Next Article in Journal
A Novel SPOTTED LEAF1-1 (SPL11-1) Gene Confers Resistance to Rice Blast and Bacterial Leaf Blight Diseases in Rice (Oryza sativa L.)
Previous Article in Journal
Why Do Farmers Disadopt Successful Innovations? Socio-Ecological Niches and Rice Intensification
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 14
Issue 10
10.3390/agronomy14102239
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effects of Microbial Biostimulants on Maize and Its Pest, the Western Corn Rootworm, Diabrotica virgifera virgifera

by
Sri Ita Tarigan
1,2,3,
Jozsef Kiss
1,
Turóczi György
1,
Nhu Phuong Y Doan
4 and
Stefan Toepfer
1,2,*
1
Department of Integrated Plant Protection, Institute of Plant Protection, Hungarian University of Agriculture and Life Sciences MATE, 2100 Gödöllő, Hungary
2
CABI, 2800 Delémont, Switzerland
3
Department of Agrotechnology, Universitas Kristen Wira Wacana Sumba, Waingapu 87113, Indonesia
4
Faculty of Agriculture, University of Szeged, 6800 Hódmezővásárhely, Hungary
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(10), 2239; https://doi.org/10.3390/agronomy14102239
Submission received: 24 August 2024 / Revised: 21 September 2024 / Accepted: 26 September 2024 / Published: 28 September 2024
(This article belongs to the Section Pest and Disease Management)
Browse Figures
Versions Notes

Abstract

:
The western corn rootworm, Diabrotica virgifera virgifera, (Coleoptera: Chrysomelidae) is a serious pest of maize in the USA and Europe. Microbial plant biostimulants such as bacteria, fungi, and algae are designed to stimulate plant nutrition and growth, with some hypothesized to also possess insecticidal properties. We tested 10 biostimulants (four bacteria, five fungi, and one alga) under laboratory and greenhouse conditions. Most biostimulants did not affect the eggs, larvae, or adults of D.v. virgifera. However, in the laboratory, 10% of biostimulants improved egg hatching, and 40% killed some larvae, including the fungi Beauveria bassiana, Rhizophagus irregularis, and Trichoderma asperellum, and the bacterium Ensifer meliloti. Under potted-plant conditions in the greenhouse, these insecticidal effects were not detectable. However, several biostimulants slightly increased height and shoot length of uninfested maize plants, but reduced volume and length of their roots as well as above-ground biomass. Interestingly, 30% of the biostimulants enhanced the plant’s defence against larvae, for example, Bacillus amyloliquefaciens, B. subtilis, and E. meliloti. These may warrant further research into their modes of action as well as field trials to better understand and optimize their potential use in sustainable and integrated pest management.

1. Introduction

The western corn rootworm Diabrotica virgifera virgifera LeConte is a chrysomelid beetle, which is one of the most important pests of maize (Zea mays L.) in the USA and Europe [1,2]. Its larvae feed on maize roots, which can lead to plant instability, reduced growth, and yield loss [1]. This pest has seven developmental stages: egg, three larval instars, pre-pupa, pupa, and adult. Adult females lay 300–400 eggs in the top 5–20 cm of soil among the roots of maize [3], and these eggs then go through a period of diapause. At the beginning of the cropping season, the first instar larvae hatch and burrow through the soil to search for and feed on the maize roots before metamorphosing into the second larval stage and finally into the third larval stage. Adults emerge from the soil feeding on young maize leaves, silk, and pollen. In order to control D.v. virgifera larvae, farmers primarily use crop rotation, transgenic maize, entomopathogenic nematodes such as Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) [4], and insecticides. Farmers apply either granular or, occasionally, liquid soil insecticides in the seed furrow or insecticide-coated seeds to control the insect larvae. Additionally, farmers occasionally spray insecticides against the adult insects to prevent silk feeding or to reduce egg laying and thus minimize the damage caused by the larvae in the following season. Some insecticides have recently been banned from use because of their toxicity to bees or other non-target effects. Some insecticides such as organophosphates, carbamates, pyrethroids, and neonicotinoids have encountered resistance in populations of D.v. virgifera [1,5,6,7,8,9]. As a result, farmers in many maize-growing areas are struggling to control soil insects such as corn rootworms.
Microbial plant biostimulants consist of microbes like bacteria, fungi, or algae. They improve plant nutritional processes without relying on their nutrient content, with the primary goal of enhancing plants’ agronomic performance [10]. They are usually applied on planting materials or in growing substrates and sometimes as foliar treatments. Furthermore, they are claimed to have the capacity to modify the physiological processes of a plant in a way that provides benefits to nutrient uptake or efficiency, and/or to plant growth or stress tolerance [10].
Commercial microbial biostimulants have been used in agriculture for decades, but the product diversity has recently increased. Interestingly, some of the microbial plant stimulants have been reported to have some insecticidal effects in addition to their plant-promoting properties [11]. For example, bacterial biostimulants, such as Bradyrhizobium japonicum (Hyphomicrobiales: Nitrobacteraceae) have been reported to attack insects such as Callosobrochus maculatus (Coleoptera: Chrysomelidae), a pest of cowpea grains, [12] or Phthorimaea operculella (Lepidoptera: Gelechiidae), a pest of potatoes [13]. Fungal biostimulants such as Rhizophagus irregularis (Glomerales: Glomeraceae) had negative effects on Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize [11,14]. Also, Trichoderma harzianum (Hypocreales: Hypocreaceae) had negative effects on Acanthoscelides obtectus (Coleoptera: Chrysomelidae), a pest of common beans [15] and on Nezara viridula (Hemiptera: Pentatomidae), a pest of soybean [16]. Another example is Trichoderma virens (Hypocreales: Hypocreaceae), which negatively affected Gryllotalpa gryllotalpa (Orthoptera: Gryllotalpidae), a pest of cowpeas, soybeans, and other crops [17]. Algae biostimulants rarely have effects on insects, but Chlorella vulgaris (Chlamydomonadales: Chlamydomonadaceae) had insecticidal effects on Chironomus riparius (Diptera: Chironomidae) [18] and Chlamydomonas reinhardtii (Chlamydomonadales: Chlamydomonadaceae) had effects on Aedes aegypti (Diptera: Culicidae), which are both pests of humans [19]. It is crucial to understand such effects, as biostimulants are supposed to only affect the plant.
Therefore, we assessed the potential insecticidal effects of a number of commonly used microbial plant biostimulants. We chose D.v. virgifera as the target pest as it is an economically important and well-studied soil pest, allowing the assessment of multiple effects of microbial biostimulants in the maize cropping system. We applied laboratory screenings on all life stages of the pest, as well as semi-field greenhouse trials. We hope our findings will reveal candidates of microbial biostimulants that can enhance maize growth and also possess insecticidal properties against D.v. virgifera. By determining which biostimulants are effective in controlling different life stages of this pest, we can provide farmers with more sustainable and integrated pest management strategies. This could potentially reduce the reliance on chemical insecticides, lower the risk of resistance development, and mitigate the negative environmental impacts associated with conventional pest control methods. Furthermore, our research will contribute to a broader understanding of the multifaceted roles that microbial biostimulants can play in modern agriculture, paving the way for more resilient and productive cropping systems.

2. Materials and Methods

2.1. The Target Pest Diabrotica virgifera virgifera

A non-diapause laboratory colony of D.v. virgifera was obtained from USDA-ARS laboratories (Brookings, SD, USA) where it had been reared for around 300 generations. The insects were reared under standardized laboratory conditions according to [20,21,22]. Ready-to-hatch 7-day-old eggs, first instar larvae, and adults were used for standardized bioassays under laboratory conditions, and ready-to-hatch eggs were injected into the soil of potted plants in the greenhouse experiments (see below).

