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Article

Germination and Agronomic Traits of Phaseolus vulgaris L. Beans Sprayed with Trichoderma Strains and Attacked by Acanthoscelides obtectus

by
Álvaro Rodríguez-González
1,*,
Marcos Guerra
2,
Daniela Ramírez-Lozano
1,
Pedro Antonio Casquero
1 and
Santiago Gutiérrez
3
1
Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Instituto de Medio Ambiente Recursos Naturales y Biodiversidad (INMARENBIO), Escuela de Ingeniería Agraria y Forestal (EIAF), Universidad de León, Avenida de Portugal 41, 24071 León, Spain
2
Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Escuela de Ingeniería Agraria y Forestal (EIAF) (Campus de Ponferrada), Universidad de León, Avenida de Astorga s/n, 24401 Ponferrada, Spain
3
Área de Microbiología, Escuela de Ingeniería Agraria y Forestal (Campus de Ponferrada), Universidad de León, Avenida de Astorga s/n, 24401 Ponferrada, Spain
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(11), 2130; https://doi.org/10.3390/agronomy11112130
Submission received: 14 September 2021 / Revised: 17 October 2021 / Accepted: 22 October 2021 / Published: 24 October 2021
(This article belongs to the Special Issue Using Biological Control Agents in Crop Protection)
Browse Figures
Versions Notes

Abstract

:
Acanthoscelides obtectus, one of the world’s most important post-harvest pests, attacks wild and cultivated common beans (Phaseolus vulgaris L.). Four Trichoderma strains, Trichoderma arundinaceum IBT 40,837 (=Ta37), a wild-type strain producer of trichothecene harzianum A (HA); two transformed strains of Ta37, Ta37-17.139 (Δtri17) and Ta37-23.74 (Δtri23); and T. brevicompactum IBT 40,841 (=Tb41), a wild-type strain producer of the trichothecene trichodermin, were evaluated to determine the effect of these compounds on the virulence of A. obtectus and the effect of these strains on the seed’s capacity of germination and on the agronomic traits of the plants grown from these seeds. Treatments of bean seeds with different Trichoderma strains provided varying survival rates in A. obtectus adults, so life survival of insects after Tb41 strain application was reduced to 15 days. Δtri17 and Tb41 strains sprayed on P. vulgaris beans resulted in low weight losses (1.21 and 1.55%, respectively). In spite of the low germination percentage of beans treated with Δtri23 strain (lower than the germination percentages of the rest of the fungal strains applied), this treatment encouraged a greater Wet Weight of Aerial Part of the plants grown from both damaged and undamaged beans. High germination rates of Ta37 and Δtri17 strains (higher than with the rest of treatments), did not turn into a greater Wet Weight Aerial Part and Wet Weight of Root System in the future plants developed. Linear regression between the number of exit holes and the wet weight aerial part on the one hand, and between the number of exit holes and the wet weight root system on the other, showed interaction, so Δtri23 and Tb41 strains behaved differently in comparison to their respective control treatments. The number of exit holes of beans treated with Δtri23 or Tb41 was negatively correlated with both the wet weight aerial part and the wet weight root system in P. vulgaris plants. Δtri23 sprayed on undamaged beans caused the greatest Wet Weight Aerial Part and wet weight root system in plants. Due to the good results obtained by Δtri23 and Tb41 strains in this work, more studies for A. obtectus control, P. vulgaris plant growth and trichothecenes production by these strains should be explored, in order to advance in the knowledge of how these fungi could be used in the field crop, together with the application of management strategies to mitigate risks for farmers and to minimize environmental contamination.

1. Introduction

Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae: Bruchidae), the bean weevil, is one of the world’s most important post-harvest pests in dry beans (Phaseolus vulgaris (L.) (Fabaceae)) [1,2]. It is mainly found in South America and Africa and in the Mediterranean area [3,4,5], where adults attack bean seeds while they are still in the field and continues to cause damage during storage. These attacks can lead to the total loss of stored bean seeds [6,7] as the larvae enter the seeds not only to feed on them but also to transform from larva to adult inside [8]. The population of this insect pest can grow exponentially and can completely destroy the stored crops in a few months [9]. Until now, the control of A. obtectus insects in storage is based on the application of synthetic insecticides [10], or is carried out through physical barriers, such as the use of hermetic packaging [11]. The continuous application of synthetic insecticides has led to concerns about pest resistance, risk to human health and environmental contamination [12]. All these reasons have motivated the search for more sustainable alternatives for pest control [13]. Recently, different sustainable alternatives to control this pest have been described, such as those through the use of essential oils [14], fungi [15,16,17,18,19] or bacteria [20]. The use of fungi to control invertebrate pests, weeds and plant diseases during the last years has grown due to the numerous commercial products that have been marketed or are still under development [21] to minimize side effects on the indigenous-beneficial organisms and on the environment.
Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya) is a well-studied fungal genus currently consisting of more than 200 molecularly defined species [22]. Trichoderma species are generally considered cosmopolitan and prevalent components of different ecosystems in a wide range of climatic zones [23]. However, some species are ubiquitous while others are limited to specific geographical areas [24]. Many Trichoderma spp. are non-pathogenic soil-borne fungi considered opportunistic, avirulent and plant symbionts which are able to colonize the roots of many plants [24]. Up to now, it is known that these fungi are highly beneficial for agriculture due to their ability to protect crops against diseases and to improve overall crop yields [25]. These Trichoderma strains can perform their biocontrol activity by several mechanisms, the most important being mycoparasitism, antibiosis and competition. Furthermore, the most efficient biocontrol strains display, simultaneously or sequentially, more than one of those biocontrol strategies [26].
Trichoderma strains hold a huge potential to produce a wide variety of secondary metabolites in their genomes [27,28], such as pyrones [6-pentyl-2H-pyran-2-one (6-PP) derivatives] [29,30], butenolides [31], peptaibols [32,33], terpenes (e.g., trichothecenes, triterpenes) [34,35,36,37] and gliotoxin, viridin, harzianopyridone and harziandione [28,38].
Terpene compounds have a variety of roles in mediating antagonistic and beneficial interactions between organisms [39]. These interactions can occur between arthropods and host plants infected with fungi, but there is a lack of information related to the impact of mycotoxins, as for example, volatile mycotoxins or intermediates of their biosynthesis, produced by saprofitic-beneficial fungi on visiting herbivores [40].
Trichothecenes, a group of non-volatile sesquiterpenoid mycotoxins, have a central core of fused cyclohexene/tetrahydropyran rings [41,42]. Most of these compounds are phytotoxic, have high antibiotic activity and high toxicity for human and animals, resulting in skin or intestinal mucosa irritation and effects in the immune and nervous system [43]. Up to now, the knowledge about trichothecenes interaction with plants is very limited, and their phytotoxic activity is widely known because they suppress the defense response in host plants [44]. However, the trichothecene harzianum A (HA), produced by T. arundinaceum (Zafari, Gräfenhan & Samuels), is not harmful for plants when assayed in vivo, and it also induces the expression of plant defense genes linked to the salicylic acid (SA) and jasmonic acid (JA) pathways [35].
The aims of this work were: (1) to determine the effect of compounds and intermediates produced by different wild-type and transformant Trichoderma strains sprayed over P. vulgaris beans against A. obtectus adults; (2) to analyse the effect of these strains on the germination capacity of beans and on the agronomic traits of the plants grown from beans treated with the selected fungal strains, and that later were damaged or undamaged by A. obtectus larvae.

