsulation, against Musa domestica (Diptera: Muscidae).
In this study, the application of seed coatings using B. Bassiana (V) and the combined application of B. Bassiana and B. amyloliquefaciens (VS), demonstrated a beneficial effect on cotton’s growth and metabolism, and the same effect has been found in other crops [59,60,61,62]. The growth characteristics of cotton, such as fresh and dry biomass, were increased in the coated treatments, especially in the case of B. bassiana. This result has also been observed in the work of [41], where the fresh weight of the roots and shoots of coated Vicia faba (Fabales: Fabaceae) seeds was increased. Moreover, in the work of Sánchez-Rodríguez et al., 2018 [63], endophytic B. bassiana, used as a seed dressing, increased the dry weight and total grain weight of bread wheat, Triticum aestivum (Poales: Poaceae). It also effectively controlled cotton leafworm larvae, Spodoptera littoralis (Lepidoptera: Noctuidae). The number of internodes was higher than on the untreated cotton seed. An equivalent result was exhibited in the study by Canassa et al., 2019 [39], where coated bean seeds (with B. Bassiana) strengthened plant growth. A similar effect on the growth of Z. mays seeds coated with B. bassiana was also observed in the study of Rivas-Franco et al., 2019 [64], in which a significant increase in the vegetation length was noted, something that was not observed to a significant extent in this experiment.
The use of B. bassiana as a seed coating for Phaseolus vulgaris L. (Fabales: Fabaceae) showed an increased number of leaves [46], while in other studies, an enlarged leaf area was observed [57]. A similar occurrence was noted in this study regarding treatment with V. The number of cotton bolls were also greater when treated with V. This has been observed in other B. bassiana application experiments [65]. In a lot of studies, the beneficial impact of seed coatings has been mentioned, regarding several metabolic traits [31,66]. The higher the amount of chlorophyll in the leaves, the more beneficial it is for the plant [67] because it is involved in various metabolic processes that relate to enhancing its robustness. The single-inoculated treatment containing B. bassiana had a higher performance on chlorophyll, compared to the co-inoculated treatment with B. bassiana + B. amyloliquefaciens. Of course, both the coating treatments showed a more significant effect compared to the uncoated seeds. A similar effect of B. bassiana has been observed on rice, Oryza sativa (Poales: Poaceae) [68]; on barley, Hordeum vulgare (Poales: Poaceae) [69]; on chili, Capsicum annuum L. (Solanales: Solanaceae) [70]; and on cucumber, Cucumis sativus L. (Cucurbitales: Cucurbitaceae) [71]. However, the low chlorophyll in the control treatment may also be related to the fact that the sucking damage caused by A. gossypii was higher.
Proline is an indicator of abiotic stress [72] and has been detected as enriched in some B. Bassiana application experiments [59], which contrasts with our experiment, where proline levels were not affected, as the same levels were presented in all treatments.
The above led us to ascertain that the coating composition using B. bassiana had a biostimulant effect due to the enhancement of growth characteristics and chlorophyll in the cotton crop. On the contrary, we could not identify the same strong biostimulant and bioinsecticidal properties in the co-inoculation treatment (VS), as this did not result in the same vigorous impact in all growth parameters. This may be attributed to the antagonistic interactions that evolved between the two microorganisms [73,74]. However, in the work of Prabhukarthikeyan et al., 2014, the combined application of Bacillus ssp. and Bassiana ssp. was effective in combating Fusarium oxysporum f., sp., lycopersici’s (Hypocreales: Nectriaceae) wilt and the fruit borer Helicoverpa armigera (Lepidoptera: Noctuidae) in tomato plants, Solanum lycopersicum (Solanales: Solanaceae) [75]. This was achieved without competing to the detriment of the plant’s metabolism. The application of Beauveria and Bacillus strains in a simultaneous mixture has been referred to as highly effective in the control of greenhouse whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) [76]; red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) [77]; and tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae) [78]. However, this performance was not repeated in our results. In addition, in a study by Wang et al., 2015 [79], co-inoculated plants were weaker than plants with individual inoculations, but no harmful effects on their growth were observed. This fact has also been reflected in other studies [80]. Quantitative PCR studies have shown that the presence of beneficial bacteria within plant tissues suppressed fungal colonization. This may explain the reduction in biomass of the co-inoculated plants, compared to the single-inoculated plants. Two potential ways could lead to the observed decrease in fungal colonization mentioned in this context. Firstly, the decline in fungal population density might be caused directly by certain substances, which are produced by bacteria that inhibit fungal growth. Research has demonstrated that alkaloids from beneficial microorganisms and the defensive chemicals produced by the host in response to endophytic invasion can reduce the colonization of fungi [81,82]. Secondly, the coexistence of symbiotic microorganism reforms how the host distributes its resources, consequently, impacting fungal colonization [83,84]. The aforementioned concept of antagonism between B. bassiana and B. amyloliquefaciens in the co-inoculated coated treatment (VS) also derives from the study of metabolic parameters, such as chlorophyll, which was higher in the treatment with B. bassiana (V) than in the co-inoculated treatment. Moreover, a similar effect was presented by the reduced population of A. gossypii in the same treatment. However, this hypothesis needs further study to define the interactions between the two microorganisms: initially, by studying the degree of their interaction during the endophytic growth of the co-inoculated coated cotton seed and, next, establishing by which factors the synergism and viability of each strain can be affected.
The typical functioning and development of a plant is generally unaffected by symbiotic microorganisms [85]. Nevertheless, EPF and PGPR may occasionally boost the host’s ability to withstand challenging environmental factors, such as drought [86] and lack of nutrients [87], or fortify the host’s defenses against pests [88,89,90]. For instance, due to EPF, plants amplify their resistance to insect feeding damage and encounter fewer biomass losses [91].

