Advances in Crop Science and Technology
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Bacillus subtilis; Biozeid; binary mixtures; Eobania vermiculata; Monacha cartusiana

Introduction

Land snails are the most severe pests attacking crops worldwide (Barker, 2002). The terrestrial snails Eobania vermiculata and Monacha cartusiana are particularly the most common and harmful snails infesting various plants in Egypt (Mahrous et al., 2002 and Gabr et al., 2006). Chemical control of these snails using synthetic organic molluscicides is costly, environmentally unfriendly, toxic to non-target organisms, and challenging to sustain (Mansour, 2005; Santos et al., 2010 and Nyandwaro, 2018). Consequently, alternative means or new strategies for controlling land snails have become necessary. Biological control of land snails by using microbial agents such as bacteria and fungi represents an alternative control method to pesticides that has recently gained greater attention (Genena and Mostafa, 2008). Bacillus spp. is a gram-negative bacteria that can produce crystalline spores and toxic chemicals for pests (Genena, 2010) [1]. Bacillus megaterium bacterium and Trichoderma album fungus have a significant molluscicidal effect against E. vermiculata and Succinea putris snails (Mona et al., 2017).

Additionally, these bio-agents are considered typical soil beneficial bio-fertilizers that increase plant growth and represent potential biocontrol agents against nematode, which dramatically impacts vegetables and crops (Affokpon et al., 2011). The binary mixtures of biological agents are considered an effective strategy for enhancing practical pest control (Qiu-Yang, 2020). Moreover, it is represented excellent, effective alternative to chemical pesticides for controlling pests (Narayanasamy, 2006) [2, 3].

The present study was conducted to assess the potential of Bacillus subtilis spore suspension and the commercial product Biozeid (Trichoderma album), applied alone and in binary mixtures against the terrestrial snails, E. vermiculata and M. cartusiana under laboratory and field conditions. The mixing patterns of these bio-agents in the binary mixtures were also analyzed to find combinations with synergistic effects that are of interest for practical applications of bio-agents [4 ].

Materials and Methods

Tested snails

Adults of the brown garden snail, Eobania vermiculata were collected from infested navel orange trees at Banadf village, Meniet El-Kamh district, Sharkia Governorate, Egypt. In contrast, adults of the glassy clover snail, Monacha cartusiana, were collected from infested clover field at Kafr al-Ashraf village, Zagazig district, Sharkia Governorate, Egypt. Adult individuals of each snail species were kept in muslin bags and transferred to the laboratory. There were kept in a glass container containing moist clay soil and covered with muslin cloth secured with a rubber band to prevent snails from escaping. Snails were supplied daily with fresh cabbage leaves for 14 days to acclimate [5, 6].

Tested bio-agents

Preparation of Bacillus subtilis inoculum

The identified isolate Bacillus subtilis was grown on a Nutrient broth medium (5 g/l bactopeptone, 5g/l beef extract, five g/l NaCl) (Ph 7.2) and incubated at 37°C. The concentration of 4 × 105 spores/ml of B. subtilis was prepared for the laboratory and field treatments in this study by adding the appropriate amount of sterilized distilled water for the bacterial spores [7].

Preparation of Biozeid (Trichoderma album) concentrations

Weights of 0.5, 1.0, and 1.5 grams of the commercial formulation of the fungus Trichoderma album were diluted in 10 ml sterilized distilled water for laboratory treatments. Other weights from the same biopesticide formulation; 50, 100, and 150 grams, were diluted in 1000 ml sterilized distilled water for the field application to get, in both cases of laboratory and field experiments, the desired concentrations of 0.5 × 106, 1 × 106 and 1.5 × 106 spores/ml, respectively (Mona et al., 2017) [8].

Preparation of bio-agents binary mixtures

The concentration of 4 × 105 spores/ml of B. subtilis was mixed with each prepared concentration of T. album formulation (0.5 × 106, 1 × 106, and 1.5 × 106 spores/ml) by dissolving the amount of B. subtilis bacterium required to obtain the appropriate concentration in the water amount which the fungal concentration prepared in it (Hend, 2013) [9 ].