2.2. Tested Microbial Biostimulants under Laboratory Conditions

In this study, ten commercially available microbial biostimulants were tested to determine their effects on D.v. virgifera life stages (eggs, larvae, and adults) under standard laboratory conditions (Table 1). These included a bacterial group of Bacillus amyloquafaciens and Bacillus subtilis (both Bacillales: Bacillaceae), Bradyrhizobium japonicum (Hyphomicrobiales: Nitrobacteraceae), Ensifer meliloti and Rhizobium leguminosarum (both Hyphomicrobiales: Rhizobiaceae), a fungal group of Trichoderma asperellum and Trichoderma harzianum (both Hypocreales: Hypocreaceae), Beauveria bassiana (Hypocreales: Cordycipitaceae), and Rhizophagus irregularis (Glomerales: Glomeraceae) as well as the alga Chlorella vulgaris (Chlorellales: Chlorellaceae). All agents were commercial products and diluted in sterile tap water to the required doses based on the label instructions. Three to six concentrations of each microbial biostimulant were tested against the eggs, larvae, and adults (Table 1). Imidacloprid was used as a positive control and sterilized tap water as a negative control. Systematic block designs with regard to treatments were used in all three bioassay types as described below.

2.2.1. Egg Bioassays

To assess the effect of microbial plant biostimulants on eggs, we applied standard screening methods under controlled semi-sterile conditions [23]. In detail, ready-to-hatch eggs were washed and placed on a 100 µm sieve. Treatments were prepared in Eppendorf tubes based on the concentrations on the product labels (Table 1). Eggs were transferred to the tubes with treatments using a stainless-steel spoon (2 cm long). The spoon was dipped into 70% ethanol and sterilized tap water for 3 s before use in another treatment. Eggs were soaked in the treatments for 1 h. Then, 20 µL of eggs were pipetted onto filter paper in Petri dishes (150 mm × 25 mm) and then 100 µL of sterilized tap water was added. Around (15 ± 8) eggs were added per dish. Eggs were incubated in the dishes at 24–25 °C for 7 days. Six Petri dishes were used per treatment. Six experimental replicates were conducted per treatment. Egg hatching and mortality of newly hatched larvae were recorded 1, 3, and 5 days after treatments.

2.2.2. Larvae Bioassays

To assess the effect of microbial plant biostimulants on first instar larvae of D.v. virgifera, we applied artificial diet-overlay bioassays under controlled semi-sterile conditions. These are standard screening methods for novel agents used by many researchers [23,24,25,26]. Sterilized tap water was used as the untreated control, and imidacloprid was used as the positive control. Each bioassay consisted of 3 to 6 96-well polystyrene plates (07-6096 of Biologix Ltd., USA, or Costar 3917 of Corning Inc., Corning, NY, USA). Each well had a 330 µL volume, was 5 mm in diameter and 10 mm in height and had a 0.34 cm2 surface. Each treatment was applied to 8 wells of each plate per bioassay. Six experimental replicates were conducted per treatment.
The larval diet for a bioassay had been prepared 1 day before treatment and infestation. The diet was prepared under semi-sterile conditions following methods validated in the scientific literature [23,27,28,29,30]. This diet formulation consisted of ground maize roots and food color, D (+) sucrose, vitamin-free casein, cellulose, Wesson’s salt mix, methylparaben fungicide, sorbic acid, cholesterol, raw wheat germ, Vanderzant’s vitamin mix, raw linseed oil, streptomycin sulphate antibiotic, and chlortetracycline antibiotic. A 190 µL volume of the diet was pipetted into each 330 µL well, filling each around two-thirds. Plates with diet were allowed to dry in a laminar flow cabinet for 45 min and then stored overnight at 3 to 5 °C. Treatments were applied the following day as follows: 17 µL of the treatment was applied to the 0.34 cm2 diet surface, achieving good coverage and therefore forcing larvae to feed through (10 to 100 µL pipette). The order of treatment was reversed on every other plate to avoid edge effects. Plates were dried for 1 to 1.5 h and then cooled in a 3 to 5 °C fridge for 1 h.
One first instar larvae larva was placed on the diet surface per well using a fine artist brush. A fast-moving, healthy-looking larva was chosen and lifted from the end of the abdomen with the brush, moved towards a well surface, and allowed to crawl off the brush onto the diet. Larvae were not placed in treatment column order but in a rectangular arrangement to avoid systemic errors. After every 12 individual larvae, the brush was cleaned in 70% ethanol followed by sterile tap water. The filled plate was sealed with an optically clear adhesive qPCR seal sheet (#AB-1170, Thermo Scientific, Waltham, MA, USA or #BS3017000, Bioleader, USA) allowing data assessment without opening the plate. Four to five holes were made with flamed 00-insect pins into the seal per well to allow aeration. Plates containing larvae were incubated at 24 ± 2 °C and 50 to 70% r.h. in the dark in a ventilated incubator for 5 days.
We assessed the mortality and stunting of larvae within 3 to 5 days. These parameters were visually assessed through the clear seals of the bioassay plates using a stereomicroscope (10× magnification, SMZ-B4, Optec, Chongqing, China). Data from a plate were only accepted if the natural mortality threshold of 37.5% in the untreated control was not reached, i.e., no more than 3 dead of 8 larvae per column of wells per treatment. This is contrary to common practices with other insects in bioassays where the quality acceptance is <10% of the natural background mortality [30]. However, this is rarely achievable for rootworm larvae as the artificial diets known to date remain suboptimal [31].

2.2.3. Adult Bioassays

To assess the effects of microbial plant biostimulants on adult D.v. virgifera, artificial diet-overlay bioassays with different dosages were conducted under controlled semi-sterile conditions. These are standard screening methods for novel agents used by many researchers [9,32]. In detail, each bioassay consisted of 6 polystyrene plates of 6 wells each. Each treatment was applied to 3 wells of each plate per bioassay. The experiments were repeated five times.
The adult diet for a bioassay had been prepared before treatment and adult infestation. The diet was prepared under semi-sterile conditions following methods validated in the scientific literature [28,33]. For example, for 200 mL of diet, 16.5 g sucrose, 9 g cellulose, 8 g casein, 6 g soybean flour, 2.5 g yeast, 0.6 g Wesson salt mix, and 0.15 g cholesterol diet were ground and added to 165 mL fluid agar at 60 to 70 °C. After mixing and cooling to 55 to 60 °C, 6 g of ground wheat germ was added. Additionally, 0.0064 g chlortetracycline and 0.0064 g streptomycin sulphate were also added. Thereafter, 5.5 mL glycerol was added to reach pH 5 with temperature between 50 and 55 °C. Then, the diet was poured out into 5–6 sterile 11 mm Petri dishes. The plates with diet were allowed to dry for up to 15 min under a laminar flow cabinet and then stored at 3 to 5 °C overnight. The following day, a core of diet was first transferred using screw iron. A core diet was placed in all 6-well plates.
Treatments of 40 µL each were applied on the surface of each diet core (0.34 cm3). Sterilized tap water was served as the untreated control, and imidacloprid as the positive control. Each treatment was applied to 3 wells of each plate per bioassay. Adults were then transferred from the rearing cage into the wells of the 6-well plates containing diet and treatments using a handheld tube aspirator (3 to 4 adults per well). Adults were then incubated in the plates at 24–25 °C for 5 days. Adult mortality was recorded 1, 3, and 5 days after treatments.