5. Conclusions

In our experiment, the seed coatings created by EPF and PGPR are of interest when focusing on their application in IPM cotton cultivation programs. The cotton seed coating treatment with B. bassiana and the co-inoculation treatment with B. amyloliquefaciens and B. bassiana showed bioinsecticidal properties, reducing the population of the cotton aphid, A. gossypii, for a long time. The application of B. bassiana enhanced the growth of cotton in several parameters, such as the total fresh and dry biomass. In addition, the larger leaf area and the higher amount of TCHL in the leaves were shown to be an effect of B. bassiana and its co-inoculation with B. amyloliquefaciens, which improved the robustness of the cotton crop. In addition, the results of our research suggest that the combined application of the two microorganisms was not as successful as the single inoculation treatment with B. bassiana. The data present, once again, the biostimulant and bioinsecticidal effect of beneficial EPF and PGPR on the growth and metabolic traits of cotton. In order to shed light on the potential antagonistic interactions between the two microorganisms, it would be relevant to explore the precise mode of action of the endophytic coatings that co-inoculate EPF B. bassiana and PGPR B. amyloliquefaciens. Our research on utilizing EPF and PGPR bacteria as seed coatings for commercially important plant species has provided viable pathways towards reducing the excessive use of agrochemicals, while boosting the metabolic resilience of plants. Considering these promising opportunities, more research must be conducted on the use of endophytic seed coatings containing B. amyloliquefaciens and B. bassiana in cotton production under realistic circumstances. The positive results of our research highlight the significance of these initiatives.

Author Contributions

Conceptualization, V.P. and S.M.; methodology, V.P.; software, V.P. and S.M.; validation, V.P., S.M., A.K., E.M.K., P.A.E., D.S., S.B. and G.P.; formal analysis, V.P., A.K. and E.M.K.; investigation, V.P., A.K. and E.M.K.; resources, V.P. and S.M.; data curation, V.P., S.M. and P.A.E.; writing—original draft preparation, V.P.; writing—review and editing, V.P., S.M., P.A.E., D.S. and S.B.; visualization, V.P. and S.M.; supervision, V.P., SM. and G.P.; project administration, V.P., S.M. and G.P. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that this study received funding from the University of Ioannina (grant number 61392). The funder had the following involvement in the study: funding and material supplies for the experiment.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors, V.P. and S.M.

Acknowledgments

The authors gratefully acknowledge the Department of Agriculture of the University of Ioannina for providing the necessary facilities to carry out the experiments.

Conflicts of Interest

Authors Dimitrios Servis and Stergios Bitivanos were employed by the company BASF Hellas S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Agronomy 14 02335 g001
Figure 1. The mean number of A. gossipii aphids on cotton plant leaves up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control.
Figure 1. The mean number of A. gossipii aphids on cotton plant leaves up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control.
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Figure 2. The mean length (cm) of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 2. The mean length (cm) of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 3. The mean shoot number of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 3. The mean shoot number of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 4. The mean number of internodes on cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 4. The mean number of internodes on cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 5. The mean number of cotton bolls up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 5. The mean number of cotton bolls up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 6. The mean number of leaves on cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 6. The mean number of leaves on cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 7. The mean stem diameter (mm) of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer-Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 7. The mean stem diameter (mm) of cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer-Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 8. The mean proline values (μmol g−1) for cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 8. The mean proline values (μmol g−1) for cotton plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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Figure 9. The mean values of the leaves’ total chlorophyll content (μg cm−2) up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control.
Figure 9. The mean values of the leaves’ total chlorophyll content (μg cm−2) up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control.
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Figure 10. The mean fresh (A) and dry (B) weight (g) of roots, shoots, leaves, and seedcotton of G. hirsutum plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
Figure 10. The mean fresh (A) and dry (B) weight (g) of roots, shoots, leaves, and seedcotton of G. hirsutum plants up to 150 days after treatment: V—Velifer; VS—Velifer–Serifel; and C—control. Different letters among treatments indicate statistically significant differences (Tukey test, p < 0.05).
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MDPI and ACS Style

Papantzikos, V.; Mantzoukas, S.; Koutsompina, A.; Karali, E.M.; Eliopoulos, P.A.; Servis, D.; Bitivanos, S.; Patakioutas, G. Use of Beauveria bassiana and Bacillus amyloliquefaciens Strains as Gossypium hirsutum Seed Coatings: Evaluation of the Bioinsecticidal and Biostimulant Effects in Semi-Field Conditions. Agronomy 2024, 14, 2335. https://doi.org/10.3390/agronomy14102335

AMA Style

Papantzikos V, Mantzoukas S, Koutsompina A, Karali EM, Eliopoulos PA, Servis D, Bitivanos S, Patakioutas G. Use of Beauveria bassiana and Bacillus amyloliquefaciens Strains as Gossypium hirsutum Seed Coatings: Evaluation of the Bioinsecticidal and Biostimulant Effects in Semi-Field Conditions. Agronomy. 2024; 14(10):2335. https://doi.org/10.3390/agronomy14102335

Chicago/Turabian Style

Papantzikos, Vasileios, Spiridon Mantzoukas, Alexandra Koutsompina, Evangelia M. Karali, Panagiotis A. Eliopoulos, Dimitrios Servis, Stergios Bitivanos, and George Patakioutas. 2024. "Use of Beauveria bassiana and Bacillus amyloliquefaciens Strains as Gossypium hirsutum Seed Coatings: Evaluation of the Bioinsecticidal and Biostimulant Effects in Semi-Field Conditions" Agronomy 14, no. 10: 2335. https://doi.org/10.3390/agronomy14102335

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