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Biological control of major pathogenic bacteria of potato by Bacillus amyloliquefaciens strains SS-12.6 and SS-38.4
Abstract
Potato is ranked as one of the most important food crops. Bacterial wilt caused by Ralstonia solanacearum and blackleg and soft rot caused by different species from genera Pectobacterium and Dickeya are considered two of the most important disease of the potato. Biological control is the optimal strategy for controlling pathogens in crops generally, including bacteria. The present study aimed to evaluate the antagonistic effects of two Bacillus amyloliquefaciens strains, SS-12.6 and SS-38.4, against bacterial pathogens isolated from the potato in Serbia, such as R. solanacearum, Pectobacterium carotovorum, Pectobacterium brasiliense, and Dickeya dianthicola. The diameter of the inhibition zones formed by ethyl acetate extracts of SS-12.6 and SS-38.4 strains show much higher values than the inhibition zones of supernatants, which implies the main power of these antagonists' potential lies in lipopeptides. The effectiveness of the treatment (19.7-44.5%), based on the difference in weight of potato tubers on the fifth and 15th day after treatment (DAT), showed that the antagonistic strains were almost equally effective in the suppression of P. carotovorum, P. brasiliense, and D. dianthicola strains. Strains SS-12.6 and SS-38.4 exhibited the efficacy in the suppression of R. solanacearum wilt from 28.64 to 60.22%. The analysis of the area under the disease progress (AUDPC) confirmed differences among pathogen control treatments and biocontrol treatments with B. amyloliquefaciens strains in all trials. This study shows that the two B. amyloliquefaciens strains, SS-12.6 and SS-38.4, can potentially be used as biocontrol agents against potato pathogens.
... Bacillus species, in particular, have garnered significant research attention for advancing future biological control strategies (Shafi et al., 2017;Etesami et al., 2023;Poulaki and Tjamos, 2023). Currently, various antagonistic Bacillus species have been isolated and screened from soil, plants, and other environments, including B. amyloliquefaciens (Zheng et al., 2018;Wu et al., 2019;Jia et al., 2023;Markovićet al., 2023), B. velezensis (Reyes-Estebanez et al., 2020Chen et al., 2022;Yan et al., 2022), B. subtilis (Bais et al., 2004;Ding et al., 2017;Qiao et al., 2023), among others. Previous investigations highlight the inhibitory and anti-infection effects of B. amyloliquefaciens against pathogenic fungi causing diseases in rice, tomato, potato, brinjal and fruit (Jiao et al., 2020;Nie et al., 2022;Markovićet al., 2023;Pradhan et al., 2023;Zhang et al., 2023). ...
... Currently, various antagonistic Bacillus species have been isolated and screened from soil, plants, and other environments, including B. amyloliquefaciens (Zheng et al., 2018;Wu et al., 2019;Jia et al., 2023;Markovićet al., 2023), B. velezensis (Reyes-Estebanez et al., 2020Chen et al., 2022;Yan et al., 2022), B. subtilis (Bais et al., 2004;Ding et al., 2017;Qiao et al., 2023), among others. Previous investigations highlight the inhibitory and anti-infection effects of B. amyloliquefaciens against pathogenic fungi causing diseases in rice, tomato, potato, brinjal and fruit (Jiao et al., 2020;Nie et al., 2022;Markovićet al., 2023;Pradhan et al., 2023;Zhang et al., 2023). B. amyloliquefaciens has demonstrated potent capabilities in crop disease control, but at present, it is only reported that B. amyloliquefaciens can promote the root growth of replanted Malus hupehensis Rehd. ...
Apple replant disease (ARD), caused by Fusarium pathogens, is a formidable threat to the renewal of apple varieties in China, necessitating the development of effective and sustainable control strategies. In this study, the bacterial strain BA-4 was isolated from the rhizosphere soil of healthy apple trees in a replanted orchard, demonstrating a broad-spectrum antifungal activity against five crucial apple fungal pathogens. Based on its morphology, physiological and biochemical traits, utilization of carbon sources, and Gram stain, strain BA-4 was tentatively identified as Bacillus amyloliquefaciens. Phylogenetic analysis using 16S rDNA and gyrB genes conclusively identified BA-4 as B. amyloliquefaciens. In-depth investigations into B. amyloliquefaciens BA-4 revealed that the strain possesses the capacity to could secrete cell wall degrading enzymes (protease and cellulase), produce molecules analogous to indole-3-acetic acid (IAA) and siderophores, and solubilize phosphorus and potassium. The diverse attributes observed in B. amyloliquefaciens BA-4 underscore its potential as a versatile microorganism with multifaceted benefits for both plant well-being and soil fertility. The extracellular metabolites produced by BA-4 displayed a robust inhibitory effect on Fusarium hyphal growth and spore germination, inducing irregular swelling, atrophy, and abnormal branching of fungal hyphae. In greenhouse experiments, BA-4 markedly reduced the disease index of Fusarium-related ARD, exhibiting protective and therapeutic efficiencies exceeding 80% and 50%, respectively. Moreover, BA-4 demonstrated plant-promoting abilities on both bean and Malus robusta Rehd. (MR) seedlings, leading to increased plant height and primary root length. Field experiments further validated the biocontrol effectiveness of BA-4, demonstrating its ability to mitigate ARD symptoms in MR seedlings with a notable 33.34% reduction in mortality rate and improved biomass. Additionally, BA-4 demonstrates robust and stable colonization capabilities in apple rhizosphere soil, particularly within the 10-20 cm soil layer, which indicates that it has long-term effectiveness potential in field conditions. Overall, B. amyloliquefaciens BA-4 emerges as a promising biocontrol agent with broad-spectrum antagonistic capabilities, positive effects on plant growth, and strong colonization abilities for the sustainable management of ARD in apple cultivation.
... This approach not only addresses the challenge of soil acidity, but also presents a sustainable solution for marginal soils. Microbial inoculants formulated from beneficial soil bacteria are becoming increasingly popular as a sustainable alternative to conventional chemical fertilisers, primarily due to the ability of selected bacteria to increase the availability of elements to plants, degrade toxic compounds, act as biocontrol agents, and alleviate the negative impact of abiotic stress (Alori & Babalola 2018;Fira et al. 2018;Marković et al. 2023;Raspor et al. 2023). In contrast to chemical fertilisers, which often consist of inorganic compounds with potential negative effects on soil and water quality, bacterial inoculants are eco-friendly alternatives which can be tailored for specific crop or soil types Kumar et al. 2022). ...
The agricultural industry is constantly searching for new solutions to increase the productivity and nutritional value of crops under various conditions. Microbial inoculants have emerged as an alternative to traditional chemical fertilisers which could enhance crop productivity in acid soils - a major problem in modern agriculture. The aim of this research was to evaluate the effects of Bacillus megaterium and Mesorhizobium sp. on the seed germination of bird?s foot trefoil and orchardgrass under low pH (in vitro), as well as the nutrient composition of plants grown in acid soil. A positive impact of bacterial inoculation on seed germination was observed at pH 5 and 6 for both plant species. The content of macro- and microelements was within the range of optimal values for both plant species. This research provides valuable insights into the potential benefits of using bacterial inoculants to improve the seed germination and nutrient composition of plants grown in acid soils.
... However, integrated pest management programs (IPMs) are being increasingly employed in the agricultural sector, with the emphasis on the use of biological products as an environmentally friendly pathogen control strategy. In this context, Bacillus species have been shown to be promising biological control agents (Berić et al., 2012;Gond et al., 2015;Marković et al., 2020Marković et al., , 2023Jelušić et al., 2021;Iličić et al., 2022;Soliman et al., 2023). They exhibit multiple modes of action and produce a wide range of biologically active compounds with antifungal potential, making them suitable for controlling different diseases. ...
Species of the genus Alternaria are significant wheat contaminants
during production, transport and storage, requiring biocontrol measures which typically rely on the bacteria from the Bacillus genera. As these are among the most beneficial and exploited biocontrol agents, in this study, the inhibitory activity of indigenous Bacillus spp. was assessed against the Alternaria alternata isolate originating from the wheat seed. Two of the
fifteen Bacillus spp. included in the study showed the inhibitory effect. Specifically, 25.0−55.0% inhibition of A. alternata growth was achieved when the isolate coded as NB11 was applied in 106
−109 cells mL-1 concentrations. On the other hand, when applied in 107−109 cells mL-1 concentrations, the isolate coded as NB16 inhibited A. alternata growth by 35.2−51.1%, but was ineffective at lower concentrations. Thus, these in vitro assays indicate that both Bacillus spp. (NB11 and NB16) isolated from the wheat rhizosphere can be applied in practice in the control of A. alternata.