Effect of the tested bio-agents used individually and as binary mixtures against both snail species under the laboratory conditions

The individual molluscicidal activity of B. subtilis spores suspension at the concentration 4 × 105 spores/ml and T. album formulation at three tested concentrations of 0.5 × 106, 1 × 106, and 1.5 × 106 spores/ ml were evaluated against E. vermiculata and M. cartusiana snails. The effect of binary mixing between B. subtilis spores suspension and each concentration of T. album was also investigated against the same snail’s species under laboratory conditions [10].

This experiment was carried out using plastic boxes (3/4 kg capacity). Each box contained 1/2 kg of moistened sterilized clay soil. Ten adults of each snail species and ten discs of cabbage leaves were provided to each box on the soil surface. The spores suspension of B. subtilis and tested concentrations of T. album were sprayed separately on the soil and cabbage discs. The effect of the binary mixing between B. subtilis and each concentration of T. album against both snail species was also tested and sprayed in the same manner. Three replicates were prepared for the treatment with each bio-agent concentration and each binary mixture for the two snail species. The other three replicates were designed as the other boxes but without any treatment as a control. It is worth noting that each tested concentration of each bio-agent and mixture was sprayed at a rate of 10 ml for each replicate, and in the case of control, 10 ml of water only without any treatment was sprayed. All boxes were tightly covered with muslin cloth and secured with a rubber band to prevent snails from escaping. Mortality percentages of adults of each snail species at the treatment with bio-agents individually and as binary mixtures were recorded daily for a month and corrected by Abbott’s formula (1925) [11, 12].

Synergism analysis of tested bio-agents against both snail species

The interaction between the two bio-agents in the mixture against E. vermiculata and M. cartusiana snails after 21 days of the experiment was expressed as the Co-Toxicity factor (C.F.) determined according to Sun and Johnson (1960) as follows:

Where the observed mortality was the visualized efficiency of the two bio-agents applied in combination, and the Expected mortality was the sum of the efficiency of each bio-agent used separately. It was considered potentiation if the Co-Toxicity factor was positive factor ≥ 20. A negative factor ≤ 20 means antagonism, while intermediate values between – 20 and + 20 indicate an additive effect [14].

Evaluation of the tested bio-agents used individually and as binary mixtures against both snail species under the field conditions

This field trial was conducted in March–2020 at two fields of navel orange trees heavily infested with each E. vermiculata and M. cartusiana snails. The first is at Banadf village, Meniet El-Kamh district, Sharkia Governorate, for E. vermiculata treatment, and the other field is located at El-Krakra village, Meniet El-Kamh district, Sharkia Governorate, for M. cartusiana treatment. Three infested trees were sprayed by each of B. subtilis spores suspension at 4 × 105 spores/ml, and T. album tested concentrations 0.5 × 106, 1 × 106, and 1.5 × 106 spores/ml individually for the two snail species. The binary mixing between B. subtilis and each concentration of T. album was also tested; three infested trees were sprayed with each mixture for both snail species. Each tested concentration of each bio-agent and mixture was poured at a rate of 1000 ml per tree, and in the case of control trees, 1000 ml of water without any treatment was sprayed per tree. Before the spraying of treatments, live snails were counted in four directions of 25×25 cm under each tested and control tree, at 1 m high of the tree trunk, and on five branches of the different directions of a tree. The number of living snails was counted in the same areas of control and tested trees after 1, 3, 7, 14, 21- and 30-days post-treatment. Reduction percentages were calculated by the formula of Henderson and Tilton (1955) [15, 16 ].

Statistical analysis

All data were statistically analyzed, and the treatment means were compared by the least significant difference (LSD) test at P ≤ 0.05 levels according to the method of Costat’s (2005) statistical program analysis, computer program software.