2.3. Testing Microbial Biostimulants under Greenhouse Conditions

In this study, ten commercial microbial biostimulants were tested at different doses in potted maize plants under greenhouse conditions to determine their effects on D.v. virgifera larvae and on the maize plants (Table 2). These were microbial biostimulants from a group of bacteria (Bacillus amyloquafaciens, Bradyrhizobium japonicum, Bacillus subtilis, Ensifer meliloti, and Rhizobium leguminosarum), a group of fungi (Trichoderma asperellum, Beauveria bassiana, Trichoderma harzianum, and Rhizophagus irregularis), and a group of algae (Chlorella vulgaris).
We examined the effects of the ten microbial biostimulants at recommended dosages as written on the labels (Table 2), and, if effects were found, at two more dosages. All agents were diluted in unsterilized tap water to the required doses. The experiment was conducted in a greenhouse at the Plant Protection and Soil Conservation Directorate of Csongrad-Csanad County in Southern Hungary, rented by CABI. The glass greenhouse consisted of closed-system compartments with ventilation capacity.

Greenhouse Experiments

To assess the insecticidal effects of microbial biostimulants against D.v. virgifera larvae under semi-field conditions, two systematic controlled experiments were conducted using potted plants in a glass greenhouse (Figure 1). The experiments were conducted for 1.5 months at 21–25 °C and 50 to 60% relative humidity. NPK served as a positive control and unsterilized tap water as a negative control, both with infested and uninfested plants. Each of the treatments included 10 to 15 potted plants infested with larvae and 10 to 15 other plants that were uninfested, all arranged in a complete block design. Two repetitions of the experiment were performed.
Maize was sown individually into 0.5 L soil in 1 L plastic cups (8 cm inner diameter, 14 cm height). The soil used was black clay loam soil from the field. Briefly, the plastic cup was first filled with 0.5 L of soil. One maize seed was placed in each plastic cup and then 20–40 mL water was applied. Treatments were applied in liquid form using a pipette directly to the surface of the maize seed on the soil. Then, 0.25 L of soil was added, burying the treatment and seeds 3 cm deep into the soil and leading to a soil surface of 9 cm diameter in the plastic cup. Plants were watered with 90–100 mL of water per week (average 20–30 mL per week, total 0.3 L over the experimental period). About 100 ready-to-hatch eggs were transferred to the plants at the 3 or 4-leaf stage and 3 weeks after sowing. A 100 µL of a 0.2% aqueous agar–egg solution was prepared and pipetted into a 5–10 cm hole in the soil next to the maize plant.
The germination rate was recorded. Plant height, leaf number, and shoot length were recorded 3 weeks after sowing and treatment. Root length, fresh root weight, root length, root damage, root volume, above-ground fresh biomass, and number of living larvae were recorded 5 weeks after sowing (Figure 1). At this period, the maize was at the 5 to 7-leaf stage, and mainly second and early third instar stages were found. In detail, each maize plant was lifted from the soil and gently shaken to remove loosely adhering soil particles from the roots. Each maize plant was cut 1 cm above the roots and fresh weight, leaf number, and plant height were measured. Roots were washed and assessed for damage under a stereomicroscope according to the modified 1.0 to 6.0 Iowa scale [34]. Then, the soil in each plastic cup was placed onto a plastic screen to dry out and allow living larvae to emerge and drop onto the wet tissue paper in the tray below, following the Berlese approach (Figure 1) [35,36]. The number of living larvae was counted after 1 and 3 days. The untreated control aimed to have a minimum of 20% infestation with second or third instar larvae to validate the results of the effect of the biostimulants. In this experiment, larvae were recovered from 100% of the infested pots in the untreated control.

2.4. Data Analysis

To allow comparisons between experimental replicates, all data were standardized to the data of corresponding negative control (sterilized tap water). Data as well as residuals were observed for normality. One-way ANOVA was used to analyse the effect of the treatments on eggs, first instar larvae, or adults in the laboratory experiments, or on plant height, leaf number, shoot length, root length, fresh root weight, root volume, above-ground fresh biomass, root damage, and the number of living larvae in the greenhouse experiment. Dunnet test was used to assess differences in treatments compared to the negative control at p < 0.05. IPM SPSS version 22 statistical software was used [37].

3. Results

3.1. Effects of Microbial Biostimulants on Diabrotica virgifera virgifera Life Stages under Laboratory Conditions

3.1.1. Effects of Microbial Biostimulants on Diabrotica virgifera virgifera Eggs

Our results from screening biostimulants on D.v. virgifera eggs showed that none were able to kill any larvae freshly hatching from treated eggs (df = 43, F = 3.08, p > 0.05, R2 = 0.17) (Figure 2 and Figure 3). Most biostimulants also did not affect the hatching rate, but B. japonicum, at 100 cfu/mL, slightly increased the rates of early hatching. None of the treatments delayed the egg-hatching period (df = 37, F = 3.97, p > 0.05, R2 = 0.21).

3.1.2. Effects of Microbial Biostimulants on Diabrotica virgifera virgifera Larvae

Our laboratory results showed that some biostimulants had an effect on larvae (df = 43, F = 3.08, p > 0.001, R2 = 0.17). Beauveria bassiana at a dose of 106 cfu/g, Ensifer meliloti at 107 cfu/mL, Rhizophagus irregularis at 2000 spores/g, and Trichoderma asperellum at 2000 cfu/mL caused slight mortality of first instar larvae in diet-overlay assays within 3 days (df = 48, F = 14.8, p < 0.001, R2 = 0.20) (Figure 4). Bradyrhizobium japonicum at a dose of 1000 cfu/mL, R. irregularis at 2000 spores/g, B. bassiana at a dose of 106 cfu/g, and T. asperellum at 2000 cfu/mL caused some mortality in the first instar larvae within 5 days post-treatment (Figure 4).
Some biostimulants caused stunting of larvae (df = 48, F = 4.08, p < 0.001, R2 = 0.15), this is, Rh. irregularis at a dose of 2 × 103 spores/g, T. asperellum at a dose of 107 cfu/mL, and T. harzianum at a dose of 107 cfu/g c within 3 days post-treatment (Figure 4).

3.1.3. Assessing the Effects of Microbial Biostimulants on Diabrotica virgifera virgifera Adults

Microbial biostimulants tested had no effect on D.v. virgifera adults (df = 39, F = 1.85, p > 0.001, R2 = 0.39) (Figure 5).