2. Materials and Methods

2.1. Fungal Strains Evaluated

Four Trichoderma strains were evaluated: Trichoderma arundinaceum IBT 40,837 (=Ta37) and T. brevicompactum IBT 40,841 (=Tb41), two wild-type strains producers of trichothecenes harzianum A (HA) and trichodermin, respectively [45], and Ta37-17.139 (Δtri17) and Ta37-23.74 (Δtri23), two transformants of Ta37, isolated in previous works, in which the genes tri17 and tri23, respectively, both involved in the HA biosynthetic pathway, were deleted [37,46]. Δtri17 and Δtri23 mutants do not produce HA, but in both cases accumulate trichodermol, one of the intermediates in the synthesis of HA [37,46].

2.2. Insect Collection and Rearing

The original population of A. obtectus was collected during the years 2017, 2018 and 2019 from storages located in the Protected Geographical Indication (PGI) “Alubia de La Bañeza-León” (EC Reg. n.256/2010 published on 26 March 2010, OJEU L880/17). The common bean (Phaseolus vulgaris L.) “Canela” variety, was used to feed the different A. obtectus stages. To keep the insects under laboratory conditions before and after experiments the methodology described by Rodríguez-González et al. [16,19,20] was used.

2.3. Fungal Culture Conditions

PPG medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used for the growth of the fungal isolates according to the methodology described by Lindo et al. [47]. In order to obtain fungal spores and to calculate the final concentration of spores used in the experiments, the methodology described by Rodríguez-González et al. [16,20] was used.

2.4. Design of Experiments

2.4.1. Experiment 1: Effects of Beans Sprayed with Trichoderma Strains on A. obtectus Insects

With a manual loading Potter Tower (Burkard Scientific Limited, Po Box 55 Uxbridge, Middx UB8 2RT, UK) (Figure 1a), one ml of the spores’ suspension (1 × 107 spores/mL) of each Trichoderma strain was directly applied on 40 P. vulgaris beans placed in a Petri dish (90 mm diameter) (Figure 1b) following the methodology described by Potter [48]. Distilled water was used as a control treatment and carrier in all the treatments with fungal isolates. Beans (treated with Trichoderma strains or theirs controls) were transferred to a structure made up of five circular plastic containers (Figure 1c). Four containers (A, B, C and D) (40 mm diameter and 70 mm high) with a central container (E) (120 mm diameter and 60 mm high) connected to the other four containers by plastic cylinders (70 mm long and 7 mm in diameter). Containers B and D were arranged diagonally and filled with the 40 beans treated with the Trichoderma strain. Containers A and C (controls) were filled with the 40 beans treated with the controls. In the central container, 20 A. obtectus adults (10 males and 10 females) were released. Four treatments (4 Trichoderma strains) with four replicates of 20 adults were used for each treatment. After 24 h once A. obtectus adults decided their location in containers A, B, C or D, beans (treated with Trichoderma strains or their controls) and insects were transferred back to Petri dishes where the treatments had been applied with the Potter tower. The insect mortality of A. obtectus adults in contact with the beans was recorded daily. After 15 days, the weight loss (2 × 40 beans) due to the insects’ attacks on beans (treated with Trichoderma strains or their controls) in each Petri dish and treatment were recorded.

2.4.2. Experiment 2: Evaluation of the Germination Capacity of Beans

One-litre polypropylene pots filled with peat (TYPical, Brill, Georgsdorf, Germany) were stored in climatic chambers under controlled conditions in order to test the germination capacity of bean seeds previously in contact with Trichoderma spp. (inoculated as described in Section 2.4.1) or control samples (sprayed with distilled water as described in Section 2.4.1) and A. obtectus insects. Each pot was filled with 250 mL of water prior to sowing. Five undamaged beans (1 seed without any hole from every Petri dish, described above; see Section 2.4.1, Experiment 1) were randomly selected and sown in five pots, each bean in an individual pot. Five damaged beans (beans with at least one hole per bean of every container caused by A. obtectus), were selected and sown in five pots, each bean in an individual pot. Controlled conditions were maintained for 45 days, which involved exposing seeds to a photoperiod of 16 h light, day/night temperatures of 25 °C/16 °C, 60% RH and brightness of 3500 lux. Irrigations were performed every 4 days with about 250 mL tap water per pot according to the methodology described by Mayo et al. [49,50]. A nutrient solution was added on the 2nd-4th week according to the methodology described by Rigaud and Puppo [51]. Plants were removed after 45 days and the parameters “wet weight” and “dry weight” (72 h in an oven, at 82 °C) of the aerial part and root system were calculated. Four replicates for each fungal isolate were used.