Results and Discussion

Molluscicidal effect of tested bio-agents when applied alone and as binary mixtures against Eobania vermiculata and Monacha cartusiana snails under the laboratory conditions

The individual and mixture effect of B. subtilis spores’ suspension and T. album concentrations against E. vermiculata snails for a onemonth exposure period is shown in (Table 1) (Figure 1) from which it can be seen that no mortality occurred after one day of exposure to each bio-agent individually. After three days of the experiment, B. subtilis at the tested concentration of 4 × 105 spores/ml recorded 53.33% mortality of snails, then slightly increased to 56.66% on the seventh day and then remained fixed until the end of the experiment. While in the case of T. album, its first molluscicidal effect was recorded on the seventh day of the investigation, with a 20% mortality of snails at the concentrations of 0.5 × 106, 1 × 106 spores/ml, and 23.33% at the concentration of 1.5 × 106 spores/ml. The mortality of snails increased with the increase in the tested concentrations and the experiment period, reached to its maximum value of 26.66, 30 and 40%, respectively, for 0.5 × 106, 1 × 106, and 1.5 × 106 spores/ml concentrations on day 21 of the experiment and after which no further increase in effect occurred until the end of the investigation [17, 18].

Table 1: Molluscicidal effect of tested bio-agents used individually and as binary mixtures against Eobania vermiculata snails under the laboratory conditions

Figure 1: Molluscicidal effect of tested bio-agents used individually and as binary mixtures against Eobania vermiculata snails under laboratory conditions.

The molluscicidal effect of B. subtilis and T. album increased in all mixtures csompared to when applied alone. The combination of B. subtilis with the highest concentration of T. album 1.5 × 106 spores/ml achieved the highest mortality of 70% after 21 days of the experiment.

At the same trend, adding B. subtilis to the other tested concentrations of T. album 0.5 × 106 and 1 × 106 spores/ml recorded 60 and 63.33% mortality in the same experiment period, respectively. No other change occurred in the molluscicidal activity of all mixtures against snails until the end of the experiment. No mortalities were recorded in control at all. Moreover, it is strongly observed that there is a highly significant difference in the means of snail mortality compared to untreated snails [19].

The molluscicidal activity of B. subtilis and T. album was also investigated against M. cartusiana snails when applied separately and in mixtures. As illustrated in (Table 2) (Figure 2), no mortalities of snails were recorded by each bio-agent until the third day of the experiment. B. subtilis at the tested concentration of 4 × 105 spores/ml caused 40% mortality of snails on the seventh day of the investigation. The mortality increased up to 63.33% on day 21 of the treatment, after which no other increase in effects occurred until the end of the experiment. On the other hand, T. album showed only 16.66% mortality at the lowest concentration of 0.5 × 106 spores/ml on the seventh day of the experiment. While the other tested concentrations of 1 × 106 and 1.5 × 106 spores/ml recorded the same mortality of 20% after the same trial period [20, 21].

Table 2:  Molluscicidal effect of tested bio-agents used individually and as binary mixtures against Monacha cartusiana snails under the laboratory conditions

Figure 2: Molluscicidal effect of tested bio-agents used individually and as binary mixtures against Monacha cartusiana snails under laboratory conditions.

The lethal effect of T. album at all tested concentrations increased gradually, reached to 23.33 and 26.66% mortality, respectively, for 0.5 × 106 and 1 × 106 spores/ml after 21 days of the experiment. A rather observed increase in the molluscicidal effect of the highest concentration, 1.5 × 106 spores/ml, occurred by achieving 33.33% mortality after the same trial period. Concerning mixing B. subtilis with each tested concentration of T. album, adding B. subtilis to 0.5 × 106 or 1 × 106 spores/ml of T. album recorded the same mortality of 40% on the first day of the experiment. At the same time, it recorded 43.33% mortality in combination with the highest concentration of T. album 1.5 × 106 spores/ml during the same experiment period. Subsequently, the molluscicidal activity of all mixtures significantly increased by record 80, 86.66 and 90% at the combination of B. subtilis with each of 0.5 × 106, 1 × 106, and 1.5 × 106 spores/ml of T. album on day 21 of the trial, respectively. The lethal effect of all mixtures remained constant until the end of the experiment. On the other hand, no mortalities have been achieved in control. All the tested treatments showed a significant difference in the mortality of snails compared to the untreated individuals [22, 23].