3.2. Effects of Microbial Biostimulants on Maize and Diabrotica virgifera virgifera Larvae under Greenhouse Conditions

3.2.1. Effects of Microbial Biostimulants on Maize

Our study revealed that some of the microbial biostimulants tested had an effect on the height of maize plants without infestation of D.v. virgifera larvae (df = 11, F = 8.63, p < 0.001, R2 = 0.38) (Figure 6). For example, B. japonicum at a dose of 2 × 109 cfu/mL, B. subtilis at 5 × 109 cfu/g, E. meliloti at 109 cfu/mL, and Rh. leguminosarum at 109 cfu/mL increased the height of maize plants compared to the untreated uninfested control. Only one microbial biostimulant affected the height of maize plants infested with larvae (df = 20, F = 2.21 p < 0.05, R2 = 0.14). This is B. subtilis (107 cfu/g), which reduced the heights of maize plants compared to the untreated infested control.
There is a general slight treatment effect on the number of leaves of uninfested maize plants (df = 11, F = 4.34, p < 0.001, R2 = 0.24). However, the multiple comparison Dunnet test could not detect any single microbial biostimulant affecting the number of leaves of uninfested maize plants. In contrast, some of the biostimulants affected the number of leaves of infested plants (df = 20, F = 4.44, p < 0.001, R2 = 0.25). Chlorella vulgaris (107 cell/mL) and Rh. leguminosarum (108 cfu/mL) caused maize plants to have fewer leaves compared to the untreated infested control.
There was a general treatment effect on the root length of uninfested maize plants (df = 11, F = 2.9, p < 0.001, R2 = 0.20). However, the multiple comparison Dunnet test could not detect any single microbial biostimulant affecting the root length of uninfested maize. In contrast, most biostimulants reduced root lengths when infested with larvae (df = 20, F = 4.34, p < 0.001, R2 = 0.30), except B. bassiana, T. harzianum, and Rh. irregularis.
Some microbial biostimulants had an effect on the root fresh weight of uninfested maize (df = 11, F = 3.6, p < 0.001, R2 = 0.21). Beauveria bassiana at a dose of 107 cfu/g, B. subtilis at 5 × 109 cfu/g, R. irregularis at 2 × 103 spores/g, and T. asperellum at 105 cfu/g reduced root weight compared to the untreated uninfested control. In addition, all biostimulants tested reduced root weights of infested maize compared to the untreated infested control (df = 20, F = 4.29, p < 0.001, R2 = 0.2), except Rh. irregularis.
One out of ten microbial biostimulants had an effect on above-ground fresh biomass in uninfested maize (df = 11, F = 3.35, p < 0.001, R2 = 0.20); that is, B. bassiana at a dose of 107 cfu/g reduces above-ground fresh biomass. Most of the microbial biostimulants tested also had an effect on the above-ground fresh biomass of infested maize plants (df = 20, F = 5.93, p < 0.001, R2 = 0.31). Specifically, B. amyloquafaciens (105, 106, 5 × 109 spores/mL), B. bassiana (107 cfu/g), B. subtilis (5 × 109 cfu/g), Ch. vulgaris (103, 2 × 107 cell/mL), E. meliloti (109 cfu/mL), R. leguminosarum (106, 108 cfu/mL), and T. asperellum (105 cfu/g) caused maize plants to have less above-ground fresh biomass compared to the untreated infested control.
None of the microbial biostimulants affected the root volume of uninfested maize plants (df = 11, F = 2.2, p > 0.05, R2 = 0.14), but most affected the root volume of infested maize (df = 20, F = 4.9, p < 0.001, R2 = 0.28). For example, B. amyloquafaciens (108 spores/mL), B. subtilis (107 cfu/g), and C. vulgaris (103 cells/mL) caused maize plants to have less root volume compared to the untreated infested control.
Some of the microbial biostimulants affected the shoot length of uninfested maize (df = 11, F = 6.7, p < 0.001, R2 = 0.32). Specifically, B. japonicum (2 × 109 cfu/mL) and E. meliloti increased the shoot length of maize plants. There was also a general treatment effect on shoot length of infested maize plants (df = 20, F = 1.69, p < 0.05, R2 = 0.12). However, multiple comparison Dunnet test could not detect a single microbial biostimulant affecting the shoot length of maize plants compared to the untreated infested control.

3.2.2. Effects of Microbial Biostimulants on Diabrotica virgifera virgifera Larvae

All microbial biostimulants tested had no effect on the number of surviving larvae compared to the untreated infested control (df = 10, F = 1.38, p > 0.01, R2 = 0. 13) (Figure 7). Among the ten tested biostimulants, we found three that prevented some of the root damage caused by D.v. virgifera larvae on maize plants (df = 20, F = 2.34, p < 0.01, R2 = 0.16); these were B. amyloliquefaciens at a dose of 5 × 109 cfu/mL, B. subtilis at 5 × 109 cfu/g, and E. meliloti at 109 cfu/mL.

4. Discussion

In this study, we successfully assessed the insecticidal effects of microbial biostimulants on the important soil insect pest, D.v. virgifera, under laboratory and greenhouse conditions. Under laboratory conditions, our study revealed that most of the ten tested biostimulants had, as expected, no effect on the different life stages of D.v. virgifera, particularly not on the adults. However, among the ten biostimulants tested, Beauveria bassiana, Rhizophagus irregularis, Trichoderma asperellum, and Ensifer meliloti, seem, in addition to their biostimulant properties, to be able to also cause some mortality in D.v. virgifera larvae. However, those effects only appeared at high doses of the used microbials. Therefore, those effects were usually not confirmed in our potted plant experiments under greenhouse conditions. This suggested that the insecticidal effects of the used strains of B. bassiana, R. irregularis, and T. asperellum against D.v. virgifera larvae are rather weak. In contrast, in our greenhouse results, E. meliloti was able to prevent some root damage caused by D.v. virgifera larvae somewhat confirming the insecticidal effects found in the laboratory assays.
Although not apparent in the greenhouse situation, our diet-overlay bioassays in the laboratory showed that B. bassiana can kill first instar larvae. This is not astonishing, as B. bassiana is a well-known biopesticide [38]. For example, B. bassiana at a dose of 5 × 107 conidia/mL reduced the total productive capacity (mean number of eggs/female), and reduced egg viability and the total egg production of D.v. virgifera [39]. Beauveria bassiana also kills larvae of Anopheles gambiae (Diptera: Culicidae) [40,41]. It is suggested to infect mosquito larvae by attaching to the larval body at the head and perispiracular lobes of the siphon [42,43,44] and by ingestion of fungal spores [44]. Infection of mosquito larvae with B. bassiana causes histological changes in the larval body, including disintegration and deformation of the larval cuticle, epidermis, and adipose tissue [43,45]. There are many other reports that show that many strains of B. bassiana have insecticidal properties and are therefore more accurately described as biopesticides rather than plant biostimulants. Unfortunately, many microbial plant biostimulants lack information on the used strain, hindering comparison with other studies.
We also showed in our bioassays that R. irregularis can cause a slight level of mortality to D.v. virgifera first instar larvae, which is consistent with another study that also reported that inoculation of maize with R. irregularis can reduce larval development of D.v. virgifera [46]. However, the mechanisms behind the interaction between R. irregularis and D.v. virgifera remain unknown.
Finally, T. asperellum also killed some D.v. virgifera larvae in our bioassays. Trichoderma asperellum is not well known as an insecticide, but another study showed that inoculation of Anopheles spp. with conidia of T. asperellum can cause 85% mortality after 72 h of treatment [47]. Another example is the study which reported that treating poplar seedlings with T. asperellum decreased the survival, body weight, body length, and head capsule width of Lymantria dispar (Lepidoptera: Erebidae) [48]. The mechanism by which T. asperellum affected the larvae involved fungal spores attached to the larval surface using specific carbohydrate-containing adhesives. The spores then grew and secreted specific enzymes that degraded the cuticle, the outer layer of the larvae. This allowed the spores to puncture the cuticle and enter the larval body, ultimately causing death. Whether this mechanism is also valid for D.v. virgifera larvae remains to be studied.
As stated above, our greenhouse experiments revealed some biostimulants to affect the maize itself. Interestingly several biostimulants reduced volume and length of roots of uninfested maize as well as biomass. In contrast, B. japonicum and E. meliloti increased maize plant height without D.v. virgifera infestation. This is consistent with [49], who reported a 22% increase in soybean plant height following inoculation with these biostimulants, and [50] showed that E. meliloti improved plant growth, including plant height, in Arabidopsis thaliana (Brassicales: Brassicaceae), particularly under nitrogen-deficient conditions. This supports the notion that E. meliloti has beneficial effects on plant growth beyond its primary association with legumes, probably through mechanisms such as enhanced nutrient uptake or stress tolerance. It therefore warrants further development as a commercial plant biostimulant in a wider range of crops.
Our results under greenhouse conditions also revealed that biostimulants in general did not directly affect the number of D.v. virgifera larvae, as assessed in our Berlese sampling system. In contrast, we found that B. amyloquafaciens, B. subtilis, and E. meliloti can prevent some root damage caused by D.v. virgifera larvae, although larvae numbers were not reduced. This suggests improvement of plant defence or tolerance rather than insecticidal effects of these microbials. Bacillus subtilis is known for its ability to produce a range of phytohormones in plants. For example, inoculation of B. subtilis strain 26D increased the level of zeatin-riboside in shoots of Solanum tuberosum (Solanales: Solanaceae). This enhanced the recovery of shoot growth and biomass of roots after attack by Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) [51]. Moreover, a consortia of B. amyloquafaciens and Lysinibacillus sphaericus (Bacillales: Bacillaceae) was shown to increase nematocidal activity and prevent root damage caused by Meloidogyne incognita (Tylenchida: Heteroderidae) [52].
Currently, the most used strategy for managing D.v. virgifera is crop rotation [53]. However, some farmers are limited in rotation possibilities and are in need of other tools. One option would be the use of biostimulants to improve the maize crop, and potentially to increase its tolerance and defence capabilities against insect pests. Most promising are, according to our findings, B. amyloquafaciens, B. subtilis, or E. meliloti, which are capable of helping the plant prevent some root damage. Farmers may use such biostimulants as seed treatments while sowing the maize seed into the soil [54,55]. Studies on clarifying the mode of action of these microbials as well as on their performance under field conditions seem important before their further consideration for potential use by farmers.

