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Pseudomonas stutzeri YPL-1 Genetic Transformation and Antifungal Mechanism against Fusarium solani, an Agent of Plant Root Rot
Applied and Environmental Microbiology
Abstract
An actively antagonistic bacterium that could be used as a biocontrol agent against Fusarium solani, which causes root rots with considerable losses in many important crops, was isolated from a ginseng rhizosphere and identified as a strain of Pseudomonas stutzeri. In several biochemical tests with culture filtrates of P. stutzeri YPL-1 and in mutational analyses of antifungal activities of reinforced or defective mutants, we found that the anti-F. solani mechanism of the bacterium may involve a lytic enzyme rather than a toxic substance or antibiotic. P. stutzeri YPL-1 produced extracellular chitinase and laminarinase when grown on different polymers such as chitin, laminarin, or F. solani mycelium. These lytic extracellular enzymes markedly inhibited mycelial growth rather than spore germination and also caused lysis of F. solani mycelia and germ tubes. Scanning electron microscopy revealed degradation of the F. solani mycelium. Abnormal hyphal swelling and retreating were caused by the lysing agents from P. stutzeri YPL-1, and a penetration hole was formed on the hyphae in the region of interaction with the bacterium; the walls of this region were rapidly lysed, causing leakage of protoplasm. Genetically bred P. stutzeri YPL-1 was obtained by transformation of the bacterium with a broad-host-range vector, pKT230. Also, the best conditions for the transformation were investigated.
... Isolate STJP was grown in chitin-peptone medium and centrifuged, and collected supernatant was taken as sample for the estimation of chitinase activity [48]. For estimation of β-1, 3-glucanase, STJP was inoculated in peptone medium amended with laminarin (0.2%, Sigma-Aldrich, St. Louis, Missouri, USA) as per Lim et al. [48]. ...
... Isolate STJP was grown in chitin-peptone medium and centrifuged, and collected supernatant was taken as sample for the estimation of chitinase activity [48]. For estimation of β-1, 3-glucanase, STJP was inoculated in peptone medium amended with laminarin (0.2%, Sigma-Aldrich, St. Louis, Missouri, USA) as per Lim et al. [48]. After incubation, medium was centrifuged, and the supernatant was taken for quantification of β-1, 3glucanase [28,48]. ...
... For estimation of β-1, 3-glucanase, STJP was inoculated in peptone medium amended with laminarin (0.2%, Sigma-Aldrich, St. Louis, Missouri, USA) as per Lim et al. [48]. After incubation, medium was centrifuged, and the supernatant was taken for quantification of β-1, 3glucanase [28,48]. Cellulase activity was checked on an optimized medium containing carboxymethyl cellulose (CMC) as per Ghose et al. [49]. ...
The excess use of chemicals by farmers in the agroecosystems degrades soil quality, disturbs soil ecology, and increases soil salinity and health hazards in humans. Stevia rebaudiana is an important medicinal and aromatic crop whose leaves contain steviol glycosides (SGs). The Bacillus safensis NAIMCC-B-02323 strain STJP from the rhizosphere of S. rebaudiana producing salicylic acid (16.80 µg/mL), chitinase (75.58 U/mL), β-1,3-glucanase (220.36 U/mL), and cellulase (170 U/mL) was taken as a plant growth-promoting rhizobacteria (PGPR). The cell-free supernatant (CFS) from strain STJP showed significant biocontrol activity against Alternaria alternata (80%), suggesting the protective role of extracellular metabolite(s) against phytopathogens. Paneer whey-based bioformulation (P-WBF) was developed to exploit B. safensis STJP to enhance the growth, nutrient uptake, soil properties, stevioside content, and SGs biosynthesis in S. rebaudiana under an A. alternata-infested field. The combined treatment of P-WBF and mycorrhiza (Glomus fasciculatum ABTEC) significantly enhanced plant growth parameters after 90 days, in comparison with control. The symbiotic action (P-WBF and mycorrhiza) displayed much better results in terms of chlorophyll a and b (improved by 132.85% and 39.80%, respectively), protein (by 278.75%), flavonoid (by 86.99%), carbohydrate (by 103.84%), antioxidant (by 75.11%), and stevioside (by 120.62%) contents in plants as compared to the untreated set. Further, the augmentation of potassium (by 132.39%), phosphorous (by 94.22%), and zinc (by 111.11%) uptake in plant tissues and soil was also observed by the application of P-WBF and mycorrhiza. The expression of UGT74G1 and UGT85C2 genes related to SG biosynthesis was upregulated (2.7- and 3.2-fold, respectively) in plants treated with P-WBF and mycorrhiza as further confirmed by the accumulation of SGs. The results suggest that the application of P-WBF and mycorrhiza not only provides an ecofriendly and sustainable solution to improve stevioside content in S. rebaudiana by a nutrient-linked mechanism but also paves the way to enhanced production of stevioside.
... Pseudomonas cell suspension was grown at 28 • C for 96 h and mixed in a rotary shaker containing 50 ml of chitin-peptone medium as per Lim et al. (1991). The centrifuged cell suspension supernatant was used as an enzyme source. ...
... A unit of chitinase was considered as 1 nmol of GlcNAc released per minute per mg of protein. The primary procedure followed in chitinase enzyme estimation, the same procedure repeated up to supernatant preparation except growing medium as peptone medium containing laminarin as per Lim et al. (1991). The reaction mixture was incubated at 40 • C for 2 h in a water bath and ß-1,3-glucanase activity was measured as 1 nmol of glucose released per minute per mg of protein. ...
Seven strains of Pseudomonas spp. were isolated from the south Gujarat region of India. Antifungal and bacterial activities of bacterial strains were evaluated against important plant pathogens in vitro , among them, PaRS was found most effective. The indole acetic acid production was recorded in all isolated Pseudomonas spp. Seed treatment with PaRS at 6 g/kg was significantly superior over other treatments for plant growth promotion (germination, shoot and root length, shoot and root weight, vigor index, and both shoot and root colonization) under standard roll towel method and pot conditions. The maximum siderophore production was observed in PaRS and medium production in PfRB, PaNS, and PfNC. PaNS and PaRS strains recorded strong HCN production but moderate production recorded in PaWP, PaWS, and PfNC. The maximum phosphate solubalization zone (22 mm) was found in PaRS. PaRS recorded maximum chitinase, ß-1,3-glucanase activity, hydrogen cyanide, and salicylic acid production as compared to other strains.
... Theisolate were grown in 250 ml conical flasks containing 50 ml of liquid Czapek-Dox both media contained laminarin (0.2%) (From Laminaria digitata; Sigma) Lim et al., (1991), incubated at 7 days on a rotary shaker incubator. The cultures then centrifuged as previously mentioned and the resulted supernatant used as crude enzyme source. ...
... Absorbance was measured at 660 nm using a Milton Roy Spectronic 1201. Standard curve of glucose was used as reference (Lim et al., 1991). β 1,3 glucanase activity was determined as µg of glucose released / ml /min. ...
Two fungal isolates from commercial sugar beet phyllospher in Kafr-Elshaikh governorate, Egypt were tested for its antagonistic activity against Cercospora beticola the causal pathogen of sugar beet cercosporia leaf spot (CLS) disease in vitro.