2.5. Statistical Analysis

Experiment 1. Mortality data of A. obtectus insects were submitted to survival analysis estimator using IBM SPSS Statistics for Windows, Version 26.0. (Armonk, NY, USA: IBM Corp.), and the functions obtained from each treatment were compared with the Wilcoxon-Gehan test (p < 0.05).
Experiment 2. A randomly completed experiment using the Generalized Linear Model (GLM) procedure, with four treatments and four replicates was subjected to ANOVA, using IBM SPSS Statistics for Windows, Version 26.0. (Armonk, NY, USA: IBM Corp.). Differences (p < 0.05) among weight losses of plants grown from damaged beans were examined by mean comparisons using Fisher least significant difference (LSD) tests.
Experiment 3. Germination capacity of beans was submitted to the Kaplan–Meier estimator and the functions obtained from each treatment were compared by the log-rank test (Mantel-Cox) (p < 0.05). Analysis of covariance (ANCOVA) was used to examine the effect of the number of exit holes (NEH) of damaged beans (fixed factor) on the Wet Weight of the Aerial Part (WWAP) of P. vulgaris plants (obtained from beans previously sprayed with different treatments) as a covariate. The linear regression coefficients of the NEH x WWAP interaction were tested using an F-test, considering a significance level at p ≤ 0.05. Analyses were conducted using IBM SPSS Statistics for Windows, version 26.0. (Armonk, NY, USA: IBM Corp.). A randomly completed experiment using the Generalized Linear Model (GLM) procedure, with four treatments and four replicates (damaged and undamaged beans) was subjected to ANOVA using IBM SPSS Statistics for Windows, Version 26.0. (Armonk, NY, USA: IBM Corp.). Differences (p < 0.05) among WWAP and WWRS were examined by mean comparisons using Fisher least significant difference (LSD) test.

3. Results

3.1. Effects of Beans Sprayed with Spores of Trichoderma Strains on the Survival Rate of A. obtectus Insects

The treatment influences the survival rate of insects (Wilcoxon-Gehan = 13.631; df = 7,141; p = 0.050) (Figure 2). Insects subjected to treatments with Tb41 and Ta37 had a significant lower survival rate after 15 days, 0 and 20% respectively, than those subjected to their respective controls.
Moreover, these strains, Tb41 and Ta37, provided a significant lower survival rate for the insect population than the rest of the fungal strains applied to the beans, Δtri17 and Δtri23, whose insects had survival rates after 15 days of 67 and 71%, respectively (Figure 2).

3.2. Weight Losses of Beans Damaged by A. obtectus Larvae Treated with Trichoderma Strains and Controls

Weight losses in beans treated with Trichoderma strains ranged from 1.21% to 1.93%, whereas untreated beans (sprayed with distilled water) showed a weight loss between 1.58% and 3.99%. While beans treated with Δtri17 (F = 8.292; df = 1,9; p = 0.037) and Tb41 F = 7.082; df = 1,10; p = 0.043) strains showed a significantly lower weight loss than their controls, differences were not significant between beans treated with Δtri23 strain and its control (Figure 3).

3.3. Effect of A. obtectus on the Germination of Beans and on Agronomic Traits of Future P. vulgaris Plants

3.3.1. Beans Sprayed with Spores of Trichoderma Strains and Damaged by A. obtectus Larvae

Table 1 shows that the type of Trichoderma strain used influences the germination capacity of damaged beans (log-rank χ2 = 15.424; df = 5,28; p = 0.009). Thus, damaged beans previously sprayed with Δtri17 had a higher germination percentage than damaged beans treated with Δtri23. In addition, Δtri17 and Tb41 treatments allowed the beans to have a significantly lower number of exit holes per bean (1 and 1.33) and a lower weight loss (1.21% and 1.55%) than their respective controls.
Although damaged beans previously sprayed with Δtri23 strain had a lower germination percentage than both the control and the damaged beans treated with the rest of the fungal strains. Δtri23 treatment provided plants with a significantly higher wet weight aerial part (26.68 g) than Δtri17 treatment.

3.3.2. Beans Sprayed with Spores of Trichoderma Strains and Undamaged by A. obtectus Larvae

Treatments with Trichoderma strains used in the present work affected the germination capacity of undamaged beans (log-rank χ2 = 40.663; df = 7,152; p < 0.001), so beans sprayed with strain Δtri23 had a germination percentage higher than beans sprayed with Tb41 strain but lower than beans sprayed with Ta37 strain (Table 2).
The wet weight aerial part of plants grown from undamaged beans subjected to Δtri23 strain was significantly higher (35.03 g) than the control (25.38 g). Additionally, the wet weight aerial part of plants grown from beans treated with this strain was significantly higher than the wet weight aerial part of plants grown from beans treated with the other Trichoderma strains.
Regarding the root system, undamaged beans sprayed with Δtri23 strain provided plants with a higher wet weight root system (4.92 g) than those grown from beans treated with the rest of the Trichoderma strains, Ta37, Δtri23 and Tb41, with weights of 3.52, 3.19 and 2.15 g, respectively (Table 2).

3.4. Agronomic Traits of Plants Grown from Damaged Beans

3.4.1. Wet Weight Aerial Part in Relation to the Number of Exit Holes in Beans

The interaction between the wet weight aerial part and the number of exit holes was not performed with the Ta37 strain due to lack of data (Figure 4a).
The wet weight aerial part of plants as function of the number of exit holes in damaged beans between beans sprayed with Δtri23 and its control was not significantly different (F = 1.251; df = 1,9; p = 0.296). The linear regression coefficients of the wet weight aerial part x number of exit holes interaction between Δtri23 and its control treatments were significantly different (F = 7.766; df = 1,9; p = 0.024). Beans treated with Δtri23 provided an increase in the wet weight aerial part of the plants when the number of exit holes was lower (Figure 4b).
The wet weight aerial part of plants as function of the number of exit holes in damaged between beans sprayed with Δtri17 and its control was not significantly different (F = 0.000; df = 1,9; p = 0.997). The linear regression coefficients of the wet weight aerial part x number of exit holes number of exit holes between Δtri17 and its control treatments were not significantly different (F = 12.916; df = 1,9; p = 0.409). The wet weight aerial part of plants and number of exit holes of beans were not correlated in beans treated with Δtri17 strain (Figure 4c).
The wet weight aerial part of plants as function of the number of exit holes in damaged beans between beans sprayed with Tb41 and its control was not significantly different (F = 0.941; df = 1,9; p = 0.357). The linear regression coefficients of the wet weight aerial part x number of exit holes interaction between Tb41 and its control treatments were significantly different (F = 3.2740; df = 1,9; p = 0.104). Beans treated with Tb41 provided plants with bigger wet weight aerial part whenever the number of exit holes declined (Figure 4d).