5. Conclusions

We confirmed that most microbial plant biostimulants are indeed solely of a plant-affecting nature and have no insecticidal effects on the soil pest D.v. virgifera. However, our study demonstrates that the bacterial biostimulants B. subtilis, B. amyloliquefaciens, and E. meliloti can prevent some root damage. These findings highlight the potential of these biostimulants not only to enhance maize growth but also to serve as a sustainable and integrated pest management option. Scientists can study these three bacterial biostimulants to investigate their efficacy and whether they should be used individually or in consortium against other pests. In the mid-term, by identifying further microbial biostimulants that improve plant defences, we hope valuable tools can be provided to farmers to improve crop resilience and reduce reliance on chemical pesticides. Future research should focus on clarifying their modes of action. On-field trials should also be conducted to validate these results and explore the broader ecological impacts of using these biostimulants in agricultural systems.

Author Contributions

S.T., T.G., J.K. and S.I.T. jointly developed the study; S.T., J.K. and T.G. supervised the study; S.I.T. conducted laboratory bioassay experiments. S.I.T. and N.P.Y.D. conducted data collection and analysis; S.I.T. wrote the manuscript with support from all authors. All authors have read and agreed to the published version of the manuscript.

Funding

We acknowledge the PhD scholarship from the Stipendium Hungaricum and Tempus Public Foundation (SHE-02988-004/2020-2024) under the Doctoral School of Plant Science of the Hungarian University of Agriculture and Life Sciences (MATE). We also acknowledge funding from the Slovenian Research and Innovation Agency (ARIS) (J4-2543) and the National Research, Development and Innovation Office of Hungary (NKFIH) (134356 SNN20). CABI is grateful for the core funding from the respective taxpayers behind the European Commission, the Department for International Development of the U.K., the Swiss Agency for Development and Cooperation, the Directorate General for International Cooperation of the Netherlands, the Irish Aid International Fund for Agricultural Development, and the Australian Centre for International Agricultural Research (https://www.cabi.org/about-cabi/who-we-work-with/key-donors/ accessed on 10 May 2022). Lastly, we acknowledge the financial support from Universitas Kristen Wira Wacana Sumba, Indonesia, during this doctoral study.

Data Availability Statement

Data are available upon request.