Three methods were used to evaluate its antagonistic activity (Conventionalstreak, culture filtrates and volatile gases). The ability of the tested isolates to produce PR-proteins and growth-promoter indole acetic acid were studied. The tested isolates Emericella nidulans (Aspergillus nidulans) and Epicoccum nigrum found to have the potential to produce chitinase and β-1, 3 glucanase while Emericella nidulansthe only found to have the potential to produce indole acetic acid (IAA). The tested fungal isolates showed a remarkable antagonistic activity in both green-house and open field against C. beticola compared with the healthy control and the infected control. Also it was found that the control efficiency of the two isolates was almost equivalent to the tested fungicide. The present data show that phyllospheric fungi can play as a good candidate to be used as a bio-control agent against sugar beet leaf spot disease.
... cucumerinum (Kim et al., 2012), respectively. The production of extracellular chitinases and endoglucanases by Pseudomonas stutzeri has also been reported to lyse the mycelium of F. solani (Lim et al., 1991); however, chitinolytic activity is not the only mechanism in bacteria such as S. plymutica with antagonistic activity against Sclerotinia sclerotiorum and B. cinerea, since they are involved proteases and other biological control mechanisms that have greater antagonistic effect (Kamensky et al., 2003). It has also been reported that the β 1.3-glucanases synthesized by Paenibacillus sp. and Streptomyces sp., lyse the cell walls of F. oxysporum f. sp. ...
... It has been reported that the inhibition of germination and sporulation can occur by the secretion of lytic enzymes or by the production of volatile compounds (Kai et al., 2007;Leelasuphakul et al., 2008). Pseudomonas stutzeri secretes chitinase enzymes and extracellular laminarinases to lyse the mycelium of F. solani (Lim et al., 1991). Chitinolytic activity seems to be less essential in bacteria such as S. plymutica in its antagonistic activity against Sclerotinia sclerotiorum and Botrytis cinerea in which proteases and other biological control mechanisms are involved (Kamensky et al., 2003). ...
Use of biotechnological potential of native microorganisms as bio-inputs is having a great impact on agricultural systems. PlantGrowth Promoting Rhizobacteria (PGPR), in addition to their beneficial effect on plant growth and on the availability of soilele-ments, also have an antagonistic effect against different pathogens. In this study, growth promotion mechanisms with emphasisonthe antagonism of PGPR isolated from sugarcane and tomato crops were evaluated. Antagonism against Fusarium oxysporumf.sp lycopersici(Fol) was determined by dual tests, inhibition of germination and production of chitinases and endoglucanases. 52 iso-lates were evaluated and according to their results in dual tests 10 were selected for further analysis. Isolate GIBI127 showed the best percentage of Inhibition Germination (IG) of Fol (59.29%). Then, a selection index was calculated using results from gi,dual tests and growth promotion mechanisms to select five best isolates. Finally, these bacteria were evaluated for chitinases anden-doglucanases production using Miller ́s method. As a result, strain GIBI419 (Burkholderia cepacia) showed a higher production of these enzymes. Selected isolates have antagonistic potential along with plant growth promotion characteristics, which can be used for the development of microbial inoculants which allow the establishment of agricultural systems for tomato cultivation thatare sustainable, efficient, and environmentally friendly.
... Trichoderma isolates were screened for their antagonism against A. solani under in vitro conditions on malt extract dextrose agar (MDA) medium by following dual culture technique as described by (Lim et al., 1991) [6] . Five mm diameter discs of Trichoderma as well as the test pathogen were cut with a sterilized cork borer from the periphery of seven-day old culture. ...
... Trichoderma isolates were screened for their antagonism against A. solani under in vitro conditions on malt extract dextrose agar (MDA) medium by following dual culture technique as described by (Lim et al., 1991) [6] . Five mm diameter discs of Trichoderma as well as the test pathogen were cut with a sterilized cork borer from the periphery of seven-day old culture. ...
Alternaria solani (Ellis and Martin) is a pathogen causing early blight of tomato resulting in heavy yield losses. Thirteen isolates of Trichoderma tested against Alternaria solani and considered for assessment of variation as well as for management. Out of 13 Trichoderma isolates tested, T4 exhibited maximum inhibition (66.72%) against A. solani followed by T6 (65.57%). The isolates varied in their antagonistic ability against A. solani. While, T4 and T6 showed >65% inhibition, all remaining isolates exhibited >50% antagonism showing their potential as biocotnrol agents.
... BCA parasitize fungal phytopathogens by excreting extracellular cell wall-degrading enzymes (CWDEs) such as chitinases, glucanases, and proteases that target the pathogen's cell wall, resulting in lysis of the phytopathogen cells. For example, P. stutzeri produces extracellular chitinase and laminarinase, which lyses the mycelia of F. solani (Lim et al., 1991). The enhancement of the natural CWDEs biosynthetic capacity of these BCA has been evaluated. ...
In agriculture, the use of plant-associated microorganisms has emerged as an alternative to reduce the excessive use of chemicals and toxic fertilizers. The plant microbiome, composed of a diversity of microorganisms such as bacteria, fungi, algae, and nematodes, plays a fundamental role in plant growth and health, and the use of these microorganisms requires bioprospecting studies to isolate them from agroecosystems similar to those in which they are intended to be applied. These microorganisms can directly promote plant growth through various mechanisms, including the production of phytohormones, nitrogen fixation, phosphate solubilization, and the production of ACC deaminase enzymes. They can also exert indirect effects by combating plant pathogens through the production of antibiotics, lipopeptides, siderophores, and lytic enzymes. Advances in omics technologies have revolutionized the analysis of microbial inoculants' mechanisms of action from different perspectives. Genomics enables the determination of the genetic potential of microorganisms as plant growth promoters. The transcriptomic analysis provides insights into the physiological and metabolic status of these microorganisms. Proteomics is used to identify and quantify proteins related to plant growth-promoting traits. The metabolomic analysis examines the metabolites resulting from the interaction between the genome, transcriptome, and proteome of microorganisms. This comprehensive set of approaches allows for a deeper understanding of the metabolic mechanisms in plant-microorganism interactions which is of vital importance for the development of sustainable agricultural practices.
... BCA parasitize fungal phytopathogens by excreting extracellular cell wall-degrading enzymes (CWDEs) such as chitinases, glucanases, and proteases that target the pathogen's cell wall, resulting in lysis of the phytopathogen cells. For example, P. stutzeri produces extracellular chitinase and laminarinase, which lyses the mycelia of F. solani (Lim et al., 1991). The enhancement of the natural CWDEs biosynthetic capacity of these BCA has been evaluated. ...
The microbial inoculant industry, encompassing formulation, commercialization, and use, has witnessed remarkable global growth in recent years. Legal regulations are necessary to ensure the quality of microbial inoculants, protect farmers' and consumers' rights, and guide the commercial promotion of microbial products. However, regulating microbial inoculants remains a worldwide challenge, as they are often classified under chemical fertilizer laws due to the lack of experience with microorganism-based products. The lack of a consensus on the definition of these products and insufficient communication between the scientific community and farmers have hindered their legal regularization. To establish a framework for the development of microbial inoculants, these issues must be addressed. This includes reporting the microbial quality of the products, such as identification, abundance, plant growth-promoting capabilities, and contamination levels. In addition, physical and chemical factors, mode of application, dosage, and other aspects impacting product quality should be considered. Numerous organizations in different countries are currently working towards regulating the production, commercialization, and use of organic inputs in agriculture. Therefore, efforts are needed to define microbial inoculants and standardize regulations across countries, emphasizing cooperation among scientists, producers, sellers, and farmers.