3.4.2. Wet Weight Root System in Relation to the Number of Exit Holes in Beans

The interaction between wet weight root system and number of exit holes was not performed with the Ta37 strain due to lack of data (Figure 5a).
The wet weight root system of plants as function of the number of exit holes in damaged beans between beans sprayed with Δtri23 and its control was not significantly different (F = 1.236; df = 1,9; p = 0.299). The linear regression coefficients of the wet weight root system x number of exit holes interaction between Δtri23 and its control were significantly different (F = 8.377; df = 1,9; p = 0.020). The number of exit holes of beans treated with Δtri23 was negatively correlated with the wet weight root system of the plants grown from seed (Figure 5b).
The wet weight root system of plants as function of the number of exit holes in damaged beans between beans sprayed with Δtri17 and its control was significantly higher on beans sprayed with the control than in beans treated with Δtri17 strain (F = 16.420; df = 1,9; p = 0.004). The linear regression coefficients of the wet weight root system x number of exit holes interaction between Δtri17 and its control treatments were not significantly different (F = 0.554; df = 1,9; p = 0.478). The wet weight root system was not correlated with the number of exit holes in beans treated with Δtri17 strain (Figure 5c).
The wet weight root system of plants as function of the number of exit holes in damaged beans between beans sprayed with Tb41 strain and its control was not significantly different (F = 0.165; df = 1,9; p = 0.694). The linear regression coefficients of the wet weight root system x number of exit holes interaction between Tb41 strain and its control were significantly different (F = 5.690; df = 1,9; p = 0.041). The number of exit holes in beans treated with Tb41 strain was negatively correlated with the wet weight root system of the plants grown from seed (Figure 5d).

3.4.3. Agronomic Traits in Relation to the Number of Exit Holes in Beans Based on the Strain Applied

The linear regression coefficients of the wet weight aerial part x number of exit holes interaction among the different Trichoderma strains applied to damaged beans were significantly different (F = 16.852; df = 2,11; p = 0.004). The greatest increase in the wet weight aerial part of plants in relation to the decrease in the number of exit holes of beans was observed in beans treated with the Δtri23 strain (Figure 6a).
The linear regression coefficients of the wet weight root system x number of exit holes interaction among the different Trichoderma strains treatments applied to damaged beans were significantly different (F = 15.556; df = 2,11; p = 0.003). The greatest increase in the wet weight root system of plants in relation to the decrease in the number of exit holes of beans was observed in beans treated with Δtri23 strain (Figure 6b).

3.5. Comparison of Agronomic Traits between Plants Obtained from Damaged or Undamaged Beans

Regarding the beans sprayed with the fungal strains used in the present work, results indicated that undamaged beans sprayed with Δtri23 strain provided plants with significantly greater wet weight aerial part and wet weight root system (35.03 and 4.92 g, respectively) than those grown from damaged beans sprayed with the same strain. Moreover, undamaged beans sprayed with Δtri23 strain provided plants with the significantly greatest wet weight aerial part and wet weight root system when compared with undamaged beans sprayed with the rest of the strains. Furthermore, plants grown from beans sprayed with Δtri17 and Tb41 strains reached significantly higher wet weight root system (3.19 and 2.15 g, respectively), than plants grown from damaged beans and sprayed with the same strains (Table 3, left side).
Regarding the beans sprayed with distilled water (controls), results indicated that undamaged beans sprayed with the control of Δtri17 provided plants with significantly greater wet weight aerial part (32.16 g) than those grown from beans sprayed with the control of Δtri17 but previously damaged by A. obtectus larvae. In addition, undamaged beans sprayed with the control of Δtri17 provided plants with the significantly greatest wet weight aerial part and wet weight root system. Furthermore, plants grown from beans in the controls of Δtri23 and Δtri17 treatments had significantly greater wet weight root system (3.70 and 3.62 g, respectively) than plants grown from damaged beans under the same control treatments. Besides, plants grown from undamaged beans in the control of Δtri23, had a significantly greater Wet Weight Root System, than plants grown from undamaged beans in Tb41 control (Table 3, right part).