Acknowledgments

We express our gratitude to Zsuzsanna Tassy from the international student relations office at the Hungarian University of Agriculture and Life Sciences (MATE), Godollo, Hungary, for her consistent support. Additionally, we would like to acknowledge Szabolcs Tóth for his help during the experiments.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Meinke, J.; Souza, D.; Siegfried, B.D. The use of insecticides to manage the western corn rootworm, Diabrotica virgifera virgifera, Le Conte: History, field-evolved resistance, and associated mechanisms. Insects 2021, 12, 112. [Google Scholar] [CrossRef] [PubMed]
  2. Bažok, R.; Lemić, D.; Chiarini, F.; Furlan, L. Western corn rootworm (Diabrotica virgifera virgifera LeConte) in Europe: Current status and sustainable pest management. Insects 2021, 12, 195. [Google Scholar] [CrossRef]
  3. Eben, A. Ecology and Evolutionary History of Diabrotica Beetles-Overview and Update. Insects 2022, 13, 156. [Google Scholar] [CrossRef] [PubMed]
  4. Toepfer, S.; Toth, S. Entomopathogenic nematode application against root-damaging Diabrotica larvae in maize: What, when, and how. Microb. Nematode Control Invertebr. Pests IOBC-WPRS Bull. 2020, 150, 185–188. [Google Scholar]
  5. Souza, D.; Peterson, J.A.; Wright, R.; Meinke, L.; Wright, R.J.; Meinke, L.J. Field efficacy of soil insecticides on pyrethroid-762 resistant western corn rootworms (Diabrotica virgifera virgifera LeConte). Pest Manag. Sci. 2020, 76, 827–833. [Google Scholar] [CrossRef]
  6. Souza, D.; Vieira, B.C.; Fritz, B.K.; Hoffmann, W.C.; Peterson, J.A.; Kruger, G.R.; Meinke, L.J. Western corn rootworm pyrethroid resistance confirmed by aerial application simulations of commercial insecticides. Sci. Rep. 2019, 9, 6713. [Google Scholar] [CrossRef] [PubMed]
  7. Furlan, L.; Kreutzweiser, D. Alternatives to neonicotinoid insecticides for pest control: Case studies in agriculture and forestry. Environ. Sci. Pollut. Res. 2015, 22, 135–147. [Google Scholar] [CrossRef]
  8. Ciosi, M.; Toepfer, S.; Li, H. European populations of Diabrotica virgifera virgifera are resistant to aldrin, but not to methyl-parathion. J. Appl. Entomol. 2009, 133, 307–314. [Google Scholar] [CrossRef]
  9. Parimi, S.; Meinke, L.J.; Nowatzki, T.M.; Chandler, L.D.; French, B.W.; Siegfried, B.D. Toxicity of insecticide-bait mixtures to insecticide resistant and susceptible western corn rootworms (Coleoptera: Chrysomelidae). Crop Prot. 2003, 22, 781–786. [Google Scholar] [CrossRef]
  10. Yakhin, O.I.; Aleksandr, A.L.; Ildus, A.Y.; Patrick, B. Biostimulants in Plant Science: A global perspective. Front. Plant Sci. 2017, 7, 2049. [Google Scholar] [CrossRef]
  11. Tarigan, S.I.; Szabolcs, T.; Mark, S.; Jozsef, K.; Gyorgy, T.; Stefan, T. Biological control properties of microbial plant bio stimulants. A review. Biocontrol Sci. Technol. 2022, 32, 1351–1371. [Google Scholar] [CrossRef]
  12. Naseri, B.; Hamzavi, F. Effects of chemicaland bio-fertilizers on cowpea resistance to Cowpea Weevil, Callosobruchus maculatus (f.) (Coleoptera: Chrysomelidae). J. Stored Prod. Res. 2021, 92, 101785–101788. [Google Scholar] [CrossRef]
  13. Murray, C.; Markwick, N.; Kaji, R.; Poulton, J.; Martin, H.; Christeller, J. Expression of various biotin-binding proteins in transgenic Tobacco confers resistance to potato tuber moth, Phthorimaea operculella (Lepidopetera: Gelechiidae). Transgenic Res. 2010, 19, 1041–1051. [Google Scholar] [CrossRef]
  14. Yan, W.; Lin, X.; Yao, Q.; Zhao, C. Arbuscular mycorrhizal fungi improve uptake and control efficacy of carbosulfan on Spodoptera frugiperda in maize plants. Pest Manag. Sci. 2021, 77, 2812–2819. [Google Scholar] [CrossRef]
  15. Gad, H.A.; Al-Anany, M.S.; Sameer, W.M.; Al-Anany, F.S. Control of Acanthoscelides obtectus with Trichoderma harzianum applied alone or in combination with diatomaceous earth on a stored common bean. Plant Prot. Sci. 2020, 56, 107–115. [Google Scholar] [CrossRef]
  16. Alınç, T.; Cusumano, A.; Peri, E.; Torta, L.; Colazza, S. Trichoderma harzianum Strain T22 modulates direct defense of tomato plants in response to Nezara viridula feeding activity. J. Chem. Ecol. 2021, 47, 455–462. [Google Scholar] [CrossRef] [PubMed]
  17. Veena-Bhamrah, H.S. Studies on pathogenicity and management of mole cricket, Gryllotalpa gryllotalpa. Ann. Plant Prot. Sci. 2007, 15, 381–383. [Google Scholar]
  18. Purushothaman, T.; Mol, I. Micro-algae as bio-pesticides for the development of sustainable agriculture. Wide Spectr. 2020, 8, 35–48. [Google Scholar]
  19. Fei, X.; Zhang, Y.; Ding, L.; Xiao, S.; Xie, X.; Li, Y.; Deng, X. Development of an RNAi-based microalgal larvicide for the control of Aedes aegypti. Parasites Vectors 2021, 14, 1–11. [Google Scholar] [CrossRef]
  20. Georce, B.W.; Ortman, E.E. Rearing the western corn rootworm in the laboratory. J. Econ. Entomol. 1965, 58, 375–377. [Google Scholar] [CrossRef]
  21. Branson, T.F.; Guss, P.L.; Krysan, J.L.; Sutter, G.R. Corn Rootworms: Laboratory Rearing and Manipulation; USDA ARS-NC: Peoria, IL, USA, 1975; Volume 28, p. 18. [Google Scholar]
  22. Singh, P.; Moore, R.F. Hancellook of Insect Rearing; Elsevier: Amsterdam, The Netherlands, 1975. [Google Scholar]
  23. Toepfer, S.; Toth, S.; Szalai, M. Can the botanical azadirachtin replace phased-out soil insecticides in suppressing the soil insect pest Diabrotica virgifera virgifera. CABI Agric. Biosci. 2021, 2, 28. [Google Scholar] [CrossRef]
  24. Geisert, R.W.; Huynh, M.P.; Pereira, A.E.; Shapiro Ilan, D.I.; Hibbard, B.E. An improved bioassay for the testing of entomopathogenic nematode virulence to the Western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae): With focus on first instar larvae insect assessments. J. Econ. Entomol. 2023, 116, 726–732. [Google Scholar] [CrossRef] [PubMed]
  25. Bernklau, E.J.; Bjostad, L.B.; Hibbard, B.E. Synthetic feeding stimulants enhance insecticide activity against western corn rootworm larvae, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). J. Appl. Entomol. 2011, 135, 47–54. [Google Scholar] [CrossRef]
  26. Gemrich, E.G.; Dennis, M. Goldsberry. Laboratory bioassay to assess toxicity of insecticides to Diabrotica larvae. J. Econ. Entomol. 1982, 75, 220–222. [Google Scholar] [CrossRef]
  27. Sutter, G.R.; Branson, T.F.; Fisher, J.R.; Elliott, N.C. Effect of insecticides on survival, development, fecundity, and sex ratio in controlled infestations of western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 1991, 84, 1905–1912. [Google Scholar] [CrossRef]
  28. Pleau, M.J.; Huesing, J.E.; Head, G.P.; Feir, D.J. Development of an artificial diet for the western corn rootworm. Entomol. Exp. Appl. 2002, 105, 1–11. [Google Scholar] [CrossRef]
  29. Moar, W.; Khajuria, C.; Pleau, M.; Ilagan, O.; Chen, M.; Jiang, C.; Price, P.; McNulty, B.; Clark, T.; Head, G. Cry3bb1-resistant western corn rootworm, Diabrotica virgifera virgifera (LeConte) does not exhibit cross-resistance to dvSnf7 dsRNA. PLoS ONE 2017, 12, e0169175. [Google Scholar] [CrossRef] [PubMed]
  30. Dulmage, H.T.; Yousten, A.A.; Singer, S.; Lacey, L.A. Guidelines for the production of Bacillus thuringiensis H-14 and Bacillus sphaericus. In UNDP/WHO/WHO Special Programme for Research and Training in Tropical Diseases; UNDP/WHO/WHO: Geneva, Switzerland, 1990. [Google Scholar]
  31. Huynh, M.P.; Bernklau, E.J.; Coudron, T.A.; Shelby, K.S.; Bjostad, L.B.; Hibbard, B.E. Characterization of corn root factors to improve artificial diet for western corn rootworm (Coleoptera: Chrysomelidae) larvae. J. Insect Sci. 2019, 19, 20. [Google Scholar] [CrossRef] [PubMed]
  32. Toepfer, S.; Gueldenzoph, C.; Ehlers, R.; Kuhlmann, U. Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe. Bull. Entomol. Res. 2005, 95, 473–482. [Google Scholar] [CrossRef]
