نوع مقاله : مقاله پژوهشی
نویسندگان
گروه گیاهپزشکی دانشکده کشاورزی، دانشکدگان کشاورزی و منابع طبیعی دانشگاه تهران، کرج، ایران
چکیده
کلیدواژهها
عنوان مقاله [English]
نویسندگان [English]
The snail Achatina fulica (Gastropoda, Achatinidae) is one of the most significant agricultural pests, characterized by its extensive distribution and broad host range among various plant species. The most common method for controlling this pest is the application of the chemical molluscicide metaldehyde. However, this approach has proven to be inefficient and environmentally harmful. Consequently, there is an urgent need to explore alternative methods, such as applying biological control agents. Bacillus thuringiensis (Bt) is a key microbial agent widely used for the control of insect pests, yet its potential for managing agricultural pest molluscs has not been extensively studied. In the present study, the effects of Bt prepared as a locally available commercial product, were evaluated against A. fulica using both direct and indirect (endophytic) approaches. Lettuce, as a preferred host plant for the snail, was cultivated in a greenhouse and used for snail feeding. For the preparation of Bt in its vegetative phase, nutrient agar (NA) was used. The endophytic potential of Bt was investigated using two methods: leaf spraying and root drenching of six-week-old lettuce plants with bacterial suspension at a concentration of 8.7 × 10⁸ CFU/mL. Data collected 10 days after inoculation indicated successful endophytic colonization exclusively through the leaf spray method, while leaves from plants treated via root irrigation did not harbor endophytic bacteria. The efficacy of Bt was assessed under some treatments, including immersion of lettuce leaves in bacterial spore-crystal suspension (5 × 10⁸ and 10⁹ CFU/mL), vegetative-phase bacterial suspension without crystals (5 × 10⁸ cells/mL), suspension of Xanthomonas translucens (control 1), and sterile distilled water (control 2). These treatments were applied to adult and non-adult snails of A. fulica. The results revealed no visible effects on adult snails under any treatment. However, non-adult snails treated with a concentration of 10⁹ CFU/mL exhibited a complete inhibition of lettuce feeding from the second day of treatment, persisting for 21 days. To confirm the presence of the bacterium in the gastrointestinal tract of non-adults that ceased feeding, the gut was aseptically dissected 14 and 22 days post-treatment. Suspensions obtained by homogenizing the gut in sterile distilled water were cultured on nutrient agar plates. After five days of incubation, the presence of Bt spores and crystals was confirmed. Molecular analysis using PCR and sequencing of the 16S rRNA gene further validated the bacterial identity through comparison with genetic databases. Although no direct mortality of A. fulica was observed, the cessation of feeding in immature stages for three weeks significantly reduced the damage caused by the snail.
کلیدواژهها [English]
Extended Abstract
Introduction
The African snail Achatina fulica is a widespread and voracious agricultural pest. It has many host plants. The most common way to control this pest is using chemical pesticides, mainly metaldehyde. However, it is ineffective against the African snail. It also has harmful effects on the environment. Thus, it is very urgent to search for another way of control, for example applying biological agents. Bacillus thuringiensis bacterium is one of the most important component of microbial control that has been well studied as a biocontrol agent against many insect pests. On the other side, this bacterium has not yet been studied in detail as a control agent for agricultural mollusks. In this research, the effect of BT is studied directly and indirectly on the snail A. fullica.
Materials and Methods
Lettuce (Lactuca sativa) was cultivated in a greenhouse and used as snail feed. Various treatments were tested, including: dipping lettuce leaves in Bt suspensions in two different concentrations and phases, Xanthomonas translucens suspension as control 1, and sterile distilled water as Control 2. Eggs, non-adult, and adult snails were assessed. To confirm the presence of the bacterium in the digestive tract of immature individuals that had ceased feeding, the digestive tract was aseptically removed at intervals of 14 and 22 days post-treatment. The suspension obtained from homogenization in a sterile mortar containing sterile distilled water was cultured on nutrient agar plates. Five days after incubation, the presence of Bt spores and crystals was examined. To confirm that the bacteria isolated from the gastrointestinal tract were indeed B. thuringiensis, bacterial DNA was extracted and subjected to PCR amplification. The resulting PCR product was sequenced for analysis. The obtained sequence was then analyzed and compared with existing sequences in NCBI database to ensure accurate identification. Additionally, in a separate experiment, the bacterium was introduced into lettuce plants through irrigation and foliar spray to test the capabilities of internal colonization. For this, 10 days post-inoculation, leaf samples were collected, surface-sterilized, and homogenized, the homogenate was plated and incubated for five days to confirm B. thuringiensis endophytic colonization. After incubation, the presence of Bt-specific spores and crystal proteins was assessed to verify successful colonization.
Results
No visible effects were observed on eggs and adult snails across all treatments. However, non-adult snails treated with the Bt suspension at a concentration of 109 CFU/ml ceased feeding from the second day of treatment onwards and continued for 21 days. Bt presence in the homogenized suspension from the gastrointestinal tracts of non-adult, starved snails was confirmed by the observation of Bt-specific spores and crystal. The bacterial identity was confirmed through sequence analysis of the 16S rRNA gene and comparison with the NCBI-BLAST database. Additionally, the study demonstrated that B. thuringiensis successfully colonized lettuce leaves endophytically when applied through foliar spraying. Ten days post-inoculation, Bt-specific spores and crystals were detected in lettuce leaves treated with a Bt suspension via spraying. In contrast, no evidence of endophytic colonization was observed in plants treated through root drenching, indicating that the method of application significantly influences Bt's ability to colonize lettuce endophytically. The effect of the bacterium in its endophytic state on both adult and non-adult snails is under investigation to study the effect caused by endophytic bacteria through a host plant on the snails.
The findings of the present study illustrate that, though B. thuringiensis does not have a direct effect on the adult A. fulica, its effect is remarkable at high concentrations on the feeding of non-adult snails. This effect on feeding behavior of snail can result in reduced and delayed development, which can have cascading effects on the overall population dynamics of A. fulica. On the other hand, the capability of Bt to establish an endophytic relationship with lettuce through foliar application enhances new possibilities for its application in integrated pest management systems. This may, therefore, provide a long-term, systemic resistance against pests and hence reduce reliance on periodic chemical applications. The potential of Bt as a biocontrol agent has great implications for sustainable agriculture and should be further studied for its long-term efficacy and various impacts on the different crops and snail species.
All authors contributed equally to the conceptualization of the article and writing of the original and subsequent drafts.
The data will be made available on request.
This research was financially supported by the UniversityCollege of Agriculture and Natural Resources, University of Tehran. We thank University of Theran for providing research facility and support.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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