... However, integrated pest management programs (IPMs) are being increasingly employed in the agricultural sector, with the emphasis on the use of biological products as an environmentally friendly pathogen control strategy. In this context, Bacillus species have been shown to be promising biological control agents (Berić et al., 2012;Gond et al., 2015;Marković et al., 2020Marković et al., , 2023Jelušić et al., 2021;Iličić et al., 2022;Soliman et al., 2023). They exhibit multiple modes of action and produce a wide range of biologically active compounds with antifungal potential, making them suitable for controlling different diseases. ...
Compared to the previous period, the vineyard area in the Republic of Serbia has decreased significantly. Although the planting of new vineyards is subsidized over a long period of time, not enough vineyards are planted to ensure self-sufficiency in domestic grapes, wine (wine products) and spirit drinks originating from grapes. This is especially case for vineyards with local grapevine varieties, which can be of great importance for the rural development and promotion. In this paper, utilized time series analysis, specifically Auto Regressive Integrated Moving Average (ARIMA) modeling was used to predict the dynamics of newly planted vineyard areas with all grapevine varieties and local grapevine varieties based on data from the previous 20 years (period from 2003 to 2022). The aim is to forecast the trends of newly planted vineyard areas, both for all grapevine varieties and vineyards with local grapevine varieties, for the period from 2023 to 2027. A time series refers to a structured sequence of observations. The structuring is frequently done in terms of time intervals. Forecasting time series data, or determining future trends, is one of the most crucial goals of time series analysis. Based on such analysis, it is possible
to forecast the expansion of vineyard areas for the upcoming period. ARIMA models helped to determine five-year trends in data on newly planted vineyard areas. The forecast made in this paper showed that vineyard area of all grapevine varieties would be planted at an an- nual range of about 230 and 300 hectares over the next five years. The forecast for future annual planting for the same period (2023–2027) of vineyards with local grapevine varieties is in the range of only about 10 to 60 hectares. The obtained ARIMA forecast results, espe- cially for the forecasts for planting vineyards with local grapevine varieties, show that it is necessary to pay special attention to this problem and to initiate numerous measures and activities in order to increase the area of vineyards with local grapevine varieties in the Republic of Serbia.
... However, integrated pest management programs (IPMs) are being increasingly employed in the agricultural sector, with the emphasis on the use of biological products as an environmentally friendly pathogen control strategy. In this context, Bacillus species have been shown to be promising biological control agents (Berić et al., 2012;Gond et al., 2015;Marković et al., 2020Marković et al., , 2023Jelušić et al., 2021;Iličić et al., 2022;Soliman et al., 2023). They exhibit multiple modes of action and produce a wide range of biologically active compounds with antifungal potential, making them suitable for controlling different diseases. ...
The starting point for every viticultural and wine-producing country with respect to local grapevine varieties is their identification, inventory, preservation and development of genetic resources of those varieties. There are currently 224 grapevine varieties cultivated in Serbia for the purpose of commercial production of grapes and wine. Out of that number, 31 wine varieties are local. Vineyards under those varieties can be differentiated by their importance for production of grapes and wine, and by the level of their endangerment, that is, sustainability in conditions caused by climate changes. This paper presents the creation, that is, the modeling of the Method for Vineyard Sustainability Classification (MVSC). The purpose of MVSC classification is: to valorize vineyards with local wine grapevine varieties on grounds of their endangerment and sustainability, based on 20 examined and categorized individual vineyard sustainability parameters; to carry out comprehensive classification into one of the four established vineyard sustainability classes (Class A – very endangered vineyards, Class B – endangered vineyards, Class C – sustainable vineyards and Class D – very sustainable vineyards); spatial identification and presentation of vineyards based on determined vineyard sustainability class through application of GIS technology; and finally, application of Network Analysis (NA), prioritization of examined parameters and, therefore, vineyards. A total of 10,402 vineyards under local grapevine wine varieties were used for modeling, and it was determined that 29 vineyards with the total
surface of 1.2 hectares should be classified in Class A, while 2,883 vineyards with the total surface of 158.2 hectares should be classified in Class B. With respect to the strength of 20
individual vineyard sustainability parameters, it was determined that the parameter Structure of the vine rootstock (SVR) has the greatest impact, and priority in selection of vineyards in
different sustainability classes should be given to vineyards without rootstocks. In accordance with the scientific justification of obtained results, the MVSC enables comprehensive classification of the potential for sustainability of genetic resources of local grapevine varieties in Serbia, and it can be applied in other countries and wine-growing areas, as well as to other groups of grapevine varieties.
... B. amyloliquefaciens showed antagonistic activity against P. carotovorum isolated from potatoes in vitro and in vivo (Marković et al., 2023). Lipopeptides produced by these two antagonists were the main power of their biocontrol potential. ...
... B. amyloliquefaciens is a nonpathogenic bacterium known for its biological control characteristics, including colonization ability, the inhibition of pathogens, and the induction of systemic resistance in plants [33]. While many reports exist on the biocontrol potential of B. amyloliquefaciens against diverse phytopathogens [50][51][52][53], there have been limited studies on its effectiveness in reducing the growth of A. solani. Herein, we isolated and identified B. amyloliquefaciens strain XJ5. ...
Early blight, caused by Alternaria solani, is an important disease affecting tomatoes. Biological control offers an environmentally friendly approach to controlling pathogens. Herein, we identified a B. amyloliquefaciens strain XJ5 and investigated its biocontrol mechanism against A. solani. A. solani growth was significantly inhibited by XJ5, with the inhibition rate of cell-free culture supernatants reaching 82.3%. Furthermore, XJ5 crude protein extracts inhibited conidia germination and altered the mycelial morphology of A. solani. To uncover the potential biocontrol mechanism of XJ5, we analyzed its genome sequence and transcriptome. The genome of XJ5 comprised a 4.16 Mb circular chromosome and two circular plasmids. A total of 13 biosynthetic gene clusters and 127 genes encoding hydrolases were identified, suggestive of the ability of XJ5 to secrete antagonistic secondary metabolites and hydrolases. Transcript analysis revealed 174 differentially expressed genes on exposing A. solani to XJ5 crude protein extracts. The expression of genes related to chitin and mannose synthesis was downregulated, indicating that XJ5 metabolites may impact chitin and mannose synthesis in A. solani. Overall, these findings enhance our understanding of the interactions between B. amyloliquefaciens and phytopathogens and pave the way for the agricultural application of this promising biocontrol agent.
Ochratoxin A (OTA) is a significant global contaminant that poses severe challenges to food safety and public health. This study aims to isolate the OTA-degrated probiotics and evaluate genetic and biological characteristic. Here, The degradation rate of a new strain named Bacillus velezensis MM35 isolated from soil was the highest (87.10% within 48 h), and its culture supernatant was the main component of OTA degradation (63.95%) by high performance liquid chromatography. Further investigation revealed that the extracellular enzyme that degrades OTA in the culture supernatant of MM35 may be a small molecule enzyme with certain heat resistance. Genome-wide analysis showed that MM35 contains a cluster of carboxypeptidases encoding OTA-degrading potential, and had good metabolic and catalytic synthesis ability, and strong application potential in the synthesis and degradation of carbohydrates and proteins. A variety of secondary metabolites with antibacterial properties, such as non-ribosomal peptide synthetase and terpenoids, were identified in its metabolites. Consistent with the predicted results, MM35 showed various enzyme production characteristics such as cellulase and xylanase. Furthermore, MM35 could inhibit the growth of a variety of pathogenic bacteria, and showed high co-aggregation ability to Escherichia coli and Salmonella typhimurium. In addition, MM35 has certain tolerance to harsh environments such as strong acid, bile salt, and high temperature. Additionally, the adhesion rate of MM35 was 5.4%, and the invasion rate was 2.1% in IPEC-J2 cells. In summary, the data suggest MM35 isolated strain has high OTA degradation efficiency, antibacterial activity and intestinal colonization, which provided a new way for the treatment of OTA contamination in food and feed industries.
Background/Objectives: Sweetpotato black rot, caused by Ceratocystis fimbriata, is a severe fungal disease in sweetpotato production. Biological control strategies represent a promising, environmentally sustainable approach to managing this disease. This study investigates the biocontrol potential of Bacillus amyloliquefaciens SFB-1 against C. fimbriata. Methods: The antagonistic activities of strain SFB-1 on C. fimbriata were assessed through in vitro assays, including evaluations of mycelial inhibition, spore germination, and mycelial morphology. Pathogenicity assays on harvested sweetpotato roots assessed lesion diameter and depth. A transcriptomic analysis of C. fimbriata exposed to strain SFB-1 was performed to explore the underlying antifungal mechanism of SFB-1 on C. fimbriata. The qRT-PCR was employed to validate the RNA-seq results. Results: In vitro assays demonstrated that strain SFB-1 inhibited C. fimbriata mycelial growth by up to 81.01%, caused mycelial swelling, and completely suppressed spore germination at 108 CFU/mL. The cell-free supernatant of strain SFB-1 also suppressed C. fimbriata growth. Pathogenicity assays revealed that strain SFB-1 treatments reduced lesion diameter and depth on harvested sweetpotato roots by over 50% compared to untreated controls. Transcriptomic analysis of C. fimbriata treated with strain SFB-1 identified 1164 differentially expressed genes, with significant alterations in genes associated with cell wall integrity, cell membrane stability, spore germination, detoxification, and antioxidant responses. The qRT-PCR validation of 16 genes confirmed the consistency with the RNA-seq results. Conclusions: B. amyloliquefaciens SFB-1 demonstrates significant biocontrol efficacy against C. fimbriata through multiple mechanisms, positioning it as a promising solution for the sustainable management of sweetpotato black rot.
Potato soft rot and wilt are economically problematic diseases due to the lack of effective bactericides. Bacteriophages have been studied as a novel and environment-friendly alternative to control plant diseases. However, few experiments have been conducted to study the changes in plants and soil microbiomes after bacteriophage therapy. In this study, rhizosphere microbiomes were examined after potatoes were separately infected with three bacteria (Ralstonia solanacearum, Pectobacterium carotovorum, Pectobacterium atrosepticum) and subsequently treated with a single phage or a phage cocktail consisting of three phages each. Results showed that using the phage cocktails had better efficacy in reducing the disease incidence and disease symptoms’ levels when compared to the application of a single phage under greenhouse conditions. At the same time, the rhizosphere microbiota in the soil was affected by the changes in micro-organisms’ richness and counts. In conclusion, the explicit phage mixers have the potential to control plant pathogenic bacteria and cause changes in the rhizosphere bacteria, but not affect the beneficial rhizosphere microbes.
The increase in the world population has generated an important need for both quality and quantity agricultural products, which has led to a significant surge in the use of chemical pesticides to fight crop diseases. Consumers, however, have become very concerned in recent years over the side effects of chemical fungicides on human health and the environment. As a result, research into alternative solutions to protect crops has been imposed and attracted wide attention from researchers worldwide. Among these alternatives, biological controls through beneficial microorganisms have gained considerable importance, whilst several biological control agents (BCAs) have been screened, among them Bacillus, Pantoea, Streptomyces, Trichoderma, Clonostachys, Pseudo-monas, Burkholderia, and certain yeasts. At present, biopesticide products have been developed and marketed either to fight leaf diseases, root diseases, or fruit storage diseases. However, no positive correlation has been observed between the number of screened BCAs and available marketed products. Therefore, this review emphasizes the development of biofungicides products from screening to marketing and the problems that hinder their development. Finally, particular attention was given to the gaps observed in this sector and factors that hamper its development, particularly in terms of efficacy and legislation procedures.
Bacterial antagonists are effective as an alternative to synthetic bactericides in the control of potato soft rot. The use of bioagents reduces the application of synthetic bactericides, which are harmful to humans and the environment. However, the mechanisms of some bioagents, such as some fungi and bacteria, are not yet understood. This paper reviews the current situation of potato soft rot, biological controls, antagonistic bioagents and their mechanisms, application strategies and future directions in today’s agriculture. These mechanisms include mycoparasitism, competition, rhizosphere colonisation, synthesis and release of metabolites. Bioagents increased the defensive system of plants by increasing the antioxidants genes, such as superoxide dismutase, peroxidase (POD) and catalase (CAT), and eventually increased the plant growth and yield production.
Recent evaluations of the microbial biological control sector indicate that implementing microbial control of pests is still slow in the EU. The top causal factors are the lengthy, expensive, and cumbersome two‐stage procedure for approval of biological agents as active substances at the EU level and authorization of formulated products at the national level, limited funding, lack of EU level integrated approaches, and slow implementation of integrated pest management. This article contributes to a better understanding of the factors that limit microbial control of pests in EU agriculture by providing the first evaluation of the evolution of microbial biological control agent (MBCA) EU‐level approval combined with that of microbial biological control product (MBCP) national‐level authorization, discusses recent trends in research and offers some policy recommendations. By 2020, the EU had caught up with the USA regarding research output, approved MBCAs, and MBCA approval procedures (first stage). Despite improvements from 2014 to 2019, the number of authorized MBCPs (second stage) has progressed slowly and unevenly across the EU. Significant progress is concentrated in countries with more extensive agricultural land and higher research intensity. The EUʼs focus on promoting more sustainable agriculture by increasing the availability of low‐risk pesticides of biological origin as alternatives to conventional chemical pesticides has gained traction in recent years. Nevertheless, more efforts to improve the capacity and expertise of laggard EU Member States to contribute to the approval of MBCA, authorization of MBCP, and stimulating market availability are needed. Furthermore, we recommend introducing more concrete measures to promote the adoption of the microbial control of pests in the National Action Plans for the sustainable use of pesticides. © 2021 Society of Chemical Industry.
Antibiotic resistance is a massive problem rising constantly and spreading rapidly since the past decade. The major underlying mechanism responsible for this problem is an overuse or severe misuse of antibiotics. Regardless of this emerging global threat, antibiotics are still being widely used, not only for treatment of human infections, but also to a great extent in agriculture, livestock and animal husbandry. If the current scenario persists, we might enter into a post-antibiotic era where drugs might not be able to treat even the simplest of infections. This review discusses the current status of antibiotic utilization and molecular basis of antibiotic resistance mechanisms acquired by bacteria, along with the modes of transmittance of the resultant resistant genes into human pathogens through their cycling among different ecosystems. The main focus of the article is to provide an insight into the different molecular and other strategies currently being studied worldwide for their use as an alternate to antibiotics with an overall aim to overcome or minimize the global problem of antibiotic resistance.
Background
Several chemical bactericides were applied for controlling soft rot bacteria, Pectobacterium carotovorum subsp. carotovorum , which causes the destructive soft rot disease to many economically important vegetables, but because of their toxic hazards on human and environment became limit. The biocontrol was applied to control many plant pathogens. Therefore, this work is aimed to study the antagonistic activity of bacterial agents, i.e . Bacillus subtilis , Bacillus pumilus , Bacillus megaterium and Pseudomonas fluorescens , and fugal agents, i.e . Trichoderma harzianum , Trichoderma viride and Trichoderma virens , to control bacterial soft rot disease under in vitro and in vivo tests.
Results
The tested treatments could protect the potato tubers against the development of soft rot. T. viride and T. virens were highly effective in reducing soft rot symptoms on inoculated potato tuber slices, when applied at the same time or 2 h before pathogen inoculation, while B. megaterium and T. harzianum were highly effective when applied at the same time or 2 h after pathogen inoculation. In whole potato tubers technique, B.pumilus highly protected the stored potato tuber under artificially infection conditions, than P. fluorescens , T. harzianum , B. subtilis , T. viride , T. virens and B. megaterium , respectively.
Conclusion
Application of fungal agents or specify the bacterial species can play an important role in controlling bacterial soft rot disease in vegetables and increase the stored periods of potato tubers under storage conditions without any toxic effects.
Biosurfactants are amphiphilic surface-active molecules that are produced by a variety of microorganisms including fungi and bacteria. Pseudomonas, Burkholderia, and Bacillus species are known to secrete rhamnolipids and lipopeptides that are used in a wide range of industrial applications. Recently, these compounds have been studied in a context of plant-microbe interactions. This mini-review describes the direct antimicrobial activities of these compounds against plant pathogens. We also provide the current knowledge on how rhamnolipids and lipopeptides stimulate the plant immune system leading to plant resistance to phytopathogens. Given their low toxicity, high biodegradability and ecological acceptance, we discuss the possible role of these biosurfactants as alternative strategies to reduce or even replace pesticide use in agriculture.