  33. Brandson, T. Resistance in the grass tribe maydeae to larvae of the western corn rootworm. Ann. Entomol. Soc. Am. 1971, 64, 861–863. [Google Scholar] [CrossRef]
  34. Hills, T.M.; Peters, D.C. A method of evaluating post planting insecticide treatments for control of western corn rootworrn larvae. J. Ecoll. Entomol. 1971, 64, 764–765. [Google Scholar] [CrossRef]
  35. Dent, D.R.; Walton, M.P. Methods in Ecological and Agricultural Entomology; CAB International: Wallingford, UK, 1998. [Google Scholar]
  36. Fischhoff, I.R.; Keesing, F.; Ostfeld, R.S. The tick biocontrol agent Metarhizium brunneum (= M. anisopliae) (strain F52) does not reduce non-target arthropods. PLoS ONE 2017, 12, e0187675. [Google Scholar] [CrossRef] [PubMed]
  37. Kinnear, P.R.; Gray, C.D. SPSS for Windows Made Simple; Psychology Press Ltd.: East Sussex, UK, 2000. [Google Scholar]
  38. Wang, Y.; Liu, H.; Shen, Z.; Miao, Y.; Wang, J.; Jiang, X.; Qirong, S.; Rong, L. Richness and antagonistic effects co-affect plant growth promotion by synthetic microbial consortia. Appl. Soil Ecol. 2022, 170, 104300. [Google Scholar] [CrossRef]
  39. Mulock, B.S.; Chandler, L.D. Effect of Beauveria bassiana on the fecundity of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Biol. Control 2001, 22, 16–21. [Google Scholar] [CrossRef]
  40. Tawidian, P.; Kang, Q.; Michel, K. The potential of a new Beauveria bassiana isolate for mosquito larval control. J. Med. Entomol. 2023, 60, 131–147. [Google Scholar] [CrossRef]
  41. Clark, T.B.; Kellen, W.R.; Fukuda, T.; Lindegren, J.E. Field and laboratory studies on the pathogenicity of the fungus Beauveria bassiana to three genera of mosquitoes. J. Invertebr. Pathol. 1968, 11, 1–7. [Google Scholar] [CrossRef]
  42. Miranpuri, G.S.; Khachatourians, G.G. Infection sites of the entomopathogenic fungus Beauveria bassiana in the larvae of the mosquito Aedes aegypti. Entomol. Exp. Appl. 1991, 59, 19–27. [Google Scholar] [CrossRef]
  43. Bitencourt, R.D.O.B.; dos Santos Mallet, J.R.; Mesquita, E.; Gôlo, P.S.; Fiorotti, J.; Bittencourt, V.R.E.P.; da Costa Angelo, I. Larvicidal activity, route of interaction and ultrastructural changes in Aedes aegypti exposed to entomopathogenic fungi. Acta Trop. 2021, 213, 105732. [Google Scholar] [CrossRef]
  44. Miranpuri, G.S.; Khachatourians, G.G. Larvicidal activity of blastospores and conidiospores of Beauveria bassiana (strain GK 2016) against age groups of Aedes aegypti. Vet. Parasitol. 1990, 37, 155–162. [Google Scholar] [CrossRef]
  45. Farida, B.; Sonia, H.; Hakima, M.K.; Fatma, B.; Fatma, H. Histological changes in the larvae of the domestic mosquito Culex pipiens treated with the entomopathogenic fungus Beauveria bassiana. Sci. Res. Essays 2018, 13, 1–10. [Google Scholar]
  46. Dematheis, F.; Kurtz, B.; Vidal, S.; Smalla, K. Multitrophic interactions among Western Corn Rootworm, Glomus intraradices and microbial communities in the rhizosphere and endorhiza of maize. Front. Microbiol. 2013, 4, 357. [Google Scholar] [CrossRef] [PubMed]
  47. Podder, D.; Ghosh, S.K. A new application of Trichoderma asperellum as an anopheline larvicide for ecofriendly management in medical science. Sci. Rep. 2019, 9, 1108. [Google Scholar] [CrossRef] [PubMed]
  48. Jiang, D.; Tan, M.; Wu, S.; Zheng, L.; Wang, Q.; Wang, G.; Yan, S. Defense responses of arbuscular mycorrhizal fungus-colonized poplar seedlings against gypsy moth larvae: A multiomics study. Hortic. Res. 2021, 8, 1–13. [Google Scholar] [CrossRef]
  49. Sharma, P.R.; Prasad, B.N. Effect of Bradyrhizobium japonicum on Chlorophyll Content, Nodulation, and Plant Growth in Soybean. Korean J. Crop Sci. 2005, 50, 265–267. [Google Scholar]
  50. Armijo, G.; Kraiser, T.; Medina, M.P.; Gras, D.E.; Zúñiga, A.; González, B.; Gutiérrez, R.A. Arabidopsis thaliana interaction with Ensifer meliloti can support plant growth under N-deficiency. bioRxiv 2020. [Google Scholar] [CrossRef]
  51. Sorokan, A.; Svetlana, V.; Galina, B.; Igor, M. Endophytic strain Bacillus subtilis 26 D increase levels of phytohormones and repairs growth of potato plants after colorado potato beetle damage. Plants 2021, 20, 923. [Google Scholar] [CrossRef]
  52. Abdel-Salam, M.S.; Ameen, H.H.; Soliman, G.M.; Elkelany, U.S.; Asar, A.M. Improving the nematicidal potential of Bacillus amyloliquefaciens and Lysinibacillus sphaericus against the root-knot nematode Meloidogyne incognita using protoplast fusion technique. Egypt. J. Biol. Pest Control 2018, 28, 1–6. [Google Scholar] [CrossRef]
  53. Levine, E.; Oloumi-Sadeghi, H. Management of diabroticite rootworms in corn. Annu. Rev. Entomol. 1991, 36, 229–255. [Google Scholar] [CrossRef]
  54. Kiss, J.; Edwards, C.R.; Berger, H.K.; Cate, P.; Cean, M.; Cheek, S.; Vahala, O. Monitoring of western corn rootworm (Diabrotica virgifera virgifera LeConte) in Europe 1992–2003. In Western Corn Rootworm: Ecology and Management; CAB International: Wallingford, UK, 2005; pp. 29–39. [Google Scholar]
  55. Meinke, L.J.; Sappington, T.W.; Onstad, D.W.; Guillemaud, T.; Miller, N.J.; Komáromi, J.; Toth, F. Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics. Agric. For. Entomol. 2009, 11, 29–46. [Google Scholar] [CrossRef]
Agronomy 14 02239 g001
Figure 1. Assessment of the effects of microbial biostimulants on Diabrotica virgifera virgifera larvae 28 days after sowing maize and treatment, and 2 weeks after infestation of approximately 100 eggs per plant. The assessment included (1) assessing living larvae after 3 weeks of treatment using the Berlese method; (2) recording the number of living larvae observed on the filter papers (coming from the Berlese traps); (3) assessing root damage under a stereomicroscope using the 1.0 to 6.0 Iowa scale [34].
Figure 1. Assessment of the effects of microbial biostimulants on Diabrotica virgifera virgifera larvae 28 days after sowing maize and treatment, and 2 weeks after infestation of approximately 100 eggs per plant. The assessment included (1) assessing living larvae after 3 weeks of treatment using the Berlese method; (2) recording the number of living larvae observed on the filter papers (coming from the Berlese traps); (3) assessing root damage under a stereomicroscope using the 1.0 to 6.0 Iowa scale [34].
Agronomy 14 02239 g001
Agronomy 14 02239 g002
Figure 2. Effects of microbial plant biostimulants on mortality of newly hatched larvae of Diabrotica virgifera virgifera in egg-dipping bioassays under standardized laboratory conditions. Six experimental replicates were conducted per treatment and dose (numbers on the x-axis represent treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality standardized to the untreated control.
Figure 2. Effects of microbial plant biostimulants on mortality of newly hatched larvae of Diabrotica virgifera virgifera in egg-dipping bioassays under standardized laboratory conditions. Six experimental replicates were conducted per treatment and dose (numbers on the x-axis represent treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality standardized to the untreated control.
Agronomy 14 02239 g002
Agronomy 14 02239 g003
Figure 3. Effects of microbial plant biostimulants on the hatching rate of larvae of Diabrotica virgifera virgifera in egg-dipping bioassays under standardized laboratory conditions. Six experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of the hatching rate of larvae standardized to the untreated control.
Figure 3. Effects of microbial plant biostimulants on the hatching rate of larvae of Diabrotica virgifera virgifera in egg-dipping bioassays under standardized laboratory conditions. Six experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of the hatching rate of larvae standardized to the untreated control.
Agronomy 14 02239 g003
Agronomy 14 02239 g004