4. Discussion

Application of Trichoderma strains and their controls (=with distilled water) on beans provided different survival rates in the insects. Tb41 significantly reduced the survival of the insects until 20 days after the treatment’s application, and it was the only treatment to obtain a survival rate of the insects of 0% at that time. On the other hand, Δtri23 was the treatment that provided the greatest survival rate of insects, so the cumulative survival rate after 15 days of the treatment was 71%. This longer life span of the insects allowed them to lengthen the reproductive period, which led to a greater weight loss of beans due to cotyledon damage caused by this insect’s larvae (see below for a detailed explanation). Previous works have addressed modification of insect development by treatment of their host (seeds in this case) with fungi. Coppola et al. [52] reported that treatments with the fungal biocontrol agent T. atroviride strain in tomato plants induce responses in insect pests with different feeding habits: as for example the noctuid moth Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and the aphid Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), assuring that the tomato plant–Trichoderma interaction has a negative impact on the development of moth larvae and on aphid longevity. Coppola et al. [52] confirmed that these effects were attributed to a plant response induced by Trichoderma that was associated with transcriptional changes of a wide array of defense-related genes. Many studies have described the potential of Trichoderma spp. as a natural control agent against some targeted insects, as for instance, its effect against the Egyptian cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae), achieving a 20% of larvae survival rate [53]. T. harzianum provided pathogenicity against pest such as the beetle larvae Tenebrio molitor (Coleoptera: Tenebrionidae) [54] or Xylosandrus crassiusculus (Coleoptera: Curculionidae) [55]. In this experiment, Tb41 proved to be able of controlling larvae of A. obtectus insect pest since it was able to decrease the survival rate of the insect to 0%. This, this fungus can be considered a highly effective tool for the control of this insect pest in the larval stage.
Application of treatments on P. vulgaris beans modified the behavior of A. obtectus adults and their attacks on exposed beans. Δtri17 and Tb41 strains conferred effective protection against the insect pest, since these strains caused the beans to have lower weight loss (1.21 and 1.55%, respectively) than their respective controls, due to the low number of larvae that ingested their cotyledons, a fact that can be confirmed by observing the lower NEH of the insects in beans treated with these strains. On the other hand, the greatest weight loss was observed in beans sprayed with Δtri23, which favoured a higher concentration of A. obtectus adults, and later the attack of their larvae once they hatched from the eggs laid on the beans. There are other references showing modification of insect development and behavior by treatment of their host’s seeds with fungi. The attraction of insects towards their plant hosts when they are infected with fungi has already been described in previous studies and can be produced by the Volatile Organic Compounds (VOC’s) emitted by them. Most VOC’s in plants are products or by products of primary metabolic pathways [56,57]. Sithobion avenue (Hemiptera: Aphididae) was attracted by the VOC’s produced by their plant host when infected with Fusarium strains that produced the trichothecene derivative nivalenol (NIV) [40]. On the opposite side, aphids were repelled by VOC’s produced by their hosts when infected with Fusarium strains producing deoxynivalenol (DON), another trichothecene derivative [40,58]. The application of T. citrinoviride and T. harzianum strains on beans “canela variety” reduced the attack of A. obtectus larvae, obtaining less damaged beans and lower number of holes on damaged beans than not treated beans [15,20]. The studies carried out by Akello and Siroka [59] reported that the inoculation of fungal isolates (one of them, T. asperellum M2RT4) in bean seeds reduced the population of Acyrthosiphon pisum Harris (Homoptera: Aphididae) compared to population growth observed in control seeds. Menjivar-Barahona [60] described the reduction of whitefly population in tomatoes inoculated with T. atroviride. According to Rodriguez-González et al. [17], the treatment of vine wood trunks with different Trichoderma strains and Beauveria bassiana (GHA strain) reduced the population of Xylotrechus arvicola (Coleoptera: Cerambycidae) larvae bored into Vitis vinifera grapevine wood. Other genera of entomopathogenic fungi, Metarhizium spp., Beauveria spp., Isaria spp., have also been shown to be highly effective for the control of other pests of seeds or stored products by applying these fungi on their hosts. For example, Metarhizium anisopliae (TR 106 strain) and Beauveria bassiana (TR 217 strain), against the adults of the cowpea weevil, Callosobruchus maculatus F. (Coleoptera: Chrysomelidae: Bruchinae) in laboratory obtained a very successful biological control of this insect pest in laboratory through the two isolated described [61]. B. bassiana (GHA strain) proved to have a high inhibition capacity in A. obtectus eggs [15]. Or the brown planthopper (Nilaparvata lugens), that is an insect pest of rice (Oryza sativa), in which granular formulation of Isaria javanica was able to control N. lugens populations in rice fields [62].
Damaged beans previously sprayed with Δtri17 and Tb41 had a higher germination percentage (100 and 66%, respectively) than those subjected to Δtri23 (62.50%) and their respective controls. Although damaged beans previously sprayed with Δtri23 had a lower germination percentage than the control and the beans treated with the rest of the fungal treatments, Δtri23 provided plants with a significantly higher wet weight aerial part (26.82 g) than Δtri17. With regards to undamaged beans, healthy beans previously sprayed with Δtri23 treatment had a high germination percentage (75%) and plants grown from seeds subjected to this treatment had a wet weight aerial part significantly larger than the control and also higher than those from other Trichoderma strains. In relation to the root system, undamaged beans sprayed with Δtri23 provided plants with a higher WWRS than plants from beans treated with the rest of the Trichoderma strains. Regardless of the seed condition, the application of Δtri23 provided plants with a great wet weight aerial part, in spite of its poor percentage of germination in comparison to the control or to the rest of the strains applied, whereas high germination rates of Ta37 and Δtri17 (greater than their controls or than the rest of the assessed strains) did not lead to great wet weight aerial part or wet weight root system values in the plants obtained. The use of biological control agents is one of the alternatives for seed treatment to reach greater sustainability in agriculture [63,64]. Trichoderma strains are widely used in seed treatment, but little is known about the possible interactions between Trichoderma spp. and seeds in the early stages of germination [65], or the dosage to be applied. Dalzotto et al. [64] obtained a high germination percentage in P. vulgaris seeds treated with Trichoderma. Another example is the inoculation of tomato seeds with T. harzianum (Rifai T-22 strain) through a conidia solution of 2 × 107 colony forming units per gram of seeds, which had a positive effect on seed germination and growth in in vitro culture [65]. The improvement in the germination of seeds treated by Trichoderma strains may be due to the production of hormones from Trichoderma [64]. T. harzianum produces harzianic acid and isoharzianic acid, which promote plant growth [66]. On the opposite, an excessive production of indole acetic acid (IAA), ethylene [67], auxins and cytokinins hormones [68] inhibit cell division and elongation, impairing both germination and the development of seedling [66].
Linear regression coefficients of both the number of exit holes x wet weight aerial part and the number of exit holes x wet weight root system interactions were significantly different between Δtri23 or Tb41 strains and their respective control treatments. The number of exit holes of beans treated with Δtri23 or Tb41 was negatively correlated with both the wet weight aerial part and the wet weight root system in P. vulgaris plants.
The application of Trichoderma strains on bean seeds provided different wet weights in the plants grown for 45 days. Undamaged beans sprayed with Δtri23 provided plants not only with significantly greater wet weight aerial part and wet weight root system (35.03 and 4.92 g, respectively) than those grown from damaged beans sprayed with the same strain, but also greater than the plants from their respective control and higher than the plants grown from the seeds treated with the rest of the strains.
The studies performed by Chang et al. [69], Hermosa et al. [70] and Studholme et al. [71] described the beneficial effects of Trichoderma in horticultural crops such as: cucumber, periwinkle, chrysanthemum and lettuce, based on an improvement of their seed germination, vegetative growth and flowering. Furthermore, works made by Björkman et al. [72], Yedidia et al. [73], Björkman [74], Harman [75], Vargas et al. [76], Azarmi et al. [77] and Pereira et al. [78] in crops such as cucumber, maize, bean and tomato, emphasized correlations between previous inoculation with Trichoderma spp. and increases of root growth or shoot biomass production (increases in weight, shoot length and leaf area). Azarmi et al. [77], explained that T. harzianum (T-969 isolate) and Trichoderma spp. directly applied to tomato seeds yielded plants with greater shoot height and diameter, and larger shoot fresh and dry weights. Application of Trichoderma inoculum at an early stage of crop growth maximizes its benefits in terms of root development and nutrient uptake [66].