The obtained results in (Tables 1-2). are strongly corroborated by Heiba et al. (2002) reporting that snails’ mortality rates increased with increased exposure to molluscicides. In this trend, Mona et al. (2017 showed that the mortality of E. vermiculata and snails caused by Bioarc (Bacillus megaterium) and Biozeid (Trichoderma album) was increased with an increase in the exposure period to these biopesticides. The same authors added that the molluscicidal activity of Bioarc was more than Biozeid against both snails’ species. Bioarc caused 20 and 26.66% mortality, respectively for E. vermiculata and S. putris snails after 21 days of treatment compared with only 6.66 and 10% mortality exhibited by Biozeid against the two snail’s species after the same period of the experiment, respectively. Ghamry (1997) also stated that Helicella sp. is more susceptible to Bacillus thuringiensis bacterium, followed by Monacha sp. and Eobania sp. In a similar study, Genena and Mostafa (2008) demonstrated that B. thuringiensis at the tested concentration 7 × 106 cfu/ml was more effective against Monacha cantiana than E. vermiculata snail, it was recorded 86.6% and 53% mortality of the two snails after four weeks of the treatment, respectively. On the other hand, Arafa (2006) reported that B. thuringiensis failed to record any molluscicidal effect against E. vermiculata snail [24, 25].

According to the report of Durel et al. (2015), mixtures of compounds used for pests control can contribute to preserve the effectiveness of existing these compounds against pests as long as possible. Little information has been published about mixing bioagents for use in the control of land snails. But in a related study, Hend (2013) showed that E. vermiculata adult snails were more susceptible to mixing Paecilomyces variotii with ammonium nitrate at the concentration 1.5%, Paecilomyces lilacinus with 1% of urea and T. album with ammonium nitrate at 2%. All these mixtures caused complete mortality of snails after 5, 6 and 8 weeks of the experiment, respectively. The same effect was achieved against M. cartusiana adults by combining P. lilacinus with each urea and ammonium nitrate at the concentration 1% after 5 and 6 weeks of the treatment, respectively [26].

On the other hand, mixing P. variotii with urea at 0.5% and T. album with each of ammonium nitrate and urea at 0.5% exhibited the lowest molluscicidal effect against the same snail species with record 60% mortality after 5 weeks of the trial. The same mortality percentage was recorded against M. cartusiana snails by the mixture of P. variotii with 0.5% ammonium nitrate after 8 weeks of the treatment. Moreover, the binary combination of urea with each of P. lilacinus and Trichoderma harzianum caused significant reduction in Meloidogyne incognita multiplication (Siddiqui and Shakeel, 2009). Selecting the relevant incision in the binary mixing is necessary for effective pest control (Levchenko and Silivanova, 2019) [27].

Synergism analysis of tested bio-agents against Eobania vermiculata and Monacha cartusiana snails

The joint action between B. subtilis and each tested concentration of T. album against E. vermiculata snails was determined. As cleared in (Table 3) after 21 days of application, the all mixtures showed an antagonistic effect against E. vermiculata snails with co-toxicity factors - 12.03, - 9.08 and – 5.40% for the combination of 4 × 105 spores/ml of B. subtilis with each of 0.5 ×106, 1 × 106 and 1.5 × 106 spores/ml of T. album, respectively. The joint action of B. subtilis with each tested concentration of T. album after 21 days of the application against M. cartusiana snails was also investigated. The obtained results in (Table 4) indicated that all mixtures of B. subtilis with T. album concentrations showed an observed additive effect against snails with co-toxicity factors 11.29, 18.54 and 19.12% for the mixing of 4 × 105 spores/ml of B. subtilis with T. album at each of 0.5 × 106, 1 × 106 and 1.5 × 106 spores/ml, consecutively [28].

Table 3: Synergism analysis of tested bio-agents against Eobania vermiculata snails

Table 4: Synergism analysis of tested bio-agents against Monacha cartusiana snails

The illustrated results in (Tables 3- 4) follow those obtained by Hend (2013) and showed that at the mixing of each of P. lilacinus, P. variotii and T. album with ammonium nitrate at the concentration 0.5% against E. vermiculata snails, there is an antagonistic effect was achieved with co-toxicity factors –76.47, -51.72 and -69.56%, respectively. The same effect of the same mixtures was also recorded against M. cartusiana snails with co-toxicity factors -75.67, -81.24 and -69.38%, respectively. On the contrary, an additive effect was recorded at the combination of P. variotii with urea at the concentration 1% against E. vermiculata snails with co-toxicity factor 15.42%. While, the variety of the same fungus with the same fertilizer at the concentration 0.5% showed an observed potentiation activity with co-toxicity factor 400.45% against the same snail species. In the same aspect, Guruswamy et al. (2017) recorded a substantial synergistic effect from mixing nerium extract with each tobacco, neem and piper extracts against Pomacea maculata snail. Similarly, a synergistic action was also determined at the combination of potassium sulphate with each copper sulphate, niclosamide and mollutox against Lymnaea natalensis snails [29 ].