Figure 4. Effect of microbial biostimulants on Diabrotica virgifera virgifera larvae in artificial diet-overlay bioassays under standardized laboratory conditions. Three to five experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality or stunting standardized to the untreated control.
Figure 4. Effect of microbial biostimulants on Diabrotica virgifera virgifera larvae in artificial diet-overlay bioassays under standardized laboratory conditions. Three to five experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality or stunting standardized to the untreated control.
Agronomy 14 02239 g004
Agronomy 14 02239 g005
Figure 5. Effects of microbial biostimulants on Diabrotica virgifera virgifera adults in artificial diet-overlay bioassays under standardized laboratory conditions. Three to five experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality standardized to the untreated control.
Figure 5. Effects of microbial biostimulants on Diabrotica virgifera virgifera adults in artificial diet-overlay bioassays under standardized laboratory conditions. Three to five experimental replicates were conducted per treatment and dose (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-imidacloprid, 12-untreated control (sterilized tap water)). Letters on bars indicate differences among treatment effects as per Tukey HSD post hoc multiple comparison tests at p < 0.05. The y-axis represents the percentage of mortality standardized to the untreated control.
Agronomy 14 02239 g005
Agronomy 14 02239 g006aAgronomy 14 02239 g006b
Figure 6. Effects of microbial plant biostimulants on maize crop uninfested or infested with larvae of D.v. virgifera under greenhouse conditions. Each of the two experiments had 10 to 15 uninfested and 10 to 15 infested potted maize plants per treatment (numbers on the x-axis represent treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-N P K, 12-untreated control (unsterilized tap water)). Error bars represent the standard deviations. Asterisks indicate significant differences in treatment effects compared to the untreated control as per the Dunnet post hoc test at p < 0.05.
Figure 6. Effects of microbial plant biostimulants on maize crop uninfested or infested with larvae of D.v. virgifera under greenhouse conditions. Each of the two experiments had 10 to 15 uninfested and 10 to 15 infested potted maize plants per treatment (numbers on the x-axis represent treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-N P K, 12-untreated control (unsterilized tap water)). Error bars represent the standard deviations. Asterisks indicate significant differences in treatment effects compared to the untreated control as per the Dunnet post hoc test at p < 0.05.
Agronomy 14 02239 g006aAgronomy 14 02239 g006b
Agronomy 14 02239 g007
Figure 7. Effects of microbial biostimulants on Diabrotica virgifera virgifera larvae under greenhouse conditions. Each of the two experiments had 10 to 15 infested potted maize plants per treatment (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-N P K, 12-untreated control (unsterilized tap water)). Asterisks indicate significant differences in treatment effects compared to the untreated control as per the Dunnet post hoc test at p < 0.05.
Figure 7. Effects of microbial biostimulants on Diabrotica virgifera virgifera larvae under greenhouse conditions. Each of the two experiments had 10 to 15 infested potted maize plants per treatment (numbers on the x-axis represent the treatments tested: 1-Bacillus amyloliquefaciens, 2-Bradyrhizobium japonicum, 3-Bacillus subtilis, 4-Ensifer meliloti, 5-Rhizobium leguminosarum, 6-Trichoderma asperellum, 7-Beauveria bassiana, 8-Trichoderma harzianum, 9-Rhizophagus irregularis, 10-Chlorella vulgaris, 11-N P K, 12-untreated control (unsterilized tap water)). Asterisks indicate significant differences in treatment effects compared to the untreated control as per the Dunnet post hoc test at p < 0.05.
Agronomy 14 02239 g007
Table 1. Specifications of common microbial biostimulants tested for their insecticidal effects against different life stages of Diabrotica virgifera virgifera under standardized laboratory conditions.
Table 1. Specifications of common microbial biostimulants tested for their insecticidal effects against different life stages of Diabrotica virgifera virgifera under standardized laboratory conditions.
NumberActive IngredientsTrade NameActive Ingredient
in Product		FormulationTested Dosage Range
Eggs Larvae AdultsUnit
Bacteria
1Bacillus amyloliquefaciensCAPITO BIO®5 × 109 spore/mLliquid102–108101–108104–109spore/mL
2Bradyrhizobium japonicumPhylazonit® NG 2 × 109 cfu/mLliquid102–108103–108105–109cfu/mL
3Bacillus subtilisAmazoN®5 × 109 cfu/ggranule2 × 103–2 × 107101–108103–109cfu/mL
4Ensifer melilotiRhizoFix® RF-501 × 109 cfu/mLliquid102–108104–108105–109cfu/mL
5Rhizobium leguminosarumRhizoFix® RF-401 × 109 cfu/mLliquid2 × 102–2 × 106105–109105–2 × 108cfu/mL
Fungi
6Trichoderma asperellumHi-spore®3.5 × 107 cfu/gliquid102–1071.103–2 × 107105–107cfu/mL
7Beauveria bassianaBora R®5 m/m %powder103–107103–106103–107cfu/g
8Trichoderma harzianumTricho Immun®2 × 108 cfu/gpowder102–108103–107103–107cfu/g
9Rhizophagus irregularisLALRISE® MAX WP2000 spore/gpowder4 × 102–2 × 1032.101–2.1072 × 101–2 × 103spore/g
Algae
10Chlorella vulgarisBioplasma Algatrágya®2 × 107 cell/mLliquid102–107103–107105–2 × 107cell/mL
Positive and negative controls
11ImidaclopridConfidor ® 200SL200 mg/mLliquid44,900140µg/mL
12Untreated control
(sterilized tap water)
Table 2. Specifications of commercial microbial biostimulants tested for their effects on maize and Diabrotica virgifera virgifera larvae under greenhouse conditions. Each of the two experiments had 10 to 15 infested and 10 to 15 uninfested potted maize plants per treatment.
Table 2. Specifications of commercial microbial biostimulants tested for their effects on maize and Diabrotica virgifera virgifera larvae under greenhouse conditions. Each of the two experiments had 10 to 15 infested and 10 to 15 uninfested potted maize plants per treatment.
NumberActive IngredientsTrade NameActive Ingredient
Concentration in Product		FormulationDose Tested
Bacteria
1Bacillus amyloliquefaciensCAPITO BIO®5 × 109 spore/mLliquid104; 106; 108 spore/mL
2Bradyrhizobium japonicumPhylazonit ® NG2 × 109 cfu/mLliquid2 × 109 cfu/mL
3Bacillus subtilisAmazoN®5 × 109 cfu/ggranule5 × 109; 107; 109 cfu/g
4Ensifer melilotiRhizoFix® RF-501 × 109 cfu/mLliquid109 cfu/mL
5Rhizobium leguminosarumRhizoFix® RF-401 × 109 cfu/mLliquid104; 106; 108 cfu/mL
Fungi
6Trichoderma asperellumHi-spore®3.5 × 107 cfu/gliquid105 cfu/g
7Beauveria bassianaBora R®5 m/m %powder107 cfu/g
8Trichoderma harzianumTricho Immun®2 × 108 cfu/gpowder2 × 108 cfu/g
9Rhizophagus irregularisLALRISE® MAX WP2000 spore/gpowder2000 spores/g
Algae
10Chlorella vulgarisBioplasma Algatrágya®2 × 107 cell/mLliquid103; 105; 107 cell/mL
Positive and negative controls
11NPKBionova® Soil SupermixNPK 7-3-6; 2 mL/Lliquid2 mL/L
12Untreated control
(unsterilized tap water)
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Tarigan, S.I.; Kiss, J.; György, T.; Doan, N.P.Y.; Toepfer, S. Effects of Microbial Biostimulants on Maize and Its Pest, the Western Corn Rootworm, Diabrotica virgifera virgifera. Agronomy 2024, 14, 2239. https://doi.org/10.3390/agronomy14102239

AMA Style

Tarigan SI, Kiss J, György T, Doan NPY, Toepfer S. Effects of Microbial Biostimulants on Maize and Its Pest, the Western Corn Rootworm, Diabrotica virgifera virgifera. Agronomy. 2024; 14(10):2239. https://doi.org/10.3390/agronomy14102239

Chicago/Turabian Style

Tarigan, Sri Ita, Jozsef Kiss, Turóczi György, Nhu Phuong Y Doan, and Stefan Toepfer. 2024. "Effects of Microbial Biostimulants on Maize and Its Pest, the Western Corn Rootworm, Diabrotica virgifera virgifera" Agronomy 14, no. 10: 2239. https://doi.org/10.3390/agronomy14102239

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop