Bt Research 2024, Vol.15 http://microbescipublisher.com/index.php.bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher.
Bt Research 2024, Vol.15 http://microbescipublisher.com/index.php.bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher MicroSci Publisher Editedby Editorial Team of Bt Research Email: edit@bt.microbescipublisher.com Website: http://microbescipublisher.com/index.php/bt Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Bt Research (ISSN 1925-1939) is an open access, peer reviewed journal published online by MicroSciPublisher. The journal is publishing high quality original research on all aspects of Bacillus thuringiensis and their toxins affecting the living organisms, as well as environmental risk and public policy relevant to Bt modified organisms. Topics include (but are not limited to) Bt strain identification, novel Bt toxin discovery and bioassay, transgenic Bt plants, insecticidal mechanism of Bt toxin as well as resistant mechanisms of target-insect to Bt toxin. All the articles published in Bt Research are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MicroSciPublisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Bt Research (online), 2024, Vol. 15 ISSN 1925-1939 http://microbescipublisher.com/index.php/bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content 2024, Vol.15, No.3 【Review Article】 Regulatory Approval and Market Adoption of Bt-based Biopesticides 131-140 Jiawei Li DOI: 10.5376/bt.2024.15.0013 【Research Insight】 Comparative Analysis of Plasmid Prfiles in Bt Islates frm Different Habitats 118-130 Yinghua Chen, Zhongqi Wu DOI: 10.5376/bt.2024.15.0012 【Research Report】 Phylogenetic Analysis of Bt Strains: Insights into Genetic Relationships and Divergence 141-153 Bing Wang, Qikun Huang DOI: 10.5376/bt.2024.15.0014 【Feature Review】 Genomic Architecture of Bacillus thuringiensis: Insights into Functional Elements 154-163 Jiamin Wang, Jin Zhang DOI: 10.5376/bt.2024.15.0015 【Review and Progress】 Integrated Pest Management Strategies Incorporating Bacillus spp. for Control of Meloidogyne enterolobii 110-117 Wenli Yin, Kexiu Lin, Yanling Huang, Yan Zhou DOI: 10.5376/bt.2024.15.0011
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 110 Review and Progress Open Access Integrated Pest Management Strategies Incorporating Bacillus spp. for Control of Meloidogyne enterolobii Wenli Yin, Kexiu Lin, Yanling Huang, Yan Zhou Guangxi Key Laboratory for Polysaccharide Materials and Modifications, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning 530008, Guangxi, China Corresponding author: yanzhou@gxun.edu.cn Bt Research, 2024, Vol.15, No.3 doi: 10.5376/bt.2024.15.0011 Received: 08 Mar., 2024 Accepted: 19 Apr., 2024 Published: 06 May, 2024 Copyright © 2024 Yin, Lin, Huang, and Zhou, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Yin W.L., Lin K.X., Huang Y.L., and Zhou Y., 2024, Integrated pest management strategies incorporating Bacillus spp. for control of Meloidogyne enterolobii, Bt Research, 15(3): 110-117 (doi: 10.5376/bt.2024.15.0011) Abstract The emergence of Meloidogyne enterolobii as a significant agricultural pest has necessitated the development of novel and sustainable pest management strategies. This study explores the potential of incorporating Bacillus spp. as a biological control agent within an Integrated Pest Management (IPM) framework for the control of M. enterolobii. Building on previous research that demonstrated the efficacy of Bacillus spp. against Meloidogyne spp., this review evaluates the specific mechanisms through which Bacillus spp. can manage M. enterolobii populations in agricultural settings. The research utilizes a combination of in planta assays, split root assays, RT-qPCR, and qPCR to assess the direct antagonistic capabilities of Bacillus spp. against M. enterolobii and their systemic effects on host plants. Results indicate that certain Bacillus strains, such as B. amyloliquefaciens QST713 and B. firmus I-1582, can effectively colonize plant roots and induce systemic resistance through the jasmonic acid (JA) and salicylic acid (SA) pathways, thereby reducing nematode population density and enhancing plant defense mechanisms. Additionally, the study compares the performance of Bacillus spp. with chemical nematicides, highlighting the potential for these bacteria to not only suppress nematode populations but also promote plant growth and yield, as evidenced in tomato plants. The findings suggest that Bacillus spp. could be a viable component of IPM strategies, offering a sustainable alternative to chemical nematicides for the management of M. enterolobii in agricultural systems. Keywords Bacillus spp.; Integrated pest management; Meloidogyne enterolobii; Biological control; Systemic acquired resistance; Sustainable agriculture 1 Introduction Meloidogyne enterolobii, commonly known as the guava root-knot nematode, is a highly virulent pest that poses a significant threat to global agriculture. Since its initial description in 1983, M. enterolobii has been recognized for its ability to infect a wide range of economically important crops, leading to substantial yield losses and jeopardizing food security, especially in regions like sub-Saharan Africa (Collett et al., 2021). The nematode's resilience and adaptability make it a formidable adversary for farmers and researchers alike, necessitating the development of effective and sustainable management strategies. The urgency to find sustainable pest management solutions is driven by the growing awareness of the environmental and health risks associated with conventional chemical nematicides. These concerns have catalyzed the search for eco-friendly alternatives that can be integrated into pest management programs with minimal ecological impact. Biological control agents, particularly those belonging to the genus Bacillus, have emerged as promising candidates in this regard (Yin et al., 2021). Bacillus spp. are well-known for their biocontrol properties, including the ability to form protective biofilms, produce antimicrobial compounds, and induce systemic resistance in plants. For instance, Bacillus cereus strain Bc-cm103 has demonstrated remarkable efficacy against Meloidogyne incognita, a close relative of M. enterolobii, by causing high mortality rates in nematode juveniles and reducing egg hatching. Moreover, this strain has been shown to activate defense-responsive genes in host plants, providing an additional layer of protection against
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 111 nematode infection (Yin et al., 2021). These findings underscore the potential of Bacillus spp. as biocontrol agents and pave the way for their application in integrated pest management (IPM) strategies against M. enterolobii. This review paper aims to explore the integration of Bacillus spp. into IPM strategies for the control of M. enterolobii. By examining the current state of research, we will assess the viability of these biological control agents and discuss their potential role in sustainable agriculture. The need for innovative and environmentally conscious approaches to pest management has never been greater, and Bacillus spp. may hold the key to safeguarding crop production against the pervasive threat of M. enterolobii. 2Bacillus spp. as Biocontrol Agents 2.1 General characteristics of Bacillus spp. that contribute to their biocontrol potential Bacillus species are well-recognized for their biocontrol potential, primarily due to their ability to produce a wide array of antimicrobial compounds. These compounds are effective against various plant pathogens, including fungi, bacteria, and nematodes. The biocontrol efficacy of Bacillus spp. is also attributed to their capacity to form endospores, which are highly resistant to environmental stresses, allowing them to survive in adverse conditions. This resilience facilitates their persistence in the soil and rhizosphere, providing long-term protection for plants against pathogens. Additionally, Bacillus spp. can promote plant growth by producing phytohormones and facilitating nutrient uptake, which indirectly enhances plant defense mechanisms against pests and diseases. 2.2 Historical perspective on the use of Bacillus spp. in biocontrol The use of Bacillus spp. as biocontrol agents has a rich history, with early reports dating back to the 20th century. Initially, the focus was on Bacillus thuringiensis due to its insecticidal properties, but over time, other Bacillus species have been explored for their nematicidal capabilities. For instance, Bacillus cereus has been identified as a potent biocontrol agent against root-knot nematodes, such as Meloidogyne incognita. Studies have shown that certain strains of B. cereus can cause significant mortality of nematode juveniles and reduce egg hatching rates, as well as form biofilms on plant roots, which protect the plants from nematode infection (Yin et al., 2021). The historical progression in the use of Bacillus spp. reflects a growing interest in sustainable and environmentally friendly pest management strategies, which are crucial in the face of increasing resistance to chemical nematicides and the need to preserve soil health. 3 Mechanisms of Action of Bacillus spp. against Meloidogyne spp. 3.1 Overview of the direct antagonistic capabilities of Bacillus spp. Bacillus spp. have demonstrated significant direct antagonistic capabilities against Meloidogyne spp., particularly Meloidogyne incognita. Studies have shown that certain strains of Bacillus, such as B. firmus I-1582, can directly manage nematode populations by increasing mortality rates of second-stage juveniles (J2s) to above 75%(Gattoni et al., 2023). Similarly, B. cereus strain S2 has been found to cause high mortality rates in M. incognita, with the production of nematicidal compounds like sphingosine contributing to this effect (Gao et al., 2016). Another strain, B. cereus Bc-cm103, has been reported to cause 100% mortality of J2s within 12 hours and to decrease egg hatching rates (Yin et al., 2021). These findings indicate that Bacillus spp. can directly antagonize Meloidogyne spp. through the production of bioactive metabolites and other mechanisms. 3.2 Systemic resistance induced by Bacillus spp. and its role in plant defense Bacillus spp. are not only capable of direct antagonism but also play a crucial role in inducing systemic resistance within host plants. B. amyloliquefaciens QST713 and B. firmus I-1582 have been shown to stimulate systemic resistance in plants, leading to the upregulation of defense-related genes (Gattoni et al., 2023). B. cereus S2 has also been reported to induce systemic resistance in tomato plants, enhancing the activity of defense-related enzymes (Gao et al., 2016). Furthermore, B. firmus I-1582 has been found to induce systemic resistance in tomato plants, with the dynamic regulation of genes related to the salicylic acid (SA) and jasmonic acid (JA) pathways (Ghahremani et al., 2020).
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 112 3.3 The role of salicylic acid and jasmonic acid pathways in systemic resistance The salicylic acid (SA) and jasmonic acid (JA) pathways are critical components of plant defense mechanisms, and Bacillus spp. have been found to interact with these pathways to enhance systemic resistance against Meloidogyne spp. For instance, B. amyloliquefaciens QST713 and B. firmus I-1582 have been shown to upregulate genes involved in the initial stages of the JA synthesis pathway, suggesting the stimulation of an intermediate molecule, likely OPDA, rather than JA itself in the short-term systemic response (Gattoni et al., 2023). Additionally, these Bacillus spp. stimulated a SA-responsive defense-related gene after one week, indicating the involvement of SA in long-term systemic defense (Gattoni et al., 2023). B. firmus I-1582 also primed SA and JA-related genes in tomato plants at different times after nematode inoculation (Ghahremani et al., 2020). These interactions with the SA and JA pathways underscore the complex role of Bacillus spp. in plant defense against nematode infections. Obviously, Bacillus spp. exhibit a multifaceted approach to managing Meloidogyne spp. through direct antagonism and the induction of systemic resistance, with the SA and JA pathways playing a significant role in the latter. These mechanisms highlight the potential of Bacillus spp. as biocontrol agents in integrated pest management strategies against root-knot nematodes. 4 Efficacy of Bacillus spp. in ControllingMeloidogyne spp. in Agricultural Settings 4.1 Case study 1: control of Meloidogyne incognita in cotton using Bacillus spp. Meloidogyne incognita represents a significant threat to cotton production, particularly in the south-eastern United States, where it is considered the most economically damaging pathogen. The nematode's wide host range, extensive geographical distribution, and the severe damage symptoms it causes, coupled with its complex biology and life cycle, make it a formidable pest to manage (Davis and Kemerait, 2021). Recent advances in integrated pest management (IPM) have highlighted the potential of Bacillus spp. as biological control agents against M. incognita. Studies evaluating the efficacy of Bacillus spp. reveal that certain strains, such as B. amyloliquefaciens QST713 and B. firmus I-1582, can manage nematode populations effectively. These strains have been shown to exhibit both direct antagonistic capabilities and systemic activity against M.incognita (Gattoni et al., 2023). The direct antagonistic effect of B. firmus I-1582, for instance, has been demonstrated through in vitro assays, where extracted metabolites from the bacterium significantly increased the mortality rate of M. incognita's second-stage juveniles. This indicates a potential for these metabolites to be used in nematode management strategies (Gattoni et al., 2023). Systemic resistance is another critical aspect of the biological control exerted by Bacillus spp. Split root assays have shown that both B. amyloliquefaciens QST713 and B. firmus I-1582 can induce systemic resistance in cotton plants, leading to a decrease in nematode population density. Interestingly, the systemic activity observed was associated with the upregulation of genes involved in the jasmonic acid (JA) synthesis pathway, suggesting that an intermediate molecule, likely OPDA, is stimulated by the bacteria rather than JA itself in the short-term response. Furthermore, after one week, a salicylic acid (SA)-responsive defense-related gene was upregulated, indicating that SA also plays a role in the long-term systemic defense response(Gattoni et al., 2023). The ability of these Bacillus spp. to colonize cotton roots effectively and maintain their population over time is crucial for their success as biological control agents. Quantitative PCR (qPCR) assays have confirmed that B. amyloliquefaciens QST713 and B. firmus I-1582 can successfully colonize cotton roots, with their concentration remaining stable over a 24-day period (Gattoni et al., 2023). This case demonstrates that the integration of Bacillus spp. into IPM strategies offers a promising avenue for the control of M. incognita in cotton. The dual action of direct antagonism and induced systemic resistance provided by strains such as B. amyloliquefaciens QST713 and B. firmus I-1582 represents a sustainable and effective approach to managing this pervasive nematode pest (Davis and Kemerait, 2021; Gattoni et al., 2023).
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 113 4.2 Case study 2: biocontrol of Meloidogyne sp. on tomato plants by selectedBacillus spp. Integrated Pest Management (IPM) strategies are crucial for sustainable agriculture, and the use of biocontrol agents, such as Bacillus spp., has gained attention for their potential to control plant-parasitic nematodes like Meloidogyne spp. This case focuses on the efficacy of selected Bacillus strains in suppressing Meloidogyne spp. on tomato plants. Research has shown that individual Bacillus strains can significantly reduce the infestation of Meloidogyne incognita on tomato roots. Strains BMH and INV, closely related to Bacillus velezensis, were individually capable of reducing the number of galls and eggs by more than 90% (Cruz-Magalhães et al., 2021). However, when these strains were combined, the suppression of M. incognita and the promotion of tomato shoot weight were not as effective as when applied separately (Cruz-Magalhães et al., 2021). This suggests that while individual strains have strong biocontrol potential, their combination does not necessarily enhance their biocontrol activity. Another study evaluated a dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002, which exhibited nematicidal properties in the pre-infection phase. This combination decreased egg hatching, juvenile survival, and attractiveness to the roots of tomato plants. Moreover, it impaired nematode establishment, gall formation, and giant cell development, indicating interference with the nematode's Morphogenetic mechanisms (Díaz-Manzano et al., 2023). The dual-strain combination also effectively reduced nematode reproduction, regardless of the application mode, and was effective against other plant-parasitic nematodes and in different crops (Díaz-Manzano et al., 2023). Furthermore, other Bacillus spp. have been identified as effective biocontrol agents against Meloidogyne spp., enhancing the growth and yield of tomato plants. These strains not only reduced the number of galls, egg masses, and nematodes in the soil but also promoted plant growth and yield, offering an alternative to chemical nematicides (Habazar et al., 2021). Although chemical treatments were more effective in controlling nematode populations, Bacillus spp. provided the added benefit of promoting plant health (Habazar et al., 2021). It can be seen from this case study that Bacillus spp. offer a promising alternative for the biocontrol of Meloidogyne spp. in tomato plants. While individual strains have shown significant biocontrol potential, the effectiveness of strain combinations may vary. The multifunctional nature of Bacillus spp., including their role as plant growth promoters, makes them an integral part of IPM strategies for sustainable agriculture (Cruz-Magalhães et al., 2021; Habazar et al., 2021; Díaz-Manzano et al., 2023). 5 Overview of Bacillus strains with Nematicidal Activity The genus Bacillus has been recognized for its role in the biological control of plant-parasitic nematodes, particularly Meloidogyne incognita. Several Bacillus strains have been identified to possess nematicidal properties, offering a sustainable alternative to chemical nematicides. Bacillus cereus strain S2 has been reported to exhibit high nematicidal activity against M. incognita, with mortality rates reaching up to 90.96% in laboratory conditions. The strain produces sphingosine, a compound that has been identified as lethal to nematodes, and has shown to induce systemic resistance in tomato plants (Gao et al., 2016). Another strain, Bacillus firmus YBf-10, has demonstrated systemic nematicidal activity, reducing nematode damage in tomato plants and promoting plant growth. The biocontrol efficacy of this strain is attributed to its secondary metabolites (Xiong et al., 2015). Bacillus cereus strain Bc-cm103 has been used as a biological control agent due to its production of volatile organic compounds (VOCs) that exhibit fumigation activity against M. incognita. The VOCs produced by Bc-cm103, including dimethyl disulfide, have shown high mortality rates in nematodes (Yin et al., 2020).
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 114 Bacillus amyloliquefaciens Y1 produces the dipeptide cyclo (d-Pro-l-Leu), which has been identified for the first time as having nematocidal activity. This strain significantly reduces the count of eggs and galls on tomato plant roots and enhances plant growth parameters (Jamal et al., 2017). Two strains of Bacillus thuringiensis, LBIT-596 and LBIT-107, have been characterized for their nematicidal activity. These strains produce spore-crystal complexes that are lethal to nematodes and have shown to decrease the number of galls caused byM. incognita in tomato plants (Verduzco-Rosas et al., 2021). Bacillus subtilis strain Bs-1, isolated from rhizospheric soil, has strong nematicidal effects, causing egg hatching inhibition and repellence of M. incognita. This strain has been effective in reducing root galls and promoting the growth of cucumber in both pot and field experiments (Cao et al., 2019). The efficacy of Bacillus cereus strain Bc-cm103 against M. incognita has been confirmed in pot, split-root, and field tests, where it significantly reduced the appearance of root galls. The strain also activates defense-responsive genes in cucumber (Yin et al., 2021). Bacillus aryabhattai MCCC 1K02966, a deep-sea bacterium, has shown nematicidal and fumigant activities against M. incognita. The VOC methyl thioacetate produced by this strain exhibits multiple nematicidal activities, including contact nematicidal, fumigant, and repellent activities (Chen et al., 2021). Native Bacillus thuringiensis strains have been investigated for their potential against M. incognita. Certain strains have been found to inhibit juvenile emergence and exhibit biocontrol potential by suppressing nematode reproduction in tomato plants (Ramalakshmi et al., 2020). Lastly, the purL gene of Bacillus subtilis has been associated with nematicidal activity. Strains OKB105 and 69 have been used to treat various nematodes, with high mortality rates observed, indicating the potential role of the purLgene in nematicidal activity (Xia et al., 2011). In conclusion, Bacillus spp. offer a diverse arsenal of biological control agents against M. incognita, with various strains producing different nematicidal compounds and mechanisms. These findings support the integration of Bacillus-based biocontrol strategies into pest management programs for sustainable agriculture. 6 Challenges and Limitations The integration of Bacillus spp. into pest management strategies, particularly for controlling the aggressive Meloidogyne enterolobii, presents a promising avenue. However, several challenges and limitations still need to be addressed to optimize their effectiveness and ensure sustainable use. 6.1 Limitations in the current understanding of Bacillus spp. mechanisms of action While Bacillus spp. are known to exert nematicidal effects, the detailed mechanisms underlying these interactions remain inadequately characterized. Several strains, such as Bacillus thuringiensis and Bacillus subtilis, have demonstrated potential in secreting bioactive compounds that affect nematodes adversely. These compounds include enzymes, toxins, and various secondary metabolites which can disrupt the nematode's cuticle, affect its digestive system, or impede its neural functions. However, the specific pathways and the molecular targets of these bioactive substances in nematodes are not fully elucidated. This gap in knowledge hampers the ability to predict and enhance the effectiveness of Bacillus-based formulations against specific nematode pests like Meloidogyne enterolobii. 6.2 Challenges in the application and consistency of Bacillus spp. as biocontrol agents The application of Bacillus-based biocontrol agents in the field faces several practical challenges. First, the environmental persistence and activity of Bacillus spores can be highly variable, influenced by soil type, moisture, temperature, and the presence of other microorganisms. These factors can lead to inconsistent results in field applications, where efficacy might not replicate the success seen in controlled, laboratory conditions. Moreover, the formulation of Bacillus products needs to ensure that the bacterial spores remain viable and capable of
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 115 germination upon application. This requires sophisticated formulation technologies that can protect these spores from desiccation, UV degradation, and other environmental stresses. 6.3 The potential for resistance development in nematodes Like any biological or chemical control agent, there is a potential for the target pests to develop resistance against Bacillus spp. Although cases of nematode resistance to microbial biocontrol agents are less documented compared to chemical nematicides, the risk cannot be ignored. The repeated use of a single strain or a specific bioactive compound could select for resistant nematode populations over time. This potential for resistance underscores the need for a diversified approach in integrated pest management strategies, incorporating multiple Bacillus strains or combining these biological agents with other control measures. This diversification could help in managing resistance development and prolonging the efficacy of biocontrol agents. Undoubtedly，the integration of Bacillus spp. into the management of Meloidogyne enterolobii presents a viable, environmentally friendly alternative to traditional nematicides. However, overcoming the outlined challenges and limitations is crucial for achieving consistent and sustainable control. Continued research into the mechanisms of action, improved formulations, and comprehensive field studies are essential to harness the full potential of Bacillus spp. as effective biocontrol agents. 7 Future Directions As the agricultural community continues to seek sustainable solutions for pest management, particularly for the resilient Meloidogyne enterolobii, the role of Bacillus spp. within Integrated Pest Management (IPM) strategies is poised for significant advancements. Addressing current research gaps and exploring innovative applications are key to enhancing the utility and effectiveness of Bacillus-based biocontrol. 7.1 Research gaps and future studies needed to optimize the use of Bacillus spp. in IPM Current research into Bacillus spp. as biocontrol agents primarily focuses on their nematicidal effects, but comprehensive studies on their interactions with plant hosts and the broader ecosystem are needed. Future studies should aim to map the interaction networks between Bacillus spp., plants, and nematodes to understand the systemic effects of these biocontrols. Additionally, there is a need for long-term field trials to evaluate the consistency and longevity of Bacillus-based treatments under various agricultural conditions. These studies should also investigate the optimal application timings, dosages, and methods to maximize efficacy and cost-effectiveness. 7.2 The potential for genetic engineering of Bacillus spp. to enhance biocontrol efficacy Advancements in genetic engineering offer promising avenues to enhance the biocontrol capabilities of Bacillus spp. By understanding the genetic basis of the bioactive compounds and mechanisms that these bacteria use to combat nematodes, researchers can potentially engineer strains with enhanced nematicidal properties or broader spectrum activity. Genetic modifications could also improve the environmental resilience and persistence of these microbes, ensuring they remain effective in the soil for longer periods. However, any genetically modified organism (GMO) approach must be rigorously tested for safety and environmental impact before deployment. 7.3 Integration of Bacillus spp. with other IPM Strategies for holistic pest management To achieve holistic and sustainable pest management, Bacillus spp. should be integrated with other IPM strategies. This includes combining biological control with cultural practices such as crop rotation, soil health enhancement, and resistant cultivars. The synergistic use of Bacillus spp. with physical controls like soil solarization and organic amendments can also improve overall pest management outcomes. Additionally, exploring the combined use of Bacillus with other biological agents, such as fungi or predatory nematodes, could provide multiple modes of action against pests, reducing the likelihood of resistance development. 8 Concluding Remarks In the pursuit of sustainable agricultural practices, the potential of Bacillus spp. as biocontrol agents against the root-knot nematode Meloidogyne enterolobii has been highlighted through various studies. The research has
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 116 consistently demonstrated the efficacy of different Bacillus strains in suppressing nematode populations and promoting plant growth. For instance, Bacillus velezensis strain YS-AT-DS1 has shown promising results in enhancing tomato growth and reducing infection rates of Meloidogyne incognita in plants (Hu et al., 2022). Similarly, Bacillus cereus strain Bc-cm103 has been effective in causing mortality of nematode juveniles and reducing egg hatching rates, alongside activating defense-responsive genes in plants (Yin et al., 2021). These findings are supported by other studies that have reported the biocontrol efficacy of Bacillus spp. against Meloidogyne spp., indicating their potential as a sustainable alternative to chemical nematicides (Seo et al., 2012; Habazar et al., 2021). The importance of further research cannot be overstated, as it is essential to fully harness the capabilities of Bacillus spp. in sustainable agriculture. While the current body of work provides a solid foundation, there are still gaps in our understanding of the mechanisms through which Bacillus spp. exert their biocontrol effects. For example, the role of secondary metabolites and the specific pathways involved in inducing systemic resistance in plants need to be elucidated (Xiong et al., 2015; Shahid et al., 2021). Additionally, the interaction between Bacillus spp. and plant hosts in various environmental conditions warrants further investigation to optimize the application of these biocontrol agents in different agricultural settings. In conclusion, Bacillus spp. represent a promising avenue for the development of integrated pest management strategies that are both effective and environmentally friendly. Continued research is crucial to refine the application of these biocontrol agents and to ensure that they can be integrated seamlessly into existing agricultural practices, thereby contributing to the sustainability and resilience of food production systems worldwide. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Cao H., Jiao Y., Yin N., Li Y., Ling J., Mao Z., Yang Y., and Xie B., 2019, Analysis of the activity and biological control efficacy of the Bacillus subtilis strain Bs-1 against Meloidogyne incognita, Crop Protection, 122: 125-135. https://doi.org/10.1016/J.CROPRO.2019.04.021 Chen W., Wang J., Huang D., Cheng W., Shao Z., Cai M., Zheng L., Yu Z., and Zhang J., 2021, Volatile organic compounds fromBacillus aryabhattai MCCC 1K02966 with multiple modes against Meloidogyne incognita, Molecules, 27(1): 103. https://doi.org/10.3390/molecules27010103 Collett R., Marais M., Daneel M., Rashidifard M., and Fourie H., 2021, Meloidogyne enterolobii, a threat to crop production with particular reference to sub-Saharan Africa: an extensive, critical and updated review, Nematology, 1: 1-39. https://doi.org/10.1163/15685411-BJA10076 Cruz-Magalhães V., Guimarães R., Silva J., Faria A., Pedroso M., Campos V., Marbach P., Medeiros F., and Souza J., 2021, The combination of two Bacillus strains suppresses Meloidogyne incognita and fungal pathogens, but does not enhance plant growth, Pest management science, 78(2): 722-732. https://doi.org/10.1002/ps.6685 Davis R., and Kemerait R., 2021, Integrated management of Meloidogyne incognita, the most economically damaging pathogen of cotton in the south-eastern United States, Integrated Nematode Management: State-of-the-art and Visions for the Future, 13: 87-93. https://doi.org/10.1079/9781789247541.0013. Díaz-Manzano F., Amora D., Martínez-Gómez Á., Moelbak L., and Escobar C., 2023, Biocontrol of Meloidogyne spp. in Solanum lycopersicumusing a dual combination of Bacillus strains, Frontiers in Plant Science, 13: 1-13. https://doi.org/10.3389/fpls.2022.1077062 Gao H., Qi G., Yin R., Zhang H., Li C., and Zhao X., 2016, Bacillus cereus strain S2 shows high nematicidal activity against Meloidogyne incognita by producing sphingosine, Scientific Reports, 6(1): 28756. https://doi.org/10.1038/srep28756 Gattoni K., Park S., and Lawrence K., 2023, Evaluation of the mechanism of action of Bacillus spp. to manage Meloidogyne incognita with split root assay, RT-qPCR and qPCR, Frontiers in Plant Science, 13: 1079109. https://doi.org/10.3389/fpls.2022.1079109 Ghahremani Z., Escudero N., Beltrán-Anadón D., Saus E., Cunquero M., Andilla J., Loza-Álvarez P., Gabaldón T., and Sorribas F., 2020, Bacillus firmus strain I-1582, a nematode antagonist by itself and through the plant, Frontiers in Plant Science, 11: 796. https://doi.org/10.3389/fpls.2020.00796.
Bt Research 2024, Vol.15, No.3, 110-117 http://microbescipublisher.com/index.php/bt 117 Habazar T., Yanti Y., Dani M., and Monica D., 2021, Biocontrol of Meloidogyne sp. on tomato plants by selected Bacillus spp., IOP Conference Series: Earth and Environmental Science, 757: 012019. https://doi.org/10.1088/1755-1315/757/1/012019 Hu Y., You J., Wang Y., Long Y., Wang S., Pan F., and Yu Z., 2022, Biocontrol efficacy of Bacillus velezensis strain YS-AT-DS1 against the root-knot nematode Meloidogyne incognita in tomato plants, Frontiers in Microbiology, 13: 1035748. https://doi.org/10.3389/fmicb.2022.1035748 Jamal Q., Cho J., Moon J., Munir S., Anees M., and Kim K., 2017, Identification for the first time of Cyclo (d-Pro-l-Leu) produced by Bacillus amyloliquefaciens Y1 as a nematocide for control of Meloidogyne incognita, Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(11): 1839. https://doi.org/10.3390/molecules22111839 Köberl M., Ramadan E., Adam M., Cardinale M., Hallmann J., Heuer H., Smalla K., and Berg G., 2013, Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas in Egypt, FEMS microbiology letters, 342(2): 168-178. https://doi.org/10.1111/1574-6968.12089 Ramalakshmi A., Sharmila R., Iniyakumar M., and Gomathi V., 2020, Nematicidal activity of native Bacillus thuringiensis against the root knot nematode, Meloidogyne incognita (Kofoid and White), Egyptian Journal of Biological Pest Control, 30(1): 629-653. https://doi.org/10.1186/s41938-020-00293-2 Seo B., Kumar V., Ahmad R., Kim B., Park W., Park S., Kim S., Lim J., and Park Y., 2012, Bacterial mixture from greenhouse soil as a biocontrol agent against root-knot nematode, Meloidogyne incognita, on oriental melon, Journal of Microbiology and Biotechnology, 22(1): 114-117. https://doi.org/10.4014/JMB.1105.05053 Shahid I., Han J., Hanooq S., Malik K., Borchers C., and Mehnaz S., 2021, Profiling of metabolites of Bacillus spp. and their application in sustainable plant growth promotion and biocontrol, Frontiers in Sustainable Food Systems, 5: 605195. https://doi.org/10.3389/fsufs.2021.605195. Sikandar A., Gao F., Mo Y., Chen Q., Ullah R., and Wu H., 2023, Efficacy of Aspergillus tubingensis GX3′ fermentation against Meloidogyne enterolobii in tomato (Solanum lycopersicumL.), Plants, 12(14): 2724. https://doi.org/10.3390/plants12142724 Verduzco-Rosas L., García-Suárez R., López-Tlacomulco J., and Ibarra J., 2021, Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita, FEMS microbiology letters, 368(5): fnaa186. https://doi.org/10.1093/femsle/fnaa186 Viljoen J., Labuschagne N., Fourie H., and Sikora R., 2019, Biological control of the root-knot nematode Meloidogyne incognita on tomatoes and carrots by plant growth-promoting rhizobacteria, Tropical Plant Pathology, 44: 284-291. https://doi.org/10.1007/s40858-019-00283-2 Xia Y., Xie S., Ma X., Wu H., Wang X., and Gao X., 2011, The purL gene of Bacillus subtilis is associated with nematicidal activity, FEMS microbiology letters, 322(2): 99-107. https://doi.org/10.1111/j.1574-6968.2011.02336.x. Xiong J., Zhou Q., Luo H., Xia L., Li L., Sun M., and Yu Z., 2015, Systemic nematicidal activity and biocontrol efficacy of Bacillus firmus against the root-knot nematode Meloidogyne incognita, World Journal of Microbiology and Biotechnology, 31: 661-667. https://doi.org/10.1007/s11274-015-1820-7 Yin N., Liu R., Zhao J., Khan R., Li Y., Ling J., Liu W., Yang Y., Xie B., and Mao Z., 2020, Volatile organic compounds of Bacillus cereus strain Bc-cm103 exhibit fumigation activity against Meloidogyne incognita, Plant disease, 105(4): 904-911. https://doi.org/10.1094/PDIS-04-20-0783-RE Yin N., Zhao J., Liu R., Li Y., Ling J., Yang Y., Xie B., and Mao Z., 2021, Biocontrol efficacy of Bacillus cereus strain Bc-cm103 against Meloidogyne incognita, Plant disease, 105(8): 2061-2070. https://doi.org/10.1094/PDIS-03-20-0648-RE
Bt Research 2024, Vol.15, No.3, 118-130 http://microbescipublisher.com/index.php/bt 118 Research Insight Open Access Comparative Analysis of Plasmid Prfiles in Bt Islates from Different Habitats Yinghua Chen, Zhongqi Wu Institute of Life Sciences, Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding author: Zhongqi.wu@jicat.org Bt Research, 2024, Vol.15, No.3 doi: 10.5376/Bt.2024.15.0012 Received: 10 Mar., 2024 Accepted: 28 Apr., 2024 Published: 15 May., 2024 Copyright © 2024 Chen and Wu, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Chen Y.H., and Wu Z.Q., 2024, Comparative analysis of plasmid prfiles in Bt islates from different habitats, Bt Research, 15(3): 118-130 (doi: 10.5376/Bt.2024.15.0012) Abstract The study investigates the genetic diversity and plasmid profiles of Bacillus thuringiensis (Bt) isolates collected from various ecological niches. Bt is a widely used biopesticide due to its insecticidal properties, which are largely attributed to the presence of plasmid-borne genes encoding Cry and Vip proteins. This research aims to compare the plasmid profiles of Bt isolates from different habitats to understand their genetic diversity and potential for biocontrol applications. The study utilized techniques such as whole genome sequencing, PCR amplification, and bioassays to characterize the isolates. Results revealed significant genetic diversity among the isolates, with multiple unique plasmid profiles identified. The findings suggest that different habitats harbor distinct Bt strains with varying plasmid compositions, which could be exploited for developing novel biopesticides. This comparative analysis enhances our understanding of Bt genetic diversity and its implications for sustainable pest management. Keywords Bacillus thuringiensis; Plasmid profiles; Genetic diversity; Biopesticides; Insecticidal proteinse 1 Introduction Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium that is widely recognized for its insecticidal properties. It produces parasporal crystal inclusions containing Cry proteins, which are toxic to a variety of insect larvae, particularly those of the orders Lepidoptera, Coleoptera, and Diptera (Dorsch et al., 2002; Singh et al., 2021). Bt has been extensively used as a biological control agent in agriculture to manage pest populations, thereby reducing the reliance on chemical pesticides (Paeket al., 2022). The bacterium's ability to produce a diverse array of Cry toxins, each targeting specific insect pests, makes it a versatile tool in integrated pest management programs (Wang et al., 2020). Plasmids play a crucial role in the genetic diversity and adaptability of Bt strains. They often carry genes encoding for insecticidal proteins, such as Cry and Vip toxins, which contribute to the bacterium's pathogenicity against insect pests (Wang et al., 2020). Understanding the plasmid profiles of Bt isolates can provide insights into the genetic mechanisms underlying their insecticidal properties and potential for resistance management. For instance, the coexistence of cry and vip genes on the same plasmid has been shown to enhance synergistic insecticidal activity, thereby delaying the development of resistance in target insect populations (Wang et al., 2020). Additionally, plasmid studies can aid in the identification and development of novel Bt strains with enhanced insecticidal properties, as demonstrated by the isolation and characterization of highly toxic Bt strains against specific pests (Park et al., 2022). The study aim to conduct a comparative analysis of plasmid profiles in Bt isolates from different habitats. By examining the diversity and distribution of plasmid-encoded insecticidal genes, we aim to elucidate the genetic factors contributing to the efficacy and adaptability of Bt strains. This study will also explore the potential applications of these findings in the development of new bio-insecticides and strategies for resistance management. Through a comprehensive analysis of existing literature, provide a deeper understanding of the role of plasmids in shaping the insecticidal capabilities of Bt and their implications for sustainable pest control practices.
Bt Research 2024, Vol.15, No.3, 118-130 http://microbescipublisher.com/index.php/bt 119 2 Overview of Bt Isolates and Habitats 2.1 Common habitats of Bt isolates Bacillus thuringiensis (Bt) is a ubiquitous bacterium found in a variety of habitats worldwide. Commonly, Bt isolates are oBtained from soil, plant surfaces (phylloplanes), and insect guts. For instance, a study conducted in Bangladesh identified Bt isolates from vegetable and crop-cultivated soils, phylloplanes, and insect guts, with the highest prevalence in soil samples (Shishir et al., 2012). Similarly, research in Qatar revealed a diverse collection of Bt isolates from soil, highlighting the soil as a rich source of Bt diversity (Figure 1) (Nair et al., 2018). In Iran, Bt strains were isolated from fields, gardens, and desert and semi-desert areas, further emphasizing the adaptability of Bt to different environmental conditions (Rashki et al., 2021). 2.2 Environmental factors influencing Bt distribution The distribution of Bt isolates is influenced by various environmental factors, including soil composition, climate, and the presence of host insects. For example, the diversity of Bt strains in Qatar was attributed to the unique soil ecology of the region, which supports a wide range of Bt isolates with different crystal morphologies and endotoxin profiles (Nair et al., 2018). In Bangladesh, the prevalence of Bt in soil samples compared to leaf and insect samples suggests that soil properties and agricultural practices may play a significant role in Bt distribution (Shishir et al., 2012). Additionally, the presence of specific insect hosts can influence the distribution and diversity of Bt isolates, as seen in the study of Brazilian Bt isolates, where genetic diversity was linked to the ability to target Aedes aegypti larvae (Fernandes et al., 2021). 2.3 Collection and isolation techniques The collection and isolation of Bt isolates involve several techniques to ensure the recovery of diverse and representative samples. Common methods include selective culturing, molecular characterization, and bioassays. In Bangladesh, selective methods were used to oBtain Bacillus cereus-like isolates, which were then identified as Bt based on hemolytic activity, parasporal crystal proteins, and plasmid profiles. In Qatar, scanning electron microscopy was employed to analyze the crystal forms of Bt isolates, revealing a high abundance of spherical crystals (Shishir et al., 2012). Molecular techniques such as 16S rDNA gene sequencing and PCR amplification are also used to confirm the identity of Bt strains and characterize their genetic profiles (Shishir et al., 2012; Rashki et al., 2021). Advanced techniques like pulsed field gel electrophoresis (PFGE) are utilized to separate and identify plasmid profiles in Bt strains. PFGE is particularly effective for separating high molecular weight plasmid DNAs, which are difficult to resolve using conventional gel electrophoresis. This method was successfully applied to analyze plasmid profiles in 10 Bt strains, providing detailed information on the number and size of plasmids (Zhou et al., 2014). Additionally, techniques like amplified fragment length polymorphism (AFLP) and repetitive element polymorphism (Rep-PCR) are used to assess genetic variability and molecular markers among Bt isolates (Valicente and Silva, 2017). Figure 1 Electrophoresis gel showing seven different plasmid patterns observed among the Bt collection (1–7) (Adopted from Nair et al., 2018) Image caption: L represents a 1 kb plus ladder; H14 is the reference strain Bacillus thuringiensis israelensis, HD1 is the reference strain Bacillus thuringiensis kurstaki; 1, QBt229; 2, QBt6; 3, QBt43; 4, QBt212; 5, QBt99; 6, QBt3; 7, QBt375 (Adopted from Nair et al., 2018)
Bt Research 2024, Vol.15, No.3, 118-130 http://microbescipublisher.com/index.php/bt 120 In conclusion, the study of Bt isolates from different habitats provides valuable insights into the diversity and distribution of this bacterium. Environmental factors such as soil composition, climate, and host insects significantly influence Bt distribution. The use of various collection and isolation techniques, including selective culturing, molecular characterization, and advanced electrophoresis methods, ensures the recovery of diverse Bt isolates with potential applications in biopesticide development and insect pest contro (Nair et al., 2018). 3 Plasmid Composition and Structure in Bt 3.1 Types of plasmids in Bt Bacillus thuringiensis (Bt) is known for its diverse plasmid content, which plays a crucial role in its adaptability and pathogenicity. Plasmids in Bt can be categorized based on their incompatibility groups and the functions they encode. For instance, IncF plasmids are prevalent and often carry multiple resistance genes, as seen in various studies (Doumith et al., 2012; Ajayi et al., 2021; Douarre et al., 2020). These plasmids are not only limited to antibiotic resistance but also include genes that confer resistance to heavy metals and other environmental stressors (Falgenhauer et al., 2017; Dolejská et al., 2018). Additionally, plasmids from different environments, such as wastewater treatment plants and livestock farms, show a wide range of resistance and virulence genes, indicating their adaptability to diverse habitats (Falgenhauer et al., 2017; Ajayi et al., 2021). 3.2 Plasmid structure and genetic elements The structure of Bt plasmids is highly complex, often comprising multiple replicons and a variety of genetic elements such as transposons, integrons, and insertion sequences. For example, the IncF/MOBF12 plasmid pFEMG (209 357 bp) isolated from wastewater treatment plants harbors a cluster of resistance genes interspersed with transposons and insertion sequences, which facilitate horizontal gene transfer (Ajayi et al., 2021). Similarly, the IncHI2 plasmids found in livestock farms carry a mosaic of resistance genes and heavy metal resistance determinants, indicating a high level of genetic recombination and evolution (Falgenhauer et al., 2017). The presence of multiple addiction systems, such as toxin-antitoxin modules, ensures the stable maintenance of these plasmids within their bacterial hosts (Doumith et al., 2012). 3.3 Functions of plasmid-encoded genes Plasmid-encoded genes in Bt serve a variety of functions that enhance the bacterium's survival and pathogenicity. These functions can be broadly categorized into antibiotic resistance, heavy metal resistance, and virulence factors. For instance, plasmids carrying the blaCMY-42, blaTEM-1β, and blaNDM-5 genes confer resistance to beta-lactam antibiotics, while genes like mphA-mrx-mphR provide resistance to macrolides (Ajayi et al., 2021). Heavy metal resistance genes such as ter, mer, and sil are also commonly found on Bt plasmids, enabling the bacteria to thrive in contaminated environments (Falgenhauer et al., 2017; Dolejská et al., 2018). Additionally, plasmids often carry virulence genes that contribute to the pathogenicity of Bt, such as those encoding for toxins and other virulence factors (García et al., 2018). In summary, the plasmid composition and structure in Bt are highly diverse and complex, reflecting the bacterium's ability to adapt to various environmental conditions. The presence of multiple resistance and virulence genes on these plasmids underscores their importance in the survival and pathogenicity of Bt. Further research into the genetic elements and functions of these plasmids will provide deeper insights into the mechanisms of horizontal gene transfer and the evolution of antibiotic resistance and virulence in Bt. 4 Methods for Plasmid Profiling 4.1 Extraction and purification techniques The extraction and purification of plasmids fromBacillus thuringiensis (Bt) isolates are critical steps in plasmid profiling. Traditional methods often struggle with separating plasmid DNAs with molecular masses greater than 25 Kb. Pulsed Field Gel Electrophoresis (PFGE) has emerged as an ideal method for separating and identifying plasmid profiles, especially for large plasmids. PFGE leverages regular changes in the direction and size of the electric field to enable the separation of high molecular weight DNAs, making it suitable for Bt strains that commonly contain multiple plasmids ranging from 10 Kb to over 600 Kb (Zhou et al., 2014).
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