... ricini and Alternaria tenuissima using maltose-dextrose agar. The dual culture approach, as described by Lim et al. (1991), was used to screen for antagonistic activity and identify prospective isolates with antagonistic activity against test pathogens. Following the aforementioned approach, isolates that inhibited the development of all test pathogenic fungi were subsequently evaluated by quantitative methods. ...
The selection and deployment of microorganisms in stressed ecosystems with biocontrol ability is a major challenge. In this investigation, we sought to isolate and identify strains of Azotobacter and Azospirillum spp., which could withstand abiotic stresses and possess the potential to serve as biological control against five phytopathogenic fungi. Stress tolerance was evidently less obvious in Azospirillum strains than in Azotobacter strains, when bacterial strains were screened for high temperature (50 °C), salt (7% NaCl), and drought (1.2 MPa). Strains Asp30 and Asp 32 of Azospirillum and Azb 19, Azb20 and Azb27 of Azotobacter were found tolerant to temperature, drought and salinity stresses. Five strains of Azotobacter viz. Azb2, Azb6, Azb10, Azb16 and Azb18 and six strains of Azospirillum viz. Asp2, Asp10, Asp22, Asp30, Asp32 and Asp39 inhibited all the five fungal phytopathogens studied. Therefore, in vitro screening provided the basis for identification and selection of strains with abiotic stress tolerance and biocontrol ability.
... ricini and Alternaria tenuissima using maltose-dextrose agar. The dual culture approach, as described by Lim et al. (1991), was used to screen for antagonistic activity and identify prospective isolates with antagonistic activity against test pathogens. Following the aforementioned approach, isolates that inhibited the development of all test pathogenic fungi were subsequently evaluated by quantitative methods. ...
The selection and deployment of microorganisms in stressed ecosystems with biocontrol ability is a major challenge. In this investigation, we sought to isolate and identify strains of Azotobacter and Azospirillum spp., which could withstand abiotic stresses and possess the potential to serve as biological control against five phytopathogenic fungi. Stress tolerance was evidently less obvious in Azospirillum strains than in Azotobacter strains, when bacterial strains were screened for high temperature (50 °C), salt (7% NaCl), and drought (1.2 MPa). Strains Asp30 and Asp 32 of Azospirillum and Azb 19, Azb20 and Azb27 of Azotobacter were found tolerant to temperature, drought and salinity stresses. Five strains of Azotobacter viz. Azb2, Azb6, Azb10, Azb16 and Azb18 and six strains of Azospirillum viz. Asp2, Asp10, Asp22, Asp30, Asp32 and Asp39 inhibited all the five fungal phytopathogens studied. Therefore, in vitro screening provided the basis for identification and selection of strains with abiotic stress tolerance and biocontrol ability.
... Notably, strain VUPf5 exhibited a profound impact on F. solani, leading to the fragmentation of fungal filaments around bacterial cells. This effect could be attributed to the production of lytic enzymes, such as chitinase and laminarinase, which hinder mycelial development and cause hyphal lysis in F. solani 28 . Pseudomonas spp. ...
Biocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp., responsible for potato soft rot disease. Discovering agents that can effectively control both fungal and bacterial pathogens in potatoes has always presented a challenge. Biological controls were investigated using 500 bacterial strains isolated from rhizospheric microbial communities, along with two promising biocontrol strains: Pseudomonas (T17-4 and VUPf5). Bacillus velezensis (Q12 and US1) and Pseudomonas chlororaphis VUPf5 exhibited the highest inhibition of fungal growth and pathogenicity in both laboratory (48%, 48%, 38%) and greenhouse (100%, 85%, 90%) settings. Q12 demonstrated better control against bacterial pathogens in vivo (approximately 50%). Whole-genome sequencing of Q12 and US1 revealed a genome size of approximately 4.1 Mb. Q12 had 4413 gene IDs and 4300 coding sequences, while US1 had 4369 gene IDs and 4255 coding sequences. Q12 exhibited a higher number of genes classified under functional subcategories related to stress response, cell wall, capsule, levansucrase synthesis, and polysaccharide metabolism. Both Q12 and US1 contained eleven secondary metabolite gene clusters as identified by the antiSMASH and RAST servers. Notably, Q12 possessed the antibacterial locillomycin and iturin A gene clusters, which were absent in US1. This genetic information suggests that Q12 may have a more pronounced control over bacterial pathogens compared to US1. Metabolic profiling of the superior strains, as determined by LC/MS/MS, validated our genetic findings. The investigated strains produced compounds such as iturin A, bacillomycin D, surfactin, fengycin, phenazine derivatives, etc. These compounds reduced spore production and caused deformation of the hyphae in F. solani. In contrast, B. velezensis UR1, which lacked the production of surfactin, fengycin, and iturin, did not affect these structures and failed to inhibit the growth of any pathogens. Our findings suggest that locillomycin and iturin A may contribute to the enhanced control of bacterial pectolytic rot by Q12. Fungal pathogens, such as Fusarium solani (FS) 1,2 , and bacterial pathogens, including Pectobacterium carotovo-rum (PC) 3,4 , P. atrosepticum (PA) 5-8 , and Xanthomonas campestris (XC) 9 , play a significant role in global plant diseases, causing root rot (dry and soft), leaf spots, and blackleg in both forest and crop ecosystems 1,7,10. The use of chemical fungicides and bactericides for disease management has adverse effects on human health and the environment. Consequently, there is an increasing reliance on biological agents to mitigate the negative impact of agrochemicals. The rhizosphere represents a competitive environment where microorganisms vie for resources to ensure their survival 11. While beneficial microorganisms are widely used in agriculture to control plant diseases, their inconsistent performance and a lack of sufficient safety data have limited the adoption of many promising biopesticides 12. Thus, there is an urgent need to identify bacteria that not only promote plant growth but also consistently demonstrate effective biological control mechanisms. Genome mining of microorganisms can provide valuable insights into the molecular-level mechanisms involved. Plant growth-promoting rhizobacteria (PGPR) employ various mechanisms for biocontrol. In addition to secondary metabolites that directly act against plant pathogens 13 , induced systemic resistance (ISR) and rhizospheric colonization are key OPEN
... β-1,3-Glucanase (GLU) activity was measured according to the methodology of Lim et al. (1991) with some modifications. To activate Pseudomonas fluorescens (Ms9N) and Stenotrophomonas maltophilia (Ll4) β-1,3-glucanase activity, they were grown on the liquid M9 medium supplemented with 0.02% laminarin (from Laminaria digitata, Sigma) at 28 °C for 72 h on a rotary shaker (140 rpm). ...
Main conclusion
PGPRs: P. fluorescens Ms9N and S. maltophilia Ll4 inhibit in vitro growth of three legume fungal pathogens from the genus Fusarium. One or both trigger up-regulation of some genes (CHIT, GLU, PAL, MYB, WRKY) in M. truncatula roots and leaves in response to soil inoculation.