Tomato is an economic crop worldwide. Many limiting factors reduce the production of tomato, with bacterial wilt caused by Ralstonia solanacearum being the most destructive disease. Our previous study showed that the disease resistance to bacterial soft rot is enhanced by Bacillus amyloliquefaciens strain PMB05. This enhanced resistance is associated with the intensification of pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). To determine whether the PTI-intensifying Bacillus spp. strains are able to confer disease resistance to bacterial wilt, their effects on PTI signals triggered by PAMP from R. solanacearum and on the occurrence of bacterial wilt were assayed. Before assay, a gene that encodes harpin from R. solanacearum, PopW, was applied as a PAMP. Results revealed that the B. amyloliquefaciens strain PMB05 was the one strain among 9 Bacillus rhizobacterial strains which could significantly intensify the PopW-induced hypersensitive response (HR) on Arabidopsis leaves. Moreover, we observed that the signals of PopW-induced reactive oxygen species generation and callose deposition were increased, confirming that the PTI was intensified by PMB05. The intensification of the PopW-triggered HR by PMB05 in Arabidopsis was reduced upon treatment with inhibitors in PTI pathways. Furthermore, the application of Bacillus spp. strains on tomato plants showed that only the use of PMB05 resulted in significantly increased resistance to bacterial wilt. Moreover, the PTI signals were also intensified in the tomato leaves. Taken together, we demonstrated that PMB05 is a PTI-intensifying bacterium that confers resistance to tomato bacterial wilt. Screening of plant immunity intensifying rhizobacteria is a possible strategy to control tomato bacterial wilt.
[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Background:
There is an urgent need to discover biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds.
Results:
Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 °C to 40 °C. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 °C and 25 °C.
Conclusions:
The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.
: Microorganisms area treasure in terms of theproduction of various bioactive compounds which are being explored in different arenas of applied sciences. In agriculture, microbes and their bioactive compounds are being utilized in growth promotion and health promotion withnutrient fortification and its acquisition. Exhaustive explorations are unraveling the vast diversity of microbialcompounds with their potential usage in solving multiferous problems incrop production. Lipopeptides are one of such microbial compounds which havestrong antimicrobial properties against different plant pathogens. These compounds are reported to be produced by bacteria, cyanobacteria, fungi, and few other microorganisms; however, genus Bacillus alone produces a majority of diverse lipopeptides. Lipopeptides are low molecular weight compounds which havemultiple industrial roles apart from being usedas biosurfactants and antimicrobials. In plant protection, lipopeptides have wide prospects owing totheirpore-forming ability in pathogens, siderophore activity, biofilm inhibition, and dislodging activity, preventing colonization bypathogens, antiviral activity, etc. Microbes with lipopeptides that haveall these actions are good biocontrol agents. Exploring these antimicrobial compounds could widen the vistasof biological pest control for existing and emerging plant pathogens. The broader diversity and strong antimicrobial behavior of lipopeptides could be a boon for dealing withcomplex pathosystems and controlling diseases of greater economic importance. Understanding which and how these compounds modulate the synthesis and production of defense-related biomolecules in the plants is a key question—the answer of whichneeds in-depth investigation. The present reviewprovides a comprehensive picture of important lipopeptides produced by plant microbiome, their isolation, characterization, mechanisms of disease control, behavior against phytopathogens to understand different aspects of antagonism, and potential prospects for future explorations as antimicrobial agents. Understanding and exploring the antimicrobial lipopeptides from bacteria and fungi could also open upan entire new arena of biopesticides for effective control of devastating plant diseases.
In the coming decades, feeding the expanded global population nutritiously and sustainably will require substantial improvements to the global food system worldwide. The main challenge will be to produce more food with the same or fewer resources. Food security has four dimensions: food availability, food access, food use and quality, and food stability. Among several other food sources, the potato crop is one that can help match all these requirements worldwide due to its highly diverse distribution pattern, and its current cultivation and demand, particularly in developing countries with high levels of poverty, hunger, and malnutrition. After an overview of the current situation of global hunger, food security, and agricultural growth, followed by a review of the importance of the potato in the current global food system and its role played as a food security crop, this chapter analyzes and discusses how potato research and innovation can contribute to sustainable agri-food systems with reference to food security indicators. It concludes with a discussion about the challenges for sustainable potato cropping considering the needs to increase productivity in developing countries while promoting better resource management and optimization.
Bacterial diseases are one of the most important biotic constraints of potato production, especially in tropical and subtropical regions, and in some warm temperate regions of the world. About seven bacterial diseases affect potato worldwide and cause severe damages especially on tubers, the economically most important part of the plant. Bacterial wilt and back leg are considered the most important diseases, whereas potato ring rot, pink eye, and common scab are the minor. Knowledge about zebra chip is extremely rare, as it occurs in a very isolated area and is an emerging disease in New Zealand, Europe, the USA and Mexico. Potato crop losses due to bacterial diseases could be direct and indirect; and they have several dimensions, some with short-term consequences such as yield loss and unmarketability of the produce and others with long-term consequences such as economic, environmental, and social. Some of them are of national and international importance and are the major constraints to clean seed potato production, with considerable indirect effects on trade. This review focuses on Clavibacter spp., Ralstonia spp., Pectobacterium spp., Dickeya spp., Streptomyces spp., and Liberibacter spp. pathogenic to potato, and looks at the respective pathogen in terms of their taxonomy and nomenclature, host range, geographical distribution, symptoms, epidemiology, pathogenicity and resistance, significance and economic losses, and management strategies. Nevertheless, the information collected here deal more with diseases known in developed and developing countries which cause severe economic losses on potato value chain.
The research aims to study the effect of Pseudomonas fluorescens, Bacillus subtilis, P. aeruginosa and Trichoderma spp. to enrich the growth and yield of potato crop and induce the resistance toward brown rot disease caused by Ralstonia solanacearum. Pot experiments were arranged in a completely randomized block design with three replicates. The selected strains were applied singly at three different intervals (before, after and zero time of planting). Three different potato cultivars were planted in soil infested with two virulent strains of R. solanacearum race 3 biovar 2. The results indicated that the soil treated with tested biological agents significantly stimulated the plant height, fresh weight, number of branches, dry weight, tuber number and potato weight/plant, up to 75.0 cm, 96.0 g, 6.0, 25.0 g, 10.0, 103.0 g, respectively, compared with control (plant only). Treatment with bio-control agents gives protection to the infected plants, resulting to an increase in growth parameters and yield of potato cultivars compared to pathogen control (infected plant). Biological treatment of infected potato cultivars also reduced the incidence of wilt by 80.50–20.63% and increased the disease reduction by 19.5–79.37% compared with pathogen control. P. fluorescens and B. subtilis were the highest for their activities against infection, followed by P. aeruginosa and then Trichoderma spp. The data showed that with the application of all antagonists, cultivars were often more sensitive to wilt infection with R. solanacearum T6 than R. solanacearum W11 strain. It was observed that the promising time for introducing these antagonistic strains grown in pots was prior to planting, in order to protect the plants from wilt infection. We can conclude that the antagonistic advantage of these strains against R. solanacearum according to in vivo results, along with their high efficacy in terms of improved plant development, suggests that these strains could be useful for biological control of potato wilt.
Background
Efficient and sustainable plant protection is of great economic and ecological significance for global crop production. A number of challenges, e.g. climate change, population growth and global trade, put increasing demands on future crop production and crop protection. This necessitates an increase in crop productivity with less environmental impact while maintaining good food quality and food security. To meet these challenges, it is essential that the recommendations provided to growers are efficient and correct, which can only be ensured by evidence-based recommendations based on outcomes from scientific studies.
Methods and output
The aim of these systematic maps is to compile scientific evidence for different plant disease protection strategies for the main arable crops grown in Sweden. Six major crops (wheat, barley, oat, potato, sugar beet and oilseed rape) have been selected based on the area under production, the annual production, the economic importance, and the amount of pesticide used against diseases in these crops in Sweden. All methods to manage diseases will be considered, including cropping system, pesticide application, biological control methods, as well as combinations of methods and integrated pest management. These systematic maps will only deal with field studies of relevance for agricultural practices in Sweden, although we expect that the results will be applicable for northern Europe as a whole. The main outcome to be used will be productivity measured as yield per area. Plant health and pathogen reduction will be included as a proxy for potential increase in crop quality and yield. This will provide a systematic overview of the plant disease protection measures that have been reported in the scientific literature. The study will result in one searchable database per crop that may be used as a catalogue of evidence for researchers and stakeholders, especially authorities and advisory organizations. The systematic maps will aid in the identification of areas that need further research and guide funding agencies and policymakers when deciding where research resources should be allocated. It will also help to select topics for future systematic reviews and meta-studies within the field of plant protection.