Moreover, the mixing of urea with each of copper sulphate, niclosamide and mollutox also caused significant reduction on the growth rate and survival of this snail (Ragab and Shoukry, 2006). On another related study, Hend (2018) reported that an additive effect was achieved at the binary mixing of citrus limon peel juice with each of citrus limon fruit and Mentha spicata juices against M. cartusiana snails with co-toxicity factors 7.92 and 9.43%, respectively. While, the binary mixing of M. spicata with Coriandrum sativum recorded an observed potentiating activity against the same snail species with a co-toxicity factor 53.26%. At the same trend, Rao and Singh (2001) confirmed that the synergistic action of Acalypha indica and Cedrus deodara oil combination was more toxic to Lymnaea acuminata snail than the single treatment with each one [30 ].

Molluscicidal activity of tested bio-agents when applied individually and as binary mixtures against Eobania vermiculata and Monacha cartusiana snails under field conditions

The molluscicidal effect of both bio-agents B. subtilis and T. album was evaluated individually and as a binary mixture against E. vermiculata and M. cartusiana snails in March 2020 under the field conditions. As shown in (Table 5) (Figure 3) the molluscicidal effect of both bio-agents when combined is greater than the effect of each one alone against the E. vermiculata snails. At the individual treatment with each bio-agent, T. album recorded the highest initial effect against snails at the highest concentration, 1.5 × 106 spores/ml, achieving 21.12% reduction of snails. It was followed by B. subtilis at its only tested concentration 4 × 105 spores/ml which caused 20.03% reduction of snails then 18.52 and 13.42% reduction of snails which recorded by 1 × 106 and 0.5 × 106 spores/ml of T. album, respectively. The decrease in snails increased for all treatments with the trial period, reaching 51.90% reduction of snails, representing the highest residual effect exhibited by 1.5 × 106 spores/ml of T. album followed by 50.45% reduction of snails achieved by B. subtilis. The lowest residual effect was recorded by 1 × 106 and 0.5 × 106 spores/ml of T. album by gave 37.04 and 26.19% reduction of snails, respectively. On the other hand, at the treatment of snails with the binary mixtures of both bio-agents, the mixing of B. subtilis with the highest concentration of T. album 1.5 × 106 spores/ ml achieved the highest initial effect against snails by record 53.61% reduction followed by the binary mixing of B. subtilis with each of 1 × 106 and 0.5 × 106 spores/ml of T. album which gave 50.26 and 43.71% reduction of snails, respectively.

Table 5: Molluscicidal effect of tested bio-agents used alone and as binary mixtures against E. vermiculata snails under field conditions

Figure 3: Molluscicidal effect of tested bio-agents used alone and as binary mixtures against E. vermiculata snails under field conditions.

These same mixtures in the same order achieved a noticeable effect against snails with increasing the experiment period by record 77.95, 72.63 and 67.21% reduction of snails as a residual effect, respectively. The obtained data also showed a significant difference in the decrease in snails treated with all tested treatments compared with the untreated snails in control.

At studying the molluscicidal activity of the same two bio-agents also separately and as binary mixtures against M. cartusiana snails under the field conditions, the results in (Table 6) (Figure 4) indicated that the effect of B. subtilis against snails at its only tested concentration 4 × 105 spores/ml was more than T. album effect at the all concentrations [31].

Table 6: Molluscicidal effect of tested bio-agents used alone and as binary mixtures against M. cartusiana snails under field conditions

Figure 4: Molluscicidal effect of tested bio-agents used alone and as binary mixtures against M. cartusiana snails under field conditions.

B. subtilis recorded the highest residual effect 39.12% reduction of snails followed by 36.50, 36.11 and 32.42% reduction which was recorded by 1.5 × 106, 1 × 106 and 0.5 × 106 spores/ml of T. album, respectively. The binary mixing of B. subtilis with the highest concentration of T. album 1.5 × 106 spores/ml caused the highest residual effect against snails by recording 89.46% reduction of snails. It was followed by the binary combination of B. subtilis with 1 × 106 and 0.5 × 106 spores/ml of T. album by record 84.12 and 79.62% reduction of snails, respectively. That is a significant difference in the decrease in treated snails with the different tested treatments compared to the untreated individuals. From the previous results, it could be concluded that the molluscicidal activity of B. subtilis and T. album as binary mixtures against both snail species is more than their effect separately and M. cartusiana snails were highly explicitly affected by the tested binary mixtures than the snails of E. vermiculata [32].

The obtained results in (Tables 5-6) are in harmony with those reported by Hend (2013) demonstrating that the microbial molluscicides were effective when sprayed on the healthy snails. T. album didn’t record observable mortality of E. vermiculata and M. cartusiana snails at all tested concentrations during the field treatment’s first period. But at the second week, the reduction of snails started to increase. It was recorded 16.66, 20 and 41.66% reduction of E. vermiculata snails at 1 × 103, 2 × 103 and 4 × 103 spores/ml concentrations after 21 days of the field treatment, consecutively. While, after the same period the same fungus at the same concentrations caused 10, 11.66 and 23.33% reduction of M. cartusiana snails, respectively. The inability of the fungus to achieve any lethal effect against pests at the beginning of the treatment is due to a few days between spraying the fungus and kill and continued of the pest in feeding (Abd El-Fattah, 2005). On the other hand, Raul et al. (2008) explained that Trichoderma species are successful fungi in field trials to control pests in general because it produced many secondary metabolites and various degradative enzymes such as chitinase. Trichoderma viride achieved a significant reduction of land snails under the field conditions (Babou and Jayakumar, 2009). At the same time, Ghamry (1997) reported that the suspension of Bacillus thuringiensis bacterium recorded higher molluscicidal impact against Helicella vestalis snail than E. vermiculata in the field. Moreover, Zedan et al. (1999) added that the formulation of the same bacterium was the more effective against Monacha obstructa snail. At a pea field highly infested with land snails, this bacterium formulation reduced 23.54% of M. cartusiana snails after one week of the treatment. The reduction of snails increased by increasing the application period to 28.03% after 21 days of the trial (Mortada et al., 2012). There is no published paper or studies conducted to support the effect of combining two microorganisms against terrestrial snails. But in this trend, Godan (1983) published that the effectiveness of fungi against terrestrial pulmonates can be increase by using other active ingredients in combination. The mixing of T. album with ammonium nitrate at the concentration of 2% achieved the highest molluscicidal activity against E. vermiculata snails by recording 53.33% reduction after 21 days of the field experiment. This mixture was followed by the combination of Paecilomyces variotii with ammonium nitrate at the concentration of 1.5% and Paecilomyces lilacinus with urea at 1% by record 37.50 and 20.83% reduction of snails after the same period of application, respectively. Whereas, at testing an impact of the same mixtures against M. cartusiana snails also in the field, the highest molluscicidal effect was recorded by P. variotii mixed with ammonium nitrate at 1.5% which caused 82.50% reduction of snails after 21 days of the application. After the same period, the combination of T. album with ammonium nitrate at 2% and P. lilacinus with urea at 1% achieve a 73.33% and 71.66% reduction in snails, respectively (Hend, 2013) [33].

Conclusion

The binary mixing of the spore suspension of B. subtilis with the biopesticide formulation Biozeid (T. album) has a strong effect against E. vermiculata and M. cartusiana snails more than the use of each bioagent individually. M. cartusiana snails were more affected by the tested mixtures than the snails of E. vermiculata under laboratory and field conditions. Mixing these bio-agents is considered a natural, effective, inexpensive molluscicide with lower environmental toxicity than the other commonly used molluscicides. Thus, its production offers a possible alternative strategy for controlling the field populations of E. vermiculata and M. cartusiana intending to design a new biopesticide formulation.

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