5. Conclusions

Treatments of bean seeds with different Trichoderma strains provided different survival rates in A. obtectus adults, so life survival of insects after Tb41 strain application was reduced to 15 days. Δtri17 and Tb41 strains decrease weight losses of P. vulgaris beans (1.21 and 1.55%, respectively). Regardless of the seed condition, the application of Δtri23 promoted plants with a great wet weight aerial part, in spite of their poor percentage of germination in comparison to their control or the rest of the strains applied, whereas high germination rates of Ta37 and Δtri17 strains (greater than their controls or than the rest of the assessed strains) did not lead to great wet weight aerial part or wet weight root system values in the plants obtained. Linear regression between number of exit holes and wet weight aerial part on one hand, and between number of exit holes and wet weight root system on the other showed interaction, so Δtri23 and Tb41 behaved differently in comparison to their respective control treatments. The number of exit holes of beans treated with Δtri23 or with Tb41 was negatively correlated with both the wet weight aerial part and the wet weight root system in P. vulgaris plants. Undamaged beans sprayed with Δtri23 strain provided plants with the greatest wet weight aerial part and Wet Weight Root System. Due to the good results obtained by Δtri23 and Tb41 in this work, more studies for A. obtectus control, P. vulgaris plant growth and trichothecenes production by these strains should be explored, in order to advance the knowledge on how these fungi could be used in the field crop, together with the application of management strategies to mitigate risks for the farmers and to minimize the environmental contamination.

Author Contributions

Conceptualization, Á.R.-G., D.R.-L. and M.G.; methodology, D.R.-L., M.G. and S.G.; formal analysis, Á.R.-G. and P.A.C.; investigation, Á.R.-G. and M.G.; writing—original draft preparation, Á.R.-G.; writing—review and editing, P.A.C. and S.G.; project administration, P.A.C. and S.G.; funding acquisition, P.A.C. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding

We want to thank to the Education Department of the Junta de Castilla y León for the project “Aplicación de cepas de Trichoderma en la producción sostenible de judía de calidad (Reference: LE251P18)”, and the Spanish Ministry of Science, Innovation and Universities for the project “Aislamiento de cepas de Trichoderma productoras de trichotecenos a partir de cultivos de judía y estudio de su efecto en la defensa de la planta frente a enfermedades fúngicas (RTI2018-099600-B-I00)”.

Data Availability Statement

Not applicable.

Acknowledgments

We want to thank the Ministry of Science, Innovation and Universities (Spain) (Resolution of 27 July 2018, BOE No. 184, of 31 July) for the financial support of this work, that was awarded with a grant to Álvaro Rodríguez González (PTA2017e14403-I) through the program Technical Support Staff (Call 2017).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. (a) Potter Spray Tower; (b) sprayed beans in Petri dishes; (c) structure of plastic containers used in the experiment.
Figure 1. (a) Potter Spray Tower; (b) sprayed beans in Petri dishes; (c) structure of plastic containers used in the experiment.
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Figure 2. Mortality of A. obtectus exposed to beans sprayed with Trichoderma strains during the 15 days. Global comparisons of mortality rate for all treatments (Wilcoxon-Gehan = 13.631; df = 7,141; p = 0.050). Pairwise comparison among all treatments (Wilcoxon-Gehan). Different lowercase letters indicate significant differences among days within each Trichoderma strain or its control; (p < 0.05). Different capital letters indicate significant differences among treatments within the same day of evaluation; (p < 0.05).
Figure 2. Mortality of A. obtectus exposed to beans sprayed with Trichoderma strains during the 15 days. Global comparisons of mortality rate for all treatments (Wilcoxon-Gehan = 13.631; df = 7,141; p = 0.050). Pairwise comparison among all treatments (Wilcoxon-Gehan). Different lowercase letters indicate significant differences among days within each Trichoderma strain or its control; (p < 0.05). Different capital letters indicate significant differences among treatments within the same day of evaluation; (p < 0.05).
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Figure 3. Weight losses (% ± SE) of treated beans (sprayed with Trichoderma strains) and their controls (sprayed with distilled water). Different lowercase letters indicate significant differences among treatments (Trichoderma strains on the one hand, and controls on the other hand) (p ≤ 0.05), LSD test at 0.05. Different capital letters indicate significant differences between treated beans with Trichoderma strains and their controls (p ≤ 0.05). Data obtained from the weight loss of 80 beans.
Figure 3. Weight losses (% ± SE) of treated beans (sprayed with Trichoderma strains) and their controls (sprayed with distilled water). Different lowercase letters indicate significant differences among treatments (Trichoderma strains on the one hand, and controls on the other hand) (p ≤ 0.05), LSD test at 0.05. Different capital letters indicate significant differences between treated beans with Trichoderma strains and their controls (p ≤ 0.05). Data obtained from the weight loss of 80 beans.
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Figure 4. Linear regression of the wet weight aerial part (WWAP) of P. vulgaris plants (x-axis) versus the number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Intense Green Point” means bean sample sprayed with Trichoderma strains: Ta37, (b) Δtri23, (c) Δtri17 and (d) Tb41. “Triangular Light Green Point” means the controls beans sample sprayed with distilled water: (a) Ta37 (control), (b) Δtri23 (control), (c) Δtri17 (control) and (d) Tb41 (control). Linear regression trendlines are coloured based on the treatment (“Continuous Intense Green Line” represents bean samples sprayed with Trichoderma strains; “Discontinuous Triangular Light Green Line” indicates bean samples sprayed with distilled water).
Figure 4. Linear regression of the wet weight aerial part (WWAP) of P. vulgaris plants (x-axis) versus the number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Intense Green Point” means bean sample sprayed with Trichoderma strains: Ta37, (b) Δtri23, (c) Δtri17 and (d) Tb41. “Triangular Light Green Point” means the controls beans sample sprayed with distilled water: (a) Ta37 (control), (b) Δtri23 (control), (c) Δtri17 (control) and (d) Tb41 (control). Linear regression trendlines are coloured based on the treatment (“Continuous Intense Green Line” represents bean samples sprayed with Trichoderma strains; “Discontinuous Triangular Light Green Line” indicates bean samples sprayed with distilled water).
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Figure 5. Linear regression of wet weight root system (WWRS) of P. vulgaris plants (x-axis) versus number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Intense Green Point” means bean samples sprayed with Trichoderma strains: Ta37, (b) Δtri23, (c) Δtri17 and d) Tb41. “Triangular Light Green Point” means the controls beans samples sprayed with distilled water: (a) Ta37 (control), (b) Δtri23 (control), (c) Δtri17 (control) and (d) Tb41 (control). Linear regression trendlines are coloured based on the treatment (“Continuous Intense Green Line” represents bean samples sprayed with Trichoderma strains; “Discontinuous Triangular Light Green Line” indicates bean samples sprayed with distilled water).
Figure 5. Linear regression of wet weight root system (WWRS) of P. vulgaris plants (x-axis) versus number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Intense Green Point” means bean samples sprayed with Trichoderma strains: Ta37, (b) Δtri23, (c) Δtri17 and d) Tb41. “Triangular Light Green Point” means the controls beans samples sprayed with distilled water: (a) Ta37 (control), (b) Δtri23 (control), (c) Δtri17 (control) and (d) Tb41 (control). Linear regression trendlines are coloured based on the treatment (“Continuous Intense Green Line” represents bean samples sprayed with Trichoderma strains; “Discontinuous Triangular Light Green Line” indicates bean samples sprayed with distilled water).
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Figure 6. (a) Linear regression of wet weight aerial part (WWAP) of P. vulgaris plants (x-axis) versus Number of Exit Hole (NEH) of P. vulgaris beans (y-axis). (b) Linear regression of wet weight root system (WWRS) of P. vulgaris plants (x-axis) versus number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Green Point” means bean samples sprayed with Ta37. ’Triangular Red Point” means bean samples sprayed with Δtri23. “Diamond-shaped Blue Point” means bean samples sprayed with Δtri17. “Square Orange Point” means bean samples sprayed with Tb41. Linear regression trendlines are coloured based on the strain (“Green Line” represents Ta37 strain, ‘Red Line’ represents Δtri23 strain, ‘Blue Line’ represents Δtri17 strain, ‘Orange Line’ represents Tb41 strain).
Figure 6. (a) Linear regression of wet weight aerial part (WWAP) of P. vulgaris plants (x-axis) versus Number of Exit Hole (NEH) of P. vulgaris beans (y-axis). (b) Linear regression of wet weight root system (WWRS) of P. vulgaris plants (x-axis) versus number of exit holes (NEH) of P. vulgaris beans (y-axis). “Round Green Point” means bean samples sprayed with Ta37. ’Triangular Red Point” means bean samples sprayed with Δtri23. “Diamond-shaped Blue Point” means bean samples sprayed with Δtri17. “Square Orange Point” means bean samples sprayed with Tb41. Linear regression trendlines are coloured based on the strain (“Green Line” represents Ta37 strain, ‘Red Line’ represents Δtri23 strain, ‘Blue Line’ represents Δtri17 strain, ‘Orange Line’ represents Tb41 strain).
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Table
Table 1. Germination capacity of damaged beans sprayed with different Trichoderma strains and agronomic traits of P. vulgaris plants after 45 days (and their controls).
Table 1. Germination capacity of damaged beans sprayed with different Trichoderma strains and agronomic traits of P. vulgaris plants after 45 days (and their controls).
TreatmentsBeans Number Exit Holes *
(Mean ± SE)		 Bean Weight Losses *
(% ± SE)		Weight Wet Aerial Part *
(Mean ± SE)		Weight Wet Root System *
(Mean ± SE)
TotalGerminatedNot
Germinated		Germination
(%)
Ta37---- -χ2 -χ2 -χ2 -χ2
Control---- -df -df -df -df
P P P P
Δtri2385362.50 2.25 ± 0.69 aA a,b0.594 2.21 ± 0.73 aA a,b0.020 26.68 ± 2.04 aA a,b0.917 1.96 ± 0.37 aA a,b0.947
Control330100.00 1.33 ± 0.33 aB(1,9) 1.58 ± 0.38 aB(1,9) 24.83 ± 1.78 aA(1,9) 1.87 ± 0.12 aA(1,9)
0.441 0.887 0.338 0.331
Δtri17330100.00 1.00 ± 0.00 bB3.225 1.21 ± 0.02 bB12.771 21.25 ± 2.45 aB1.247 1.15 ± 0.36 aA3.109
Control87187.50 4.25 ± 2.01 aA(1,9) 4.71 ± 2.30 aA(1,9) 21.24 ± 2.88 aA(1,9) 1.79 ± 0.48 aA(1,9)
0.050 ≤0.001 0.264 0.078
Tb4132166.66 1.33 ± 0.27 bAB3.400 1.55 ± 0.31 bAB4.752 26.55 ± 0.95 aA0.222 1.59 ± 0.18 bA8.333
Control96366.66 2.66 ± 0.66 aAB(1,10) 2.91 ± 0.57 aAB(1,10) 27.00 ± 2.90 aA(1,10) 2.65 ± 0.34 aA(1,10)
0.047 0.043 0.637 0.004
Trich.Ctrl. Trich.Ctrl. Trich.Ctrl. Trich.Ctrl.
χ25.9714.230χ24.7044.454χ29.2901.901χ25.0181.241
df(2,11)(2,17)df(2,11)(2,17)df(2,11)(2,17)df(2,11)(2,17)
P0.0370.048P0.0440.045P0.0100.387P0.0810.538
* Test for equality of germination capacity of damaged beans between treatments and controls (Log Rank; Mantel-Cox). a Different lowercase letters indicate significant differences between treated and untreated bean (control) within each strain; (p < 0.05). b Different capital letters indicate significant differences among fungal strains (on one hand) and among their controls (on the other hand); (p < 0.05).
Table
Table 2. Germination capacity of undamaged beans sprayed with different Trichoderma strains and agronomic traits of P. vulgaris plants after 45 days (and their controls).
Table 2. Germination capacity of undamaged beans sprayed with different Trichoderma strains and agronomic traits of P. vulgaris plants after 45 days (and their controls).
TreatmentsBeans Weight Wet Aerial Part *
(Mean ± SE)		 Weight Wet Root System *
(Mean ± SE)
Total GerminatedNot
Germinated 		Germination
(%)
Ta372016480.00 24.73 ± 1.77 bB a,bχ2 = 3.573 3.52 ± 0.36 aB a,bχ2 = 0.029
Control2014670.00 29.30 ± 1.67 aAdf(1,38) 3.62 ± 0.40 aAdf(1,38)
p = 0.05 p = 0.865
Δtri232015575.00 35.03 ± 1.63 aAχ2 = 12.342 4.92 ± 0.53 aAχ2 = 2.262
Control2015575.00 25.38 ± 2.11 bBdf(1,38) 3.70 ± 0.46 aAdf(1,38)
p ≤ 0.001 p = 0.133
Δtri172016480.00 23.38 ± 2.31 bBχ2 = 7.487 3.19 ± 0.84 aBχ2 = 1.081
Control2014670.00 32.16 ± 2.66 aAdf(1,38) 3.62 ± 0.56 aAdf(1,38)
p = 0.006 p = 0.298
Tb412091145.00 26.98 ± 1.68 aBχ2 = 1.790 2.15 ± 0.24 aCχ2 = 1.011
Control2013765.00 23.58 ± 1.22 aBdf(1,38) 2.50 ± 0.24 aBdf(1,38)
p = 0.181 p = 0.315
Trich.Ctrl. Trich.Ctrl.
χ222.17316.672 χ221.4758.454
df(3,76)(3,76) df(3,76)(3,76)
pp ≤ 0.001p ≤ 0.001 pp ≤ 0.001p = 0.037
* Test for equality of germination capacity of undamaged beans between treatments and controls (Log Rank; Mantel-Cox). a Different lowercase letters indicate significant differences between treated and untreated beans (control) within each strain; (p < 0.05). b Different capital letters indicate significant differences among fungal strains (on one hand) and among their controls (on the other hand); (p < 0.05).
Table
Table 3. Comparison of agronomic traits (grams; mean ± SE) of P. vulgaris plants grown for 45 days from damaged and undamaged beans sprayed with Trichoderma strains and their controls.
Table 3. Comparison of agronomic traits (grams; mean ± SE) of P. vulgaris plants grown for 45 days from damaged and undamaged beans sprayed with Trichoderma strains and their controls.
TreatmentsSeed
Condition		 Agronomic TraitTreatmentsSeed Condition Agronomic Trait
Wet Weight Aerial Part Wet Weight Root System Wet Weight
Aerial Part		 Wet Weight
Root System
Ta37U.* 24.73 ± 1.77 BF
df
P		 3.52 ± 0.36 BF
df
P		ControlU. * 29.30 ± 1.67 AB F
df
P		 3.62 ± 0.40 ABF
df
P
Ta37D.** - -ControlD. ** - -
Δtri23U. 35.03 ± 1.63 aA a,b7.082
(1,18)
0.016		 4.92 ± 0.53 aA a,b9.775
(1,18)
0.006		ControlU. 25.38 ± 2.11 aBC a,b0.012
(1,16)
0.912		 3.70 ± 0.46 aA a,b3.329
(1,16)
0.046
Δtri23D. 26.68 ± 2.04 bA 1.96 ± 0.37 bAControlD. 24.83 ± 1.78 aA 1.87 ± 0.12 bA
Δtri17U. 23.38 ± 2.31 aB0.073
(1,17)
0.790		 3.19 ± 0.84 aB5.858
(1,17)
0.038		ControlU. 32.16 ± 2.66 aA6.705
(1,18)
0.019		 3.62 ± 0.56 aAB4.560
(1,18)
0.047
Δtri17D. 21.25 ± 2.45 aA 1.15 ± 0.36 bAControlD. 21.24 ± 2.88 bA 1.79 ± 0.48 bA
Tb41U. 26.98 ± 1.68 aB0.098
(1,8)
0.762		 2.15 ± 0.24 aB4.921
(1,8)
0.043		ControlU. 23.58 ± 1.22 aC1.213
(1,17)
0.286		 2.50 ± 0.24 aB0.027
(1,17)
0.872
Tb41D. 26.55 ± 0.95 aA 1.59 ± 0.18 bAControlD. 27.00 ± 2.90 aA 2.65 ± 0.34 aA
Trich.
(U.)		Trich.
(D.)		 Trich.
(U.)		Trich.
(D.)		 Ctrl.
(U.)		Ctrl.
(D.)		 Ctrl.
(U.)		Ctrl.
(D.)
F7.8131.448 F6.5640.969 F3.8551.141 F1.8801.256
df(3,50)(2,6)df(3,50)(2,6) df(3,53)(2,13)df(3,53)(2,13)
p≤0.0010.307p≤0.0010.432 p0.0140.350p0.1440.317
a Different lowercase letters indicate significant differences between damaged and undamaged beans (seed condition) within each treatment and agronomic trait (wet weight aerial part or Wet Weight Root System); LSD test at 0.05. b Different capital letters indicate significant differences among fungal strains (on one hand) or among control treatments (on the other hand) within each seed condition and agronomic trait (wet weight aerial part or wet weight root system); LSD test at 0.05. * Seed condition: Undamaged (U.); ** Seed condition: Damaged (D.).
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Rodríguez-González, Á.; Guerra, M.; Ramírez-Lozano, D.; Casquero, P.A.; Gutiérrez, S. Germination and Agronomic Traits of Phaseolus vulgaris L. Beans Sprayed with Trichoderma Strains and Attacked by Acanthoscelides obtectus. Agronomy 2021, 11, 2130. https://doi.org/10.3390/agronomy11112130

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Rodríguez-González Á, Guerra M, Ramírez-Lozano D, Casquero PA, Gutiérrez S. Germination and Agronomic Traits of Phaseolus vulgaris L. Beans Sprayed with Trichoderma Strains and Attacked by Acanthoscelides obtectus. Agronomy. 2021; 11(11):2130. https://doi.org/10.3390/agronomy11112130

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Rodríguez-González, Álvaro, Marcos Guerra, Daniela Ramírez-Lozano, Pedro Antonio Casquero, and Santiago Gutiérrez. 2021. "Germination and Agronomic Traits of Phaseolus vulgaris L. Beans Sprayed with Trichoderma Strains and Attacked by Acanthoscelides obtectus" Agronomy 11, no. 11: 2130. https://doi.org/10.3390/agronomy11112130

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