In this study, samples were collected from the leaves and stems of healthy wild Pistachio trees (Pistacia atlantica L.) from various locations of Baneh and Marivan regions, Iran. In total, 61 endophytic bacteria were isolated and grouped according to phenotypic properties. Ten selected isolates from each group were further identified by partial sequencing of the 16S rRNA gene. Based on the results, isolates were identified as bacteria belonging to Pseudomonas, Stenotrophomonas, Bacillus, Pantoea and Serratia genus. The ability of these isolates was evaluated to phytohormone production such as auxin and gibberellin, siderophore production, phosphate solubilization, atmospheric nitrogen fixation, protease and hydrogen cyanide production. All strains were able to produce the plant growth hormone auxin and gibberellin in different amounts. The majority of strains were able to solubilize phosphate. The results of atmospheric nitrogen fixation ability, protease and siderophore production were varied among strains. Only Ba66 could produce a low amount of hydrogen cyanide. The results of biocontrol assay showed that Pb78 and Sp15 strains had the highest and lowest inhibition effects on bacterial plant pathogens, Pseudomonas syringae pv. syringae Pss20 and Pseudomonas tolaasii Pt18 under in vitro condition. Pb3, Pb24 and Pb71 strains significantly promote root formation on carrot slices. To our knowledge this is the first report of the isolation of endophytic bacterial strains belonging to Pantoea, Bacillus, Pseudomonas, Serratia and Stenotrophomonas genus from wild pistachio trees with plant growth promoting potential and biocontrol activity.
Bacterial wilt caused by plant pathogenic Ralstonia spp. is one of the most important diseases affecting the production of many important crops worldwide. In China, a large scientific community has been dedicated to studying bacterial wilt and its causative agent, Ralstonia pseudosolanacearum and R. solanacearum. Most of their work was published in Chinese, which has hindered international communication and collaboration in this field. In this review, we summarize the status of knowledge on geographical distribution, diversity, and host range of Ralstonia spp., as well as, the impact of bacterial wilt on important crops and disease control approaches, in China. We present areas of research and publications by Chinese scientists and propose the promotion of collaborative research within China and with the international community.
Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.
In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction—was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.
In vitro interactions between Ralstonia solanacearum and Bacillus subtilis, B. cereus, Pseudomonas fluorescens and Rhizobium phaseoli indicated that the first was affected by the tested antagonistic bacteria. P. fluorescens had the most growth potential and great inhibition to R. solanacearum on King's B agar medium, followed by R. phaseoli, B. cereus and B. subtilis, respectively. R. solanacearum showed a slight antagonistic effect against B. subtilis and B. cereus, while P. fluorescens and R. phaseoli did not respond to such effect. The interaction among the different tested bioagents revealed that P. fluorescens had a good growth potential and antagonism with B. subtilis and B. cereus. Biocontrol efficacy against R. solanacaerum and the effect on growth promotion of potato plants were investigated. B. subtilis, B. cereus, P. fluoresnces and R. phaseoli, when used individually caused different changes in biocontrol efficacy and growth promotion efficacy, being 50.0 and 4.7, 60.7and 12.0, 78.6 and 16.5, and 70.0 and 16.3%vv, respectively. Besides, not all the mixtures of antagonistic organisms can give good results, therefore detailed studies should be done first.
Fourteen bacteriocin-producing strains from the genera Lactobacillus, Leuconostoc, Pediococcus, and Lactococcus were evaluated for their ability to inhibit the growth of eight strains of Listeria monocytogenes. Seven strains of lactic acid bacteria were antagonistic toward L. monocytogenes by deferred antagonism testing on agar. Cell-free supernatants from cultures of three of the seven bacteriocin-producing strains which inhibited growth of L. monocytogenes in deferred antagonism testing also inhibited growth in well diffusion assays. The eight strains of L. monocytogenes were identical in their sensitivity or resistance to bacteriocins. The action of the bacteriocins was eliminated by proteolytic enzymes.
Fifty seven rhizobacteria were isolated from rhizospheric soil of wilted tomato plants and among them two strains of rhizobacteria, having better antagonistic and plant growth promoting ability were characterized them as Bacillus amyloliquefaciens DSBA-11 and DSBA-12 based on morphological, biochemical, partial gene sequence analysis of 16S rRNA and fatty acid methyl ester analysis. Antagonistic activity of these strains DSBA-11, DSBA-12 was compared with other Bacillus species such as B. subtilis DTBS-5, B. cereus JHTBS-7, B. pumilus MTCC-7092 strains, against Ralstonia solanacearum race 1, bv 3, phylotype I, inciting bacterial wilt of tomato underin vitro conditions. B. amyloliquefaciens DSBA-11 showed maximum growth inhibition of R. solanacearum (4.91cm2) followed by strains DSBA-12 (3.31cm2) and B. subtilis (3.07 cm2). Moreover, strains DSBA-11 was also have better phosphorus solubilizing ability (42.6 μg/ml) and indole acetic acid (95.4 μg/ml) production than other strains of Bacillus spp. in vitro conditions. Biocontrol efficacy and plant growth ability of these bacterial antagonists was tested against bacterial wilt of tomato cv. Pusa Ruby under glasshouse conditions. Minimum bacterial wilt disease incidenceincultivar Pusa Ruby (17.95%) was recorded in B. amyloliquefaciens DSBA-11followed by B. amyloliquefaciens DSBA-12 after 30 days of inoculation.The bio-control efficacy was higher in B. amyloliquefaciens DSBA -12 treated plants, followed by B. pumilus MTCC- 7092.
The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants has remained unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of its virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to the biofilm formation by OE1-1, which is required for OE1-1 virulence. This article is protected by copyright. All rights reserved.
Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases.
A lot of crops are destroyed by the phytopathogens such as fungi, bacteria, and yeast leading to economic losses to the farmers. Members of the Bacillus genus are considered as the factories for the production of biologically active molecules that are potential inhibitors of growth of phytopathogens. Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and thus cause extended environmental pollution. Moreover, an increasing number of phytopathogens have developed resistance to antimicrobial agents. The lipopeptides have been tried as potent versatile weapons to deal with a variety of phytopathogens. All the three families of Bacillus lipopeptides, namely, Surfactins, Iturins and Fengycins, have been explored for their antagonistic activities towards a wide range of phytopathogens including bacteria, fungi, and oomycetes. Iturin and Fengycin have antifungal activities, while Surfactin has broad range of potent antibacterial activities and this has also been used as larvicidal agent. Interestingly, lipopeptides being the molecules of biological origin are environmentally acceptable.
The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.
Bacillus strains are extensively studied for their beneficial role in plant growth and biological control of tomato bacterial wilt (TBW), however their underlying mechanisms remained unexplored. In this study, four rhizobacterial strains, Bacillus amyloliquefaciens D29, B. amyloliquefaciens Am1, B. subtilis D16 and B. methylotrophicus H8 were investigated for their antibacterial activity against (TBW) pathogen and their ability to stimulate Tomato growth. Results revealed that all four strains were able to form robust biofilm, produce Indole acetic acid (IAA) and siderophores, while only D29, Am1 and H8 have capability to solubilize phosphate. The culture filtrate of each strain significantly suppressed the growth and biofilm of Ralstonia solanacearum, where, the cell wall was severely disrupted, which resulted into cell lysis and subsequent leakage of intracellular cytosolic contents. PCR analysis revealed that all four strains are harboring the antimicrobial associated genes for biosynthesis of Bacyllomicin, Fengycin, Iturin, Surfactin and Bacylisin. Subsequent real-time qPCR analysis revealed that the expression of ituC and srfAA genes in Am1 and D16 was remarkably up-regulated during in vitro interaction with R. solanacearum. This suggest that the potential antibacterial and anti-biofilm related mechanisms are associated to their ability to secret the corresponding lipopeptides in surrounding niche. In greenhouse, a positive correlation (0.777 and 0.686) was noted between the IAA amount produced by Bacillus strains and fresh/ dry weight of bacterized tomato plants. This the first report demonstrated the mode of antibacterial effect of Bacillus strains against R. solanacearum, moreover this study will help in under-standing the mode of action of Bacillus strains during biological management of TBW and promoting the growth of tomato plants.
Background
European Community (EC) legislation has limited the availability of pesticide active substances used in effective plant protection products. The Pesticide Authorisation Directive (PAD) 91/414/EEC, introduced the principle of risk assessment for approval of pesticide active substances. This principle was modified by the introduction of Regulation (EC) 1107/2009, which applies hazard, the intrinsic toxicity of the active substance, rather than risk, the potential for hazard to occur, as approval criterion.ResultsPotential impacts of EC pesticide legislation on agriculture in Ireland are summarised and whilst these will significantly impact pesticide availability in the medium- to long-term, regulations associated with water quality (Water Framework Directive (2000/60/EC) (WFD) and the Drinking Water Directive (1998/83/EC) (DWD)) have the potential to restrict pesticide use more immediately, as concerns regarding public health and economic costs associated with removing pesticides from water increase.Conclusion
This rationale will further reduce availability of effective pesticide active substances, directly affecting crop protection and increasing pesticide resistance within pest and disease populations. In addition, water quality requirements may also impact on important active substances used in plant protection in Ireland. The future challenge for agriculture in Ireland is to sustain production and profitability using reduced pesticide inputs within a framework of IPM.
Prophylactic use of broad-spectrum insecticides is a common feature of broad-acre grains production systems around the world. Efforts to reduce pesticide use in these systems have the potential to deliver environmental benefits to large areas of agricultural land. However, research and extension initiatives aimed at decoupling pest management decisions from the simple act of applying a cheap insecticide have languished. This places farmers in a vulnerable position of high reliance on a few products that may lose their efficacy due to pests developing resistance, or be lost from use due to regulatory changes. The first step towards developing Integrated Pest Management (IPM) strategies involves an increased efficiency of pesticide inputs. Especially challenging is an understanding of when and where an insecticide application can be withheld without risking yield loss. Here, we quantify the effect of different pest management strategies on the abundance of pest and beneficial arthropods, crop damage and yield, across five sites that span the diversity of contexts in which grains crops are grown in southern Australia. Our results show that while greater insecticide use did reduce the abundance of many pests, this was not coupled with higher yields. Feeding damage by arthropod pests was seen in plots with lower insecticide use but this did not translate into yield losses. For canola, we found that plots that used insecticide seed treatments were most likely to deliver a yield benefit; however other insecticides appear to be unnecessary and economically costly. When considering wheat, none of the insecticide inputs provided an economically justifiable yield gain. These results indicate that there are opportunities for Australian grain growers to reduce insecticide inputs without risking yield loss in some seasons. We see this as the critical first step towards developing IPM practices that will be widely adopted across intensive production systems.
A survey of ware potatoes (a total of 1127 samples) from localities in Serbia during two
consecutive years resulted in detection and identification of R. solanacearum in 17 tuber
samples. The monitoring detected the causal agent of bacterial wilt and brown rot of potato
in three districts of Vojvodina province. In 2011, the infection by R. solanacearum was confirmed
in 7 samples of ware potato tubers (varieties – Saturna, Pirol, Hermes, Panda) in West
Bačka and South Bačka Districts. In 2012, the infection by R. solanacearum was confirmed in
10 potato tuber samples (Lady Claire, Desiree, Panda, Red Fantasy and Vineta varieties) from
two districts: South Bačka and Central Banat. Bacterial strains obtained from positive samples
were identified as R. solanacearum biovar 2 using PCR/RFLP analysis, pathogenicity test
on tomato transplants, and nutritional, enzymatic and biovar determination tests. To our
best knowledge, these are the only findings of R. solanacearum infection in ware potatoes
in Serbia. R. solanacearum was not detected in tomato or any other host plant tested in this
study. Furthermore, the bacterium was not found in any of the water samples tested, including
those originating from areas in which the bacterium was found in ware potato samples.
The present study provides a new insight into the existing Xanthomonas arboricola pv. pruni (Xap) bacterial population originating from peach and apricot in two Western Balkans countries (Serbia and Montenegro). Multilocus sequence typing and analysis of the sequences of nine housekeeping genes revealed homology between the tested Xap strains as well as with the European population of this bacterium. The tested strains share the same haplotype (haplotype I) with the Xap strains from Italy, France, Spain, the USA, Australia, and Brazil. The revealed single nucleotide change (G ↔ C) in the sequences of gyrB1 gene differentiates haplotype I from haplotype II (Xap from South Korea, New Zealand, Argentina, and Uruguay). The detached-leaf bioassay results confirm differences in virulence between strains originating from peach and apricot toward P. armeniaca (apricot), indicating host specialization of the apricot strain toward this host. For the first time, immunity of P. fruticosa (European ground cherry) to Xap was established. According to the AUDPC, PCA and cluster analysis, other Prunus spp. were classified as having low susceptibility (P. mahaleb, P. cerasus, and P. avium), susceptible (P. domestica) and highly susceptible (P. persica, P. dulcis, P. cerasifera, and P. spinosa). Xap strains were also found to be susceptible to ten tested antibiotics. This study provides valuable knowledge on the Xap population from stone fruit grown in the Western Balkans region as well as the source of immunity, which could serve as a starting point for breeding Prunus cultivars and could be used as the main control strategy.
Soft rot and blackleg are common diseases of potato (Solanum tuberosum) in Serbia. Opportunistic pectinolytic plant pathogens in the genus Pectobacterium cause soft rot and wilt diseases by cell wall degradation. They cause major economic losses in potato crops worldwide and are among the top 10 plant pathogenic bacteria (Mansfield et al. 2012). Potato plants (‘VR808’) with symptoms of wilting, slow growth, stem blackening, and tuber softening were collected from a commercial field in Zobnatica, Serbia, in July 2019 and analyzed. All symptoms occurred in the same field, and ∼5% of plants were symptomatic. Isolation was performed from 10 randomly chosen symptomatic plants and tubers. Plant tissue was surface disinfected, and 1-cm sections from the margins of lesions were macerated in sterile distilled water (SDW) for 25 min and streaked on nutrient agar. After 48 h of incubation at 26°C, predominant shiny, cream-colored, round colonies were obtained from all samples. Three representative isolates (MMZKVR1, MMZCVR2, and MMZKVR3) from independent samples were selected randomly for biochemical and pathogenicity tests. Isolates were gram-negative, nonfluorescent facultative anaerobes exhibiting pectinolytic activity on potato tuber slices and hypersensitive response on tobacco leaves. They expressed catalase activity but did not express oxidase or acid phosphatase activity or produce indole. All strains grew at 37°C in 5% NaCl and reduced nitrate. Pathogenicity was tested on healthy 3-week-old potato plants (VR808 and ‘Kiebitz’) grown in Baltic Tray Substrate (Hawita) in a greenhouse and tubers of the same varieties. Three stems per isolate were inoculated by toothpick piercing (Duarte et al. 2004) using a bacterial suspension (∼1 × 108 CFU/ml) and incubated under plastic bags in a greenhouse at 25 ± 2°C. Blackleg symptoms and stem wilting developed within 48 h. No symptoms were observed on plants inoculated with sterile toothpicks dipped in SDW. The pathogen was reisolated from symptomatic plants, fulfilling Koch’s postulates, and sequencing of 16S rDNA confirmed the original pathogen. Three tubers per isolate were inoculated by toothpicks dipped in bacterial suspension (∼1 × 108 CFU/ml) and placed in a sealed plastic container at 25 ± 2°C. Treatment with SDW was used as a control. Tissue softening around the inoculation point developed within 48 h; no symptoms developed on controls. For molecular analyses, total DNA of isolates was extracted with a DNeasy Plant Mini Kit (Qiagen). The isolates were not detected in diagnostic PCR assays using specific primers Br1F/L1R for the detection of P. brasiliense (Duarte et al. 2004) and primers EXPCCF/EXPCCR for P. carotovorum subsp. carotovorum (Kang et al. 2003). The 16S rDNA PCR amplification was done using the universal PCR primer pair 27F/1492R (Fredriksson et al. 2013) followed by Sanger sequencing (Macrogen Europe BV). BLASTn analysis of sequences (GenBank nos. MZ048661, MZ048662, and MZ157274) revealed 100% query coverage and 100% identity to P. punjabense sequences in NCBI (MT242589 and CP038498) isolated from potato in China and Pakistan, respectively (Sarfraz et al. 2018). All three obtained isolates were proposed to belong to P. punjabense sp. nov. To further validate identification, MMZCVR2 was used for multilocus sequence analyses of five housekeeping genes: gyrA, recA, recN, rpoA, and rpoS. The gyrA (MZ161817), recA (MZ161818), recN (MZ161819), rpoA (MZ161820), and rpoS (MZ161821) sequence analysis had the highest nucleotide identity (99.44 to 100%) with P. punjabense strain SS95 (Sarfraz et al. 2018) in the NCBI GenBank database. To our knowledge, this is the first report of blackleg and soft rot caused by P. punjabense on potato in Serbia. P. punjabense causes soft rot and blackleg disease in potatoes (Sarfraz et al. 2018). Its distribution is poorly known but important because soft rot bacteria are easily transported long distances in latently infected seed tubers and can cause major economic losses.
Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20−25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates’ ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as ‘Candidatus Pectobacterium maceratum’ (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.
Ralstonia solanacearum, as a causative agent of potato wilt disease, is one of the deadliest diseases worldwide, and also in the Kurdistan province, Iran. Given the importance of the disease, the aim of the present study was to isolate endophytic bacteria from potato plant tissues such as the tuber, root, stem, and leaf, to investigate their antagonistic effects on R. solanacearum. In this regard, 236 endophytic bacteria were isolated and screened in vitro. As a result, 31 isolates were found as potential antagonists against potato wilt pathogen. For phentoypic and genotypic characterization, biochemical and pathogenicity tests as well as 16S rRNA gene sequence analyses were performed. 11 isolates as representatives of different taxa were selected for further investigations. These were identified as: Bacillus pumilus Bp91, B. pumilus Bp1, B. pumilus Bp49, B. licheniformis Bl17, Paenibacillus peoriae Pa86, Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, P. putida Ps52, Chryseobacterium indologenes Ch54, C. lathyri Chl98, and Microbacterium phyllosphaerae Mi41. Notably, the maximum inhibitory effects were observed by Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, Paenibacillus peoriae Pa86, Pseudomonas putida Ps52, and Bacillus licheniformis Bl17, by forming 17.6, 17.4, 17.3, 15.5, 15.2 mm diameter inhibition zones against R. solanacearum on nutrient agar medium, respectively. Based on the results of the greenhouse test, all 11 selected isolates simultaneously reduced the disease by 27 to 55% and also significantly increased plant growth
Among these, the five strains Pseudomonas brassicacearum Psb101, P. brassicacearum Ps169, P. putida Ps52, Paenibacillus peoriae Pa86, and Bacillus licheniformis Bl17 were introduced hereby as the most effective antagonists and growth enhancers under both laboratory and greenhouse conditions.
Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During six years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from five cultivars among 12 monitored, and in seven localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLY-1/Y-2), as well as the following tests: RFLP analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdh, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato are homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2 to 22 times in vascular necrotic tissues compared to nonnecrotic ones, and depended on disease severity but not on cultivar. Native PAGE analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.
Invasive pathogens can be a threat when they affect human health, food production or ecosystem services, by displacing resident species, and we need to understand the cause of their establishment. We studied the patterns and causes of the establishment of the pathogen Dickeya solani that recently invaded potato agrosystems in Europe by assessing its invasion dynamics and its competitive ability against the closely‐related resident D. dianthicola species. Epidemiological records over one decade in France revealed the establishment of D. solani and the maintenance of the resident D. dianthicola in potato fields exhibiting blackleg symptoms. Using experimentations, we showed that D. dianthicola caused a higher symptom incidence on aerial parts of potato plants than D. solani, while D. solani was more aggressive on tubers (i.e. with more severe symptoms). In co‐infection assays, D. dianthicola outcompeted D. solani in aerial parts, while the two species co‐existed in tubers. A comparison of 76 D. solani genomes (56 of which having been sequenced here) revealed balanced frequencies of two previously uncharacterized alleles, VfmBPro and VfmBSer, at the vfmB virulence gene. Experimental inoculations showed that the VfmBSer population was more aggressive on tubers while the VfmBPro population outcompeted the VfmBSer population in stem lesions, suggesting an important role of the vfmB virulence gene in the ecology of the pathogens. This study thus brings novel insights allowing a better understanding of the pattern and causes of the D.solani invasion into potato production agrosystems, and the reasons why the endemic D.dianthicola nevertheless persisted.
Blackleg outbreaks were noticed on three fields (total c. 100 ha) during two consecutive years (2018, 2019) in one of the main potato growing areas in Serbia (Bačka region, Vojvodina). The percentage of infected plants reached 40-70% with 10.5% to 44.7% yield reductions. From the three fields out of 90 samples Pectobacterium carotovorum subsp. brasiliensis was most frequently identified and diagnosed as causal agent of potato blackleg in Serbia for the first time (29 isolates). Dickeya dianthicola was a less frequently causative bacterium, which was also noticed for the first time (nine isolates). A total of 38 isolates were characterized based on their phenotypic and genetic features, including a pathogenicity test on potato. The repetitive element Polymerase Chain Reaction (rep-PCR) using BOX, REP and ERIC primer pairs differentiated five genetic profiles among 38 tested isolates. Multilocus sequence analysis (MLSA) of four housekeeping genes, acnA, gapA, icdA and mdh, revealed the presence of three so far unknown P. c. subsp. brasiliensis multilocus genotypes and confirmed clustering into two main genetic clades as determined in other studies. MLSA also revealed the presence of a new genotype of D. dianthicola in Serbia.
Outbreaks of stem and root rot disease of sweet potato caused by Dickeya dadantii recently occurred in major sweet potato planting areas in China. This study aimed to screen effective Bacillus strains in biocontrol of the bacterial soft rot pathogen. We screened plant-associated Bacillus strains capable of inhibiting D. dadantii using an in vitro overlay culture assay and an in vivo tuber slice assay. All Bacillus strains inhibited D. dadantii growth and maceration of sweet potato tubers. Two strains B. amyloliquefaciens A3 and B. velezensis A2 showed significantly higher rates of in vitro inhibition (55% and 50%) on D. dadantii growth and in vivo reduction (82% and 77%) of the maceration of sweet potato tuber slices by D. dadantii than other Bacillus strains did. They showed broad-spectrum inhibition on growth of soft rot Dickeya and Pectobacterium strains. They protected sweet potato seed tubers from infection by D. dadantii and promoted seedling growth. Their culture supernatants inhibited D. dadantii growth, swimming and biofilms and damaged D. dadantii cells. B. amyloliquefaciens A3 is more effective than B. velezensis A2 in producing surfactins, well-known lipopeptide antibiotics and biosurfactants, and inhibiting D. dadantii. Together, we developed an effective method to screen effective Bacillus strains to control the soft rot pathogen D. dadantii and screened out a strong surfactin producer B. amyloliquefaciens A3, which can be used as a reference strain for further screening of more effective Bacillus strains and to develop an agent to control the stem and root rot disease of sweet potato.
Aims
The aims of this study were to identify the structure of antibacterial lipopeptide produced by Bacillus amyloliquefaciens strain FJAT‐2349, to analyze the effects of the culture medium and temperature on lipopeptide production and to assess the biocontrol efficiency of the lipopeptides against tomato bacterial wilt.
Methods and Results
Lipopeptides were extracted by acid precipitation and resolved in methanol and their structure was identified through LC‐QTOF‐MS/MS method. The antibacterial activities of the lipopeptides were evaluated through inhibition zone experiments. The biocontrol efficiency of the lipopeptides against tomato bacterial wilt was examined by a pot test. The lipopeptides were composed of iturin (C14–C17 iturin A), fengycin (C14/C16 fengycin A, C14 fengycin B2, C16 fengycin A2/B2, C16–C17 fengycin B, C15fengycin A derivatives and C15 fengycin B derivatives) and surfactin (C12–C16 surfactin A). Moreover, the composition of the lipopeptides was significantly influenced by the culture medium and temperature; the contents of iturin, fengycin and surfactin varied within the range from 0·41–5·89, 4·54–181·67 and 2·05–19·65 mg l⁻¹ in the different culture media and from 0·39–11·04, 1·45–215·14 and 7·79–24·43 mg l⁻¹ under different culture tempeatures, respectively. The results indicated that the contents of the lipopeptide mixture, fengycin, and surfactin secreted from FJAT‐2349 all decreased along with an increasing culture temperature. The fermentation supernatants and lipopeptide extracts had the strongest inhibition activities of R. solanacearum when strain FJAT‐2349 was incubated at 25 °C using potato dextrose broth as the culture medium among all the assayed culture conditions. The purified fengycin was found to be the active antibacterial compound against R. solanacearum, but the purified surfactin was not. The pot experiments domonstrated that the lipopeptides secreted from the strain FJAT‐2349 could effectively control the tomato bacterial wilt with a biocontrol efficiency of 97·6%.
Conclusions
The lipopeptides secreted from strain FJAT‐2349 could serve as potential biocontrol agents against tomato bacterial wilt.
Significance and Impact of the Study
The lipopeptides exhibited good potential applications in the biocontrol of tomato bacterial wilt.
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Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4°C. The population dynamics of the best low‐temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real‐time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. A potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47%−75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.
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Copper has been used in agriculture to control oomycetes, fungi and bacteria for over a century. It plays
important roles in integrated pest management, but is essential in organic farming, where disease management depends
almost exclusively on its use. However, the use of this heavy metal may have log-term consequences due to its
accumulation in the soil, which appears incompatible with organic farming’s objectives. This awareness led the European
Union to establish maximum limits on copper in organic farming since 2002 (Commission Regulation 473/2002),
and further decisions on its use in crop protection are to be taken soon. At present, copper compounds are approved
as plant protection products until 31 January 2019. This review examines the current state of copper use, the regulatory
framework, and limits set for copper in organic farming. Strategies to reduce copper inputs are also considered,
including: preventive phytosanitary measures, innovative formulations with reduced copper content, optimization of
copper dosages, the use of forecasting models, the use of resistant varieties, optimization of agriculture management,
and natural alternatives to copper-based products. This review also examines the main research projects exploring
farming practices and appropriate alternatives to copper use for the control of plant pathogens. The review highlights
that, while there is currently no replacement for this heavy metal having the same plant protection effectiveness, agronomic
measures and management practices can be combined to reduce the amounts of copper used for this purpose.
Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU/mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC–MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection against potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti-Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato.
Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.
Aim
Assessment of biological control of P. syringae pv. aptata using crude lipopeptide extracts (CLEs) of two Bacillus amyloliquefaciens strains (SS‐12.6 and SS‐38.4) and one Bacillus pumilus strain (SS‐10.7).
Methods and Results
The minimum inhibitory concentration (MIC) of CLEs and their combinations against the pathogen and potential interaction between the extracts were determined in vitro. The most effective antibacterial activity was achieved with the CLE from B. amyloliquefaciens SS‐12.6, with an MIC value of 0.63 mg ml⁻¹. Interactions between CLE combinations were mostly indifferent. The biocontrol potential of CLEs, mixtures of CLEs, and cell culture of B. amyloliquefaciens SS‐12.6 was tested on sugar beet plants inoculated with P. syringae pv. aptata P53. The best result in inhibiting the appearance of tissue necrosis (up to 92%) was achieved with B. amyloliquefaciens SS‐12.6 cell culture.
Conclusion
This work demonstrated significant biocontrol potential of the CLE and cell culture of B. amyloliquefaciens SS‐12.6 which successfully suppress leaf spot disease severity on sugar beet plants.
Significance and Impact of the Study
The findings of biocontrol of sugar beet emerging pathogen will contribute to growers in terms of alternative disease control management. This study represent first assessment of biological control of P. syringae pv. aptata.
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Broadening the spectrum of action of microbial bioactive compounds is a priority nowadays. From a collection of 1493 aerobic endospore forming bacteria, 3.1% (47) inhibited Serratia marcescens and were highly active against R. solanacearum. Thirty-six of these strains were identified as part of the ‘Operational Group B. amyloliquefaciens’ denoting the potential of strains from these species to produce antibacterial substances. Specifically, the strain Bacillus sp. EA-CB0959 was selected for further trials. Three families of lipopeptides: surfactins, iturins and fengycins were found as the active compounds produced by this strain. The highest bioactivity, produced by fengycins, had a minimal inhibitory concentration of 32 μg/mL. Treating greenhouse banana plants with a mixed fraction of lipopeptides reduced by 35% the incidence of Moko disease caused by R. solanacearum. Here we provide first time evidence of in vitro antibacterial activity of purified fengycins and in vivo activity of mixed lipopetides against Moko disease in banana plants.
Antibiotics have been used for the management of relatively few bacterial plant diseases and are largely restricted to high-value fruit crops because of the expense involved. Antibiotic resistance in plant-pathogenic bacteria has become a problem in pathosystems where these antibiotics have been used for many years. Where the genetic basis for resistance has been examined, antibiotic resistance in plant pathogens has most often evolved through the acquisition of a resistance determinant via horizontal gene transfer. For example, the strAB streptomycin-resistance genes occur in Erwinia amylovora, Pseudomonas syringae, and Xanthomonas campestris, and these genes have presumably been acquired from nonpathogenic epiphytic bacteria colocated on plant hosts under antibiotic selection. We currently lack knowledge of the effect of the microbiome of commensal organisms on the potential of plant pathogens to evolve antibiotic resistance. Such knowledge is critical to the development of robust resistance management strategies to ensure the safe and effective continued use of antibiotics in the management of critically important diseases. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
In the present study, a collection of 235 Bacillus strains, which had been isolated from the potato rhizosphere, was screened with the aim of controlling Pectobacterium carotovorum subsp. carotovorum (Pcc), a dominant potato pathogen known for causing soft rot in Iran. Fifteen strains inhibited the known Pcc strains in vitro and these were chosen for further studies. The maceration capacity of Pcc strains was decreased by the antagonistic strains in the range of 1.6–4 times. Based on 16S rRNA sequencing, five Bacillus species were characterized. Conducting various supplementary tests, the current work evaluated all antagonistic strains for biosurfactant production, mobility, enzymatic activities, production and inactivation of acyl-homoserine lactones, and the ability to produce auxin. Among the five antimicrobial peptide biosynthetic genes tested, bmyB was the less frequent gene, while three genes, namely srfAA, fenD, and ituC, were the most distributed genes. In addition, the interaction of four promising Bacillus species in the potato-Pcc system was studied. Along with its discussion of the potency for the biocontrol of Pcc by B. pumilus IrB8 and B. amyloliquefaciens IrB12, the present study’s test results also indicated these two suppressed tuber maceration by 63.7 and 47.8% respectively. The high levels of phenylalanine ammonia-lyase, polyphenol oxidase, peroxidase and total phenols were obtained in a single application of IrB8 or IrB12 after 8 h. In addition, the discontinuous usage of Bacillus and Pcc strains was achieved in comparison to other treatments. Consequently, the current work’s findings show that IrB8 and IrB12 strains have the potential to be used for potato soft rot control.
Extensive employment of plant resins and propolis, as an antiseptic agents dating from ancient times in numerous cultures indicating that it may have antimicrobial and other biological properties. Resins from deciduous trees from the Populus and Salix genera, several fruit trees from the genus Prunus and a few other species were analyzed for their phenolic composition and antibacterial activity. Phenolic profiling of the plant resins was performed by high-performance thin-layer chromatography (HPTLC) and ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid mass spectrometry. Antimicrobial activity against seven bacterial species was determined by minimum inhibitory concentration (MIC) assays and bioautography. The synergism, additivism, and antagonism of phenolic compounds were used to define the nature and type of interactions. Populus spp. showed higher amounts of p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, chrysin, apigenin, quercetin, pinocembrin, pinobanksin and galangin, which confirmed the botanical origin of the orange and blue types of propolis. In addition, Gram-positive bacteria exhibited high susceptibility to poplar samples while being resistant to samples from other origins. Cherry bud samples had high amounts of naringenin and showed strong activity against Bacillus subtilis and Listeria monocytogenes. The combinations of tested phenolics showed mainly additive or indifferent effects.
Epidemics of downy mildew of onion, caused by Peronospora destructor, severely damaged commercial onion fields in western and central New York during the 19771979 growing seasons. On greenhouse-grown plants, when applied to onion leaves 24 hr before inoculation with P. destructor, chlorothalonil and mancozeb at rates equivalent to 1.07 L/ha (1.5 pt/acre) and 3.54 kg/ha (1 lb/acre) of formulated material, respectively, completely prevented infection. Greenhouse-grown plants were less susceptible to infection by P. destructor than outdoor-grown plants. Chlorothalonil and mancozeb at formulated rates equivalent to 3.08 L/ha (4.25 pt/acre) and 7.08 kg/ha (2 lb/acre), respectively, differed greatly in their residual effectiveness against infection of outdoor-grown onion plants. Mancozeb at that relatively low rate provided superior control over chlorothalonil at that relatively high rate when applied 2, 4, 7, or 10 days before inoculation. During 1980, onion growers in western and central New York generally used mancozeb to control downy mildew, and the incidence and severity of the disease was greatly reduced. From 1981 to 1984, incidence was nil when mancozeb was used.
Bacterial wilt of potato caused by Ralstonia solanacearum is one of the most destructive diseases in Kurdistan province, Iran. The objective of the present study was to evaluate antagonistic effects of some rhizobacteria isolated from the rhizosphere of potato plants against R. solanacearum, the agent of potato bacterial wilt. A total of 52 rhizobacteria were isolated and screened for in vitro antagonistic activity against R. solanacearum. Seven isolates with inhibiting effects of the pathogen were identified by phenotypic properties and partial sequencing of 16s rRNA as Pseudomonas fluorescens Pf11, P. fluorescens Pf16, Pseudomonas putida Pp17, Paenibacillus sp. Pb28 and Enterobacter sp. En38, Pseudomonas fluorescens Pp23 and Serratia sp. Se40. Strains Pf11, Pf16, Pp17 and Pb28 significantly inhibited the growth of the pathogen. Strains En38, Pp23 and Se40 showed a moderate or weak inhibition. During greenhouse study, strains were evaluated for their effects in reducing of disease and increasing biomass of potato plants. In according to greenhouse experiment results, isolates Pb28, Pp17 and Pf11significantly reduced disease by 55.56%, 51.50% and 38.58%, respectively. In addition, plant biomass significantly increased in plants treated with Pb28, Pp17, Pf11 and Pf16, compared to the control. Therefore, this study shows that these four strains have potential to be used as biocontrol agents against R. solanacearum. To confirm their effectiveness as commercial biocontrol agent, it is necessary to evaluate their efficiency in the field conditions in the next studies.