Abstract
Pseudomonas fluorescens (referred to as Ms9N; GenBank accession No. MF618323, not showing chitinase activity) and Stenotrophomonas maltophilia (Ll4; GenBank accession No. MF624721, showing chitinase activity), previously identified as promoting growth rhizobacteria of Medicago truncatula, were found, during an in vitro experiment, to exert an inhibitory effect on three soil-borne fungi: Fusarium culmorum Cul-3, F. oxysporum 857 and F. oxysporum f. sp. medicaginis strain CBS 179.29, responsible for serious diseases of most legumes including M. truncatula. S. maltophilia was more active than P. fluorescens in suppressing the mycelium growth of two out of three Fusarium strains. Both bacteria showed β-1,3-glucanase activity which was about 5 times higher in P. fluorescens than in S. maltophilia. Upon soil treatment with a bacterial suspension, both bacteria, but particularly S. maltophilia, brought about up-regulation of plant genes encoding chitinases (MtCHITII, MtCHITIV, MtCHITV), glucanases (MtGLU) and phenylalanine ammonia lyases (MtPAL2, MtPAL4, MtPAL5). Moreover, the bacteria up-regulate some genes from the MYB (MtMYB74, MtMYB102) and WRKY (MtWRKY6, MtWRKY29, MtWRKY53, MtWRKY70) families which encode TFs in M. truncatula roots and leaves playing multiple roles in plants, including a defense response. The effect depended on the bacterium species and the plant organ. This study provides novel information about effects of two M. truncatula growth-promoting rhizobacteria strains and suggests that both have a potential to be candidates for PGPR inoculant products on account of their ability to inhibit in vitro growth of Fusarium directly and indirectly by up-regulation of some defense priming markers such as CHIT, GLU and PAL genes in plants. This is also the first study of the expression of some MYB and WRKY genes in roots and leaves of M. truncatula upon soil treatment with two PGPR suspensions.
... The bacterial endophytes can adopt hyperparasitism and/or predation by synthesizing lytic enzymes such as chitinase, glucanase, cellulose, etc., which can degrade the cell wall of fungal pathogens. The extracellular chitinase and laminariase produced by Pseudomonas stutzeri digest and lyse mycelia of F. solani (Lim et al., 1991). Β-1,3 glucanase enzyme synthesized by B. 3azandar can destroy the integrity of R. solani, S. rolfsii, and Pythium ultimum (Fridlender et al., 1993). ...
... Pseudomonas isolates such as P. putida and P. stutzeri used in the present study were reported previously to inhibit soil-borne pathogens' growth [13]. Pseudomonas stutzeri isolated from ginseng rhizosphere produced extracellular enzymes such as: chitinase and laminarinase that lyse the mycelium of Fusarium solani to prevent the fungus from causing root rot in different crops [14]. Lalucat et al. [15] reported that P. stutzeri had a LysRtype regulator involved in controlling oxidative stresses. ...
Background:
Fusarium wilt and Ascochyta blight are the most important diseases of chickpea. The current study was designed to investigate the individual and combined effect of salicylic acid (SA) with Pseudomonas stutzeri and Pseudomonas putida to suppress Fusarium wilt and promote growth of chickpea varieties: Thal-2006 and Punjab-2008.
Methods:
At the time of sowing, inoculum of Fusarium oxysporum was applied to the soil and the incidence of Fusarium wilt was recorded after 60 days. The seeds were inoculated with Pseudomonas stutzeri and Pseudomonas putida prior to sowing. Chickpea plants were treated with salicylic acid at seedling stage.
Results:
The combination of P. stutzeri and SA significantly increased root length (166% and 145%), shoot height (50% and 47%) and shoot biomass (300% and 233%) in cv. Thal-2006 and cv. Punjab-2008, respectively, in infected plants. Similarly, the combined treatment of P. putida + SA, also enhanced the plant growth parameters of chickpea varieties. Maximum reduction in disease severity was observed in both P. stutzeri + SA (90% and 84%) and P. putida + SA (79% and 77%) treatments in cv. Thal-2006 and Punjab-2008, respectively. Both P. putida + SA and P. stutzeri + SA treatments resulted in increased leaf relative water and total protein content, peroxidase, superoxide dismutase, phenylalanine ammonia-lyase and polyphenol oxidase activities in both resistant (cv. Thal-2006) and susceptible (cv. Punjab-2008) cultivars. Both treatments also significantly reduced malondialdehyde (MDA) and proline content in cv. Thal-2006 and Punjab-2008. Cultivar Thal-2006 was more effective than cv. Punjab-2008.
Conclusions:
The results suggested that, in combination, salicylic acid and P. stutzeri may play an important role in controlling Fusarium wilt diseases by inducing systemic resistance in chickpea.
... Pseudomonas flouorescens has been widely used for controlling sheath blight of rice (Vidhyasekaran and Muthamilan, 1999). The bacterium has the ability to produce certain antifungal compounds such as cell lytic and degrading enzymes (3-glucanase and chitanses) (Lim et al., 1991;Velazhahan et al., 1999). Through siderophore production it suppresses the chlamydospores growth (Bakker et al., 1986) and Wheat foliar pathogens (Septoria tritici, Puccinia recondita) tobacco black root by HCN production (Voisard et al., 1989;Flaishman, 1996). ...
Rice (Oryza sativa L.) is one of the most important staple food crop of Pakistan as well as in the whole world. A number of phyto-pathogens including bacteria, fungi, nematodes and viruses along with abiotic stresses such as drought, salinity, nutrient deficiency and etc. have a detrimental effect on rice yield. In order to overcome the attack of pathogenic microbes, use of endophytes as bio-control agent is an attractive way to minimize the use of toxic agrochemicals. In this study, we screened seven strains of bacterial endophytes from the rhizosphere of rice plants. A colony PCR was performed using universal primers to characterize the isolated bacterial endophytes followed by sequence analysis. On the basis of molecular characterization, endophytes were identified as Pantoea sp., Burkholderiasp Bacillus megaterium, Pseudomonas flourescens, Lysinibacillus fusiformis, Delftia sp., and Acinetobacter baumnii. Additionally, endophytes were assayed for In vitro effects against Fusarium moniliforme and Rhizoctonia solani causing bakanae and sheath blight disease of rice, respectively. Pantoea sp., Burkholderia sp., Bacillus megaterium and Delftiasp moderately suppressed Fusarium moniliforme but Pseudomonas flourescens, Lysinibacillus fusiformis both showcased a strong inhibiting activity against Fusarium moniliforme. On the other hand, Pantoea sp., Burkholderia sp., Bacillus megaterium, Pseudomonas flourescens and Lysinibacillus fusiformis had a strong inhibitory effect against Rhizoctonia solani. The isolated endophytic bacteria were also found to be good producers of phyto-hormones such as hydrogen cyanide (HCN) and catalase. On the basis of our results, we conclude that the endophytic bacteria from rice rhizosphere possess antifungal activity against economic important pathogenic fungi.
... 129 (7) By reducing sodium uptake and increasing potassium uptake by roots, biochar can maintain ion homeostasis in plants. 173,174 Potassium (K) is another important macronutrient needed in large amounts for adequate plant growth and development. However, approximately 90−98% of soil K is in forms that are often considered "nonexchangeable" limiting the availability of K for plant use. ...
The contamination of soil with organic pollutants has been accelerated by agricultural and industrial development and poses a major threat to global ecosystems and human health. Various chemical and physical techniques have been developed to remediate soils contaminated with organic pollutants, but challenges related to cost, efficacy, and toxic byproducts often limit their sustainability. Fortunately, phytoremediation, achieved through the use of plants and associated microbiomes, has shown great promise for tackling environmental pollution; this technology has been tested both in the laboratory and in the field. Plant−microbe interactions further promote the efficacy of phytoremediation, with plant growth-promoting bacteria (PGPB) often used to assist the remediation of organic pollutants. However, the efficiency of microbe-assisted phytoremediation can be impeded by (i) high concentrations of secondary toxins, (ii) the absence of a suitable sink for these toxins, (iii) nutrient limitations, (iv) the lack of continued release of microbial inocula, and (v) the lack of shelter or porous habitats for planktonic organisms. In this regard, biochar affords unparalleled positive attributes that make it a suitable bacterial carrier and soil health enhancer. We propose that several barriers can be overcome by integrating plants, PGPB, and biochar for the remediation of organic pollutants in soil. Here, we explore the mechanisms by which biochar and PGPB can assist plants in the remediation of organic pollutants in soils, and thereby improve soil health. We analyze the cost-effectiveness, feasibility, life cycle, and practicality of this integration for sustainable restoration and management of soil.
... Another enzyme of interest is extracellular chitinase, which can also have several biotechnological applications. In the agricultural field, its use as biocontrol of plant diseases caused by pathogenic fungi such as Fusarium have been reported (Lim et al. 1991). Two isolates from A. taxiformis, closely related to P. celer and P. putida presented chitinase production ability. ...
The red alga Asparagopsis taxiformis, considered as an introduced and invasive species in the Mediterranean Sea, can strongly contribute to habitat modification. Among the most effective measures in the management of marine invasive species, commercial use of their biomass is prioritized. Thus, we investigated the biological properties (antibacterial, algicidal and radical scavenging capacity) of Asparagopsis taxiformis collected from northern coast of Tunisia in order to determinate its potential biotechnological uses. Organic extracts were prepared by maceration in dichloromethane (C), dichloromethane/methanol (CM) and methanol/water (MW) solvents. Antibacterial activity has been tested on three human pathogen bacterial strains. While cytotoxicity was determined by algicidal activity on microalgae cultured cells of Navicula sp. Antioxidant potential of Asparagopsis extracts have been determined by assaying target molecules such as polyphenols, flavonoids and tannins, in addition to DPPH antiradical scavenging capacity measure. Furthermore, culturable heterotrophic bacteria associated with the red alga, were isolated and identified based on their16S rRNA. Biochemical characterization, antibiotic-resistance and enzymatic production were also determined for isolated strains. Results showed that only dichloromethane extract exhibited antibacterial activity and significant algicidal inhibition have been obtained for CM extract. The determination of total polyphenols, flavonoids and tannins, in MW extract, gave 85.8 ± 4.5 mg equivalents of gallic acid per g of dry weight (mg EGA g−1 DW), 73.44 ± 8.6 mg equivalents of catechin per g of dry weight (ECat mg−1 DW) and 15.63 ± 3.9 mg ECat g−1 DW respectively. Isolation of culturable bacterial strains associated with A. taxiformis, led to the obtaining of 18 isolates belonging mainly to Proteobacteria. The majority of the strains were Gram-negative and produced more than one enzyme activity.
... Tolerance against plant pathogen: Screening for biocontrol activity was carried out using the bangle method as described by (Lim et al., 1991) to identify antagonistic potential of isolates against common root pathogen Macrophomina phaseolina, fusarium oxysporium and Sclerotium rolfsii pathogens. These pathogens were obtained from Central Research Institute for Dryland Agriculture (CRIDA), Hyderabad, India. ...
Organic and inorganic pollutants have deleterious effect in the environment, crops productivity, microbiomeand soil fertility. The persistence uses of toxic compound like pesticides not only affect soil health but alsoagitated ecosystem. Use of Plant Growth Promoting rhizobacteria can play an important role towardsachieving the objectives of sustainable ecosystem. Pseudomonas group of bacteria are known for theirremediation properties but are lesser explored for their growth promotion attributes and to alleviate otherabiotic and biotic stress in the agriculture system. The paper presents multifarious potential of Pseudomonasisolates obtained from agriculture fields in the vicinity of Ghaziabad, U.P (PGPM2, 3, 4, 5). Biochemicaltests for identification suggested that these isolates belong to Pseudomonads group of organisms. Further,molecular characterization through 16S RNA gene sequence analysis confirmed their identity as PGPM2-Pseudomonas species, PGPM3- Pseudomonas plecoglosida, PGPM4- Pseudomonas and PGPM5- Pseudomonasrespectively. Production of essential hormones such as indole acetic acid, gibberellic acid and phosphatesolubilisation activity have endorsed the plant growth promotion attribute by these isolates. Production ofammonia and secretion of lytic enzymes like lipase by PGPM2 and PGPM3 indicate stronger bio-controlabilities in these isolates as compared to other isolates, all isolates showed positive for the bio-film formation,a positive attribute that helps in colonising and surviving harsh environments. Bangle method establishedthe strong antagonistic activity that these isolates possess against 3 important plant root pathogens namelyMacrophominaphaseolina (ï¯41%) followed by Sclerotium rolfsii (ï¯36-15%) and Fusarium oxysporum (ï¯21%)respectively. Isolates PGPM2 & PGPM3 showed maximum tolerance against salt (500MM) while isolatesPGPM2 and PGPM4 showed better tolerance to Pesticides monocrotophos and dimethoate (PGPM2- MICand LD50 and PGPM3 âMIC AND LD50). Host plant bioassay with pearl millets (bajra) revealed maximumgrowth promoting efficiency (root length and shoot length) by PGPM2. The manuscript provides abiochemical and physiological evidence that native Pseudomonas isolates not only promote plant growthbut also possess unique ability to tolerate adverse abiotic stress conditions such as tolerance to salt, organic(pesticides) and inorganic pollutants thus offering multiple growth supporting advantages to the planthost.
... Cucumerinum, respectively (Ordentlich et al., 1988). Lim et al. (1991) reported that Pseudomonas stutzeri secretes extracellular chitinase and laminarinase to lyse and degrade the mycelia of Fusarium solani. Moreover, protease facilitates the growth suppression of Sclerotinia sclerotiorum and Botrytis cinerea (Kamensky et al., 2003). ...
Relationship Between Microbes and Environment for Sustainable Ecosystem Services, Volume One: Microbial Products for Sustainable Ecosystem Services promotes advances in sustainable solutions, value-added products, and fundamental research in microbes and the environment. Topics include advanced and recent discoveries in the use of microbes for sustainable development. Users will find reference information ranging from the description of various microbial applications for sustainability in different aspects of food, energy, the environment and social development. Volume One includes the direct and indirect role of bacteria, fungi, actinomycetes, viruses, mycoplasma and protozoans in the development of products contributing towards sustainable. The book provides a holistic approach to the most recent advances in the application of various microbes as a biotechnological tool for a vast range of sustainable applications, modern practices, exploring futuristic strategies to harness its full potential.
... Cucumerinum, respectively (Ordentlich et al., 1988). Lim et al. (1991) reported that Pseudomonas stutzeri secretes extracellular chitinase and laminarinase to lyse and degrade the mycelia of Fusarium solani. Moreover, protease facilitates the growth suppression of Sclerotinia sclerotiorum and Botrytis cinerea (Kamensky et al., 2003). ...
The increasing phytopathogens have become a chronic threat to food production intimidating food security. The practice of chemical pesticides on a large-scale worldwide has although ensured massive killing of pests but has also shown alarming impact on the nontarget organisms disturbing the microbial interactions as well as affecting the soil health enormously. Bacterial endophytes inhabiting the plant tissues have emerged as a more environmentally friendly alternative. The beneficial effects of these endophytes are similar to those of the rhizosphere bacteria. It can suppress the diseases in pathosystems and can also increase phytoimmunity to resist the disease. It is also established as a prospective growth-promoting agent resulting in soil and plant health restoration. A variety of mechanisms including competition for an ecological niche, antibiosis, parasitism, induction of systemic resistance, and inhibitory allelochemicals production in the host system, are the ways of bacterial endophytes to control the infection caused by broad-spectrum phytopathogens. However, still numerous challenges remain before exploiting, as the inherent characteristics of the endophyte and the environmental conditions they are adapted to. Understanding the mechanisms of their action and its regulation through the emerging genomic technologies and interdisciplinary research approaches can be helpful in targeting potent biocontrol bacterial endophytes. This chapter focuses on the recent works on the mechanisms and the principle of actions of bacterial endophytes against the phytopathogens and emphasizes the use and commercialization of biocontrol agents for treating plant diseases.
... Bacterial isolates were grown in 250 ml conical flasks containing 50 ml of peptone medium contained laminarin (0.2%) (From Laminariadigitate Sigma-Aldrich) and incubated at 35°Cfor 5 days on a rotary shaker incubator according to Lim et al., (1991). ...
In the is study, the endophytic bacteria were isolated from sugarcane stalks and tested for its in vitro antagonistic activity against Sporisorium scitamineum, the causal pathogen of sugarcane smut disease.
Also some physiological characteristics such as its ability to produce some secondary metabolites i.e. PR-proteins and growth-promoters were studied in both in vitro and in vivo. Only, 62 isolates among the total 240 isolates were found to have a bioactivity against the pathogen in vitro. Six isolates from the bioactive isolates were selected for its high antagonistic activity to study its potential to produce the enzymes (chitinase, and β 1,3 glucanase,) and growth-promoters (indole acetic acid (IAA) as well as siderophores and salicylic acid (SA)) in vitro. The selected isolates showed different degrees of antagonistic activity. The selected isolates were identified using the sequencing of the 16S ribosomal RNA (rRna) gene and subjected to mega blast-n in NCBI and proved to be Enterobacter sp.( LUX 27), Kosakonia radicictans (LUX41), Kosakonia radicictans(BAN 33), Klabsiella oxytoca (BAN 39), Bacillus subtilis subsp. Inaquosorum(Q17), Pantoea sp.(SOH 29). In vitro studies proved that, all selected endophytic bacteria produced Nitrogenase and indole acetic acid (IAA) while, Kosakonia radicictans, Klabsiella oxytoca and Bacillus subtilis subsp. Inaquosorum produced salicylic acid. Not one of the selected isolated prove to produce Siderophores. Pantoea sp., Kosakonia radicictans and Klabsiella oxytoca produced chitinase. While, the isolates Kosakonia radicictans and Bacillus subtilis subsp. Inaquosorum were the most effective in producing β 1,3glucanase. The bio-control activity of selected isolates was also studied in vivo under greenhouse conditions on sugarcane infected plants. All the tested isolates were completely reduced the disease compared to the infected control. All the selected endophytic bacteria increased Peroxidase, Chitinase and β-1, 3 glucanaseactivities in the treated plants while the isolates Kosakonia radicictans and Enterobacter sp. didn’t produce Polyphenol-oxidase in treated infected plants. The treated sugarcane plants showed a range of positive increase in Leaf Area, Stalk length, Stalk Diameter, number of tillers and total chlorophyll compared to the untreated healthy and infected control. The present study show the efficiency of using endophytic bacteria as a bio-control system against sugarcane smut.
... Cucumerinum. Extracellular chitinase and laminarinase synthesized by Pseudomonas stutzeri degraded the mycelia of F. solani (Lim et al., 1991). These and other related studies that are not included in this review suggests that, in the absence of high level of genetic resistance in high-yielding sorghum varieties, the PSM as bio-antagonists could safely be used to effectively manage the biotic stresses of sorghum and hence to reduce losses in yield and quality of sorghum cultivated in different regions of the world. ...
Sweet sorghum [Sorghum bicolor (L.) Moench] is a highly productive, gluten-free cereal crop plant that can be used as an alternative energy resource, human food, and livestock feed or for biofuel-ethanol production. Phosphate fertilization is a common practice to optimize sorghum yield but because of high cost, environmental hazards, and soil fertility reduction, the use of chemical P fertilizer is discouraged. Due to this, the impetus to search for an inexpensive and eco-friendly microbiome as an alternative to chemical P biofertilizer has been increased. Microbial formulations, especially phosphate solubilizing microbiome (PSM) either alone or in synergism with other rhizobacteria, modify the soil nutrient pool and augment the growth, P nutrition, and yield of sorghum. The use of PSM in sorghum disease management reduces the dependence on pesticides employed to control the phytopathogens damage. The role of PSM in the sorghum cultivation system is, however, relatively unresearched. In this manuscript, the diversity and the strategies adopted by PSM to expedite sorghum yield are reviewed, including the nutritional importance of sorghum in human health and the mechanism of P solubilization by PSM. Also, the impact of solo or composite inoculations of biological enhancers (PSM) with nitrogen fixers or arbuscular mycorrhizal fungi is explained. The approaches employed by PSM to control sorghum phytopathogens are highlighted. The simultaneous bio-enhancing and biocontrol activity of the PS microbiome provides better options for the replacement of chemical P fertilizers and pesticide application in sustainable sorghum production practices.
... The antagonistic activity of selected twelve bacterial endophytes was assessed against major fungal plant pathogens of maize namely Macrophomina phaseolina and Fusarium oxysporum by dual culture method [13] . 5 mm of a fungal disc of actively grown Macrophomina phaseolina and Fusarium oxysporum were placed on PDA plates. ...
... Identification of bacterial strain using 16S rRNA genes in the current study is in coherence with other research works (Dey et al. 2019;Tian et al. 2017). 16S rRNA genes have been considered to be specific and reliable genetic markers for prokaryotes identification and assessing phylogenetic relationships where biochemical methods fail to distinguish (Lim et al. 1991;Yarza et al. 2014;Egamberdieva et al. 2017a). ...
Agricultural crops are susceptible to abiotic and biotic factors. Among biotic factors, plant pathogens attacks are prominent and significantly reduce the crop productivity and production. Long term application of synthetic pesticides in agriculture not only causes resistance development in pathogens but also deteriorates the soil quality and pollutes the ecosystem. In the current study, an investigation has been made with an objective to isolate an endophytic bacterium from Withania somnifera seed and evaluate its potential biocontrol activity against soil-borne pathogens like Fusarium oxysporum var. ciceri and Rhizoctonia solani. The molecular approach of bacterium identification using 16S rRNA sequencing methods includes kit based DNA extraction and PCR amplification and the isolated endophytic bacterium was identified as Pseudomonas stutzeri. In dual culture assay, the endophytic bacterium P. stutzeri PSIISS-1 effectively suppressed growth of pathogenic fungi F. oxysporum var. ciceri (61.0%) and R. solani (53.3%), respectively. In vivo studies were conducted with initial microbial inoculum size of 10⁸cell/ml and the studies revealed that the bacterium P. stutzeri PSIISS-1 co-inoculated with R. solani had significantly inhibited the sheath blight disease with the efficacy of 76% in susceptible paddy MTU-7029. Simultaneously, co-inoculation of P. stutzeri PSIISS-1 along with the pathogen F. oxysporum var. ciceri in chickpea JG-14 variety resulted in reduction of wilt symptom up to 80.45%. Bacterial inhibitions against the pathogens were corroborated with chitinase, protease and lipase enzymatic activities. Based on the results obtained from the study, P. stutzeri showed biocontrol activity against plant pathogens which can be used as potential bio-pesticides.
... The root colonizing capacity of antagonistic rhizobacteria and their successful survival depends on their production capacity of allele chemicals such as different antibiotics, lytic enzymes, iron-chelating substances etc. [48]. It is one of the well-established facts that the bio-control agents can able to control plant diseases by the production of extracellular hydrolytic enzymes to breakdown the fungal cell wall and inactivate themselves [49][50][51]. The inoculation of chitinase producing strain of B. cereus to soil give protection to cotton seedlings from R. solani mediated root rot disease [52]. ...
Soil bacteria are now-a-days familiar for plant disease prevention in comparison to chemical control measures in an eco-friendly way. Antagonistic soil bacteria have created tremendous interests among scientists to be an alternative means of plant disease control. The aim of our study was to isolate soil bacteria with a multifaceted role, both in the aspects of plant nutrition and disease prevention. To achieve our goal, rhizobacteria from crop plants, cultivated in different regions of West Bengal were isolated and from a total of 250 isolates, only eight were primarily selected based on their initial growth suppression capabilities against the phytopathogen Fusarium oxysporum. Finally, the two bacterial isolates, NC1 and NC2, showed in vitro broad-spectrum antifungal activity against seven potent phytopathogenic fungi, i.e., Fusarium oxysporum, Alternaria alternata, Colletotrichum capsici, Macrophomina phaseolina, Sclerotium hydrophilum, Rhizoctonia solani and Penicillium digitatum, where they suppressed mycelial growth up to 69% over untreated control. The two potent antagonists were screened in vitro for their plant growth promoting (PGP) and antifungal properties where they showed their efficacy to produce many lytic enzymes, indole acetic acid (IAA) and ammonia. The isolates were detected as Gram-positive, endospore, and capsule forming rod-shaped bacteria. On the basis of morphological, physiological, biochemical and BLAST analysis of 16s rDNA sequences, NC1 and NC2 were identified as Bacillus amyloliquefaciens KY568716 and Bacillus velezensis KY568715. When applied on eggplant, both the isolates suppressed Fusarium wilt symptoms and enhanced plant growth significantly. The effects were observed up to 30 days after pathogen inoculation and both the isolates were effective in reducing Fusarium wilt disease symptoms by 57.23% and 64.19% respectively, as compared to pathogen treated plants. They also showed significant enhancement of all plant growth parameters such as root and shoot length, biomass and chlorophyll content. Thus, the two isolates can be used to develop an effective dual function bio-control agents to control plant diseases as well as promoting plant growth.
... Crude chitinase from Serratia marcescens has been reported to cause the lysis of hyphal tips of pathogenic fungi S. rolfsii up to 60% [36]. This biological control activity is also shown by Pseudomonas stutzeri against F. solani, involving bacterial hydrolytic enzyme, chitinase, and laminarinase [37]. ...
The contribution of endophytic bacteria to the wellbeing of plants as biocontrol agents may be due to endophytic bacteria growing in the same niche as phytopathogens. This work was conducted to study the antagonistic activity of endophytic bacteria recovered from sweet sorghum against Sclerotium rolfsii, Fusarium solani, Fusarium oxysporum, Colletotrichum gloeosporioides in vitro and evaluate the mechanisms of these fungal inhibitions. We selected 78 endophytic bacteria from the stem and root of sweet sorghum plants. They were tested for antagonist activity by direct confrontation method. Antifungal compound production and lytic enzyme activity were examined to determine their mechanisms in inhibiting fungal pathogens. Antifungal compound production was checked by detecting the presence of NRPS and PKS genes. Lytic enzyme activity of the bacteria was evaluated by their ability to produce cellulase, chitinase, and protease. Selected bacteria were identified using molecular analysis based on the 16S rRNA gene. 14 out of the 78 tested isolates showed antagonistic activity and two were able to inhibit all four tested fungal strains. Four bacteria, designated as ACIL1, ACNM4, ACNM6, and ATNM4, produced natural products via NRPS pathway, but only one bacterial extract, designated as ACNM4, showed fungal inhibition. Ten isolates were able to produce hydrolytic enzymes. Endophytic bacteria identified as Burkholderia were revealed to have potential as a biocontrol agent.
... and Pseudomonas spp. have been shown to have the ability to produce different degrading enzymes and metabolites that may cause septicaemia on different insects [9][10][11][12][13]. A variety of toxins and extracellular degradative enzymes, such as protease, lipase, carbohydrase and chitinase, have been reported to be secreted by S. marcescens that allow this bacterium to completely degrade and exploit the whole body of the insect host [14,15]. ...
The success of microbial termiticides in controlling termites depends on the ability of microbes to grow in different media and the functionality of the microbes as a resistant barrier or toxic bait. This study was conducted to understand the mortality rate and behaviour changes of the subterranean termite Coptotermes curvignathus Holmgren introduced with different concentrations of Serratia marcescens strain LGMS 1 and Pseudomonas aeruginosa strain LGMS 3 using wood and soil as bacterial transfer medium. In general, higher concentration of bacteria in soil caused a reduction in tunnelling activity and wood consumption and an increase in mortality. However, application on wood revealed a different outcome. Wood treated with S. marcescens of 10 ⁶ CFU ml ⁻¹ concentration proved to be more efficient as bait than higher concentration applications as it caused a high mortality rate while still highly palatable for termites. Wood or soil treated with S. marcescens concentration higher than 10 ⁹ CFU ml ⁻¹ creates a high toxicity and repellent barrier for termites. Pseudomonas aeruginosa of 10 ⁹ CFU ml ⁻¹ concentrations applied on wood served as a slow-acting toxic bait. However, the ability for S. marcescens and P. aeruginosa to survive on wood is low, which made the bait unable to retain a useful level of toxicity for a long period of time and frequent reapplication is needed.
... Frankowski et al. (2001) showed that the extracellular chitinase enzyme produced by Serratia plymuthica C48 inhibited the rate of sporulation and germination in Botrytis cinerea. Moreover, Lim et al. (1991) showed that chitinase and laminarinase that were produced extracellularly by Pseudomonas stutzeri destroyed the mycelia of F. solani. Some scientists have also shown that the GacA/GacS two-component signal transduction system regulates genes encoding proteases and chitinases involved in the lysis of pathogenic microorganism (Corbell and Loper, 1995;Gaffney et al., 1994;Natsch et al., 1994;Sacherer et al., 1994), or in the regulatory system of GrrA/GrrS (Ovadis et al., 2004); and in colony phase variation (Lugtenberg et al., 2001). ...
The application of biological control agents for the management of plant pathogens and pests requires sustainable methods that can increase the global rates of food production and food safety. This strategy has contributed to minimizing various detrimental effects of agrochemicals that not only contaminate our environment, but also present many potential health hazards as well as contributing to climate change. Recent advancement in biotechnology have led to the isolation of novel microorganisms, characterization of their gene products and subsequent cloning in plants with a view to increasing their tolerance to both biotic and abiotic stresses. In addition to improving intimate interactions between microbes and plants, these technological advancements have also allowed manipulation of modes of action of different biological control agents toward enhancing their biocontrol potentials. Therefore, this review emphasizes already existing and recent advancements in molecular techniques that have been adopted in the management of plant diseases and pests for enhanced food security and improved agricultural productivity for providing safe food and a healthy environment.
... Some strains of P. stutzeri synthesize siderophores [69,70]; for example, P. stutzeri RC7 produces a catechol-like siderophore [71]. However, no siderophores were detected in P. stutzeri YPL-1 [72]. Interestingly, our P. stutzeri QD1 was the best producer of both siderophores and HCN, supporting its anti-Fusarium oxysporum activity. ...
Introduction of quinoa (Chenopodium quinoa willd.), a gluten-free nutritious pseudo-cereal, outside its traditional growing areas exposed it to seedling damping-off. Here, we isolated eleven phosphate-solubilizing bacteria from the quinoa rhizosphere and assessed their effect on germination and seedlings growth. All isolates solubilized phosphate, produced indole3-acetic acid, hydrocyanic acid, siderophores, and ammonia. Genotypic analysis revealed that our strains are related to the genus of Bacillus, Pseudomonas, and Enterobacter. Strains Enterobacter asburiae (QD14, QE4, QE6, and QE16), Enterobacter sp. QE3, and Enterobacter hormaechei QE7 withstood 1.5 mg·L−1 of cadmium sulfate, 0.5 mg·mL−1 of nickel nitrate, and 1 mg·mL−1 of copper sulfate. Moreover, all strains solubilized zinc from ZnO; P. Stutzeri QD1 and E. asburiae QD14 did not solubilize Zn3(PO4)2 and CO3Zn, whereas CO3Zn was not solubilized by E. asburiae QE16. Bacillus atrophaeus S8 tolerated 11% NaCl. P. frederiksbergensis S6 and Pseudomonas sp. S7 induced biofilm formation. Anti-fusarium activity was demonstrated for E.asburiae QE16, P. stutzeri QD1, P. frederiksbergensis S6, Pseudomonas sp. S7, and B. atrophaeus S8. Lastly, inoculation of quinoa seeds with B. atrophaeus S8 and E. asburiae QB1 induced the best germination rate and seedling growth, suggesting their potential use as inoculants for salty and heavy metal or zinc contaminated soils.
... suppress Fusarium oxysporum while Pseudomonas sp. suppresses Fusarium solani, the commonly known plant pathogen (Lim et al. 1991). Many endophytic fungi like Alternaria alternate, Hymenoscyphus ericae, and Aspergillus terreus also produce extracellular enzyme xylanase producers including those found in Table 11.3. ...
Endophytes reside within internal tissues of living plants without causing any harm to the host. The influence of these microbial communities on plant growth, yield, stress, and disease resistance, has been identified as potential research priorities in agriculture. In this chapter, we aim to explore the diverse host–endophyte interactions for plant growth promotion and health. Initially, the colonization of endophytes in specific plant tissues is discussed along with their mechanism of entry, habitat selection, response to stimuli, and evasion of the plant immunity. Endophytic microbes promote plant growth through different types of direct and indirect mechanisms. Plant growth-promoting endophytes (PGPE) play a vital role in phytohormone production, nutrient acquisition, nitrogen fixation, and solubilization of minerals. Further, indirect mechanisms (like suppression of plant pathogens by producing volatile organic compounds, antagonizing agents, and quorum quenchers) are also discussed in detail. Siderophores production and the secretion of different hydrolytic enzymes like chitinases, glucanases, and proteases also help in the induction of systemic resistance and protection of the host plants. Bioactive metabolites derived from endophytes serve as excellent therapeutic agents and have potential applications in agriculture, cosmetics, pharmaceutical, and food industries. Hereby, this chapter highlights the scientific rationale behind using endophytic microbiomes as potential biofertilizers, biopesticides, and biocontrol agents.
... Thus, UV and chemical mutation induced higher yield of chitinase from the wild-type Bacillus licheniformis SSCL-10 by 5-6fold. Others have reported twofold enhanced chitinase production in Serratia marcescens QMB 1466 [36] and in Pseudomonas stutzeri YPL-M26 with N-methyl-N ′ -nitro-N-nitrosoguanidine (NTG) mutagenesis [37]. The partially purified chitinase was identified as a 66 kDa ( Figure 5) molecular weight protein by SDS-PAGE. ...
Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.
... They include competition for nutrients and space [14,15], antibiosis by producing antibiotics viz., pyrrolnitrin, pyocyanine, pyoluteorin, phenazines and 2, 4diacetyl phoroglucinol [16] and production of siderophores (fluorescent yellow-green pigment), viz., pseudobactin which confines the accessibility of iron required for the growth of pathogens [17,18]. The production of lytic enzymes such as chitinases and β-1, 3 glucanases which degrade chitin and glucan present in the cell wall of fungi [19,20,21,22], HCN production [23] and degradation of toxin produced by pathogen are some key mechanisms exist in PGPR [24,25]. Several species of Pseudomonas are known to protect plant through eliciting induced systemic resistance (ISR) in plants [26,2,27,28,29]. ...
Thirty well-characterized pseudomonad isolates for plant growth-promoting traits were screened for their antagonistic activities against 20 isolates of Sclerotium rolfsii. Out of the 30 pseudomonad isolates, PUR46 was found to be best against all 20 isolates of Sclerotium rolfsii, because of its unique ability to suppress the growth of mycelia as well as the sclerotia formation of most of the S. rolfsii isolates in vitro conditions. In our previous study, PUR46 was also found to be positive for growth promoting traits like phosphorus solubilization and ammonification. The results suggested that expression of one or more of the traits like antagonistic activity against S. rolfsii and solubilization of tri-calcium phosphate may help in controlling the pathogen besides enhancement of plant growth. In this study, our investigations clearly indicate that PGPR isolates PUR 46 may be exploited to be used as potential biocontrol agents against S. rolfsii in agriculture system.
... The inhibitory effect of antagonistic bacteria such as B. subtilis and P. fluorescens against growth reduction of phytopathogenic fungi may be due to the production of hydrolytic enzymes that can degrade cell walls, ironchelating siderophores and several cyclic lipodepsipeptides (Lim et al., 1991). Saravanakumar et al. (2007a) also showed that inoculation with fluorescent Pseudomonas induced a significant increase in root and shoot length over the uninoculated control. ...
... Pseudomonas stutzeri has been shown to produce chitinase and laminarinase, which caused the lysis of Fusarium solani germ tube and its mycelia. Markedly, this effect is more on mycelial growth inhibition rather than spore germination (Lim et al., 1991). The prevalence of diseases caused by R. solani, Sclerotium rolfsii, and Pythium ultimum was declined due to the enzymatic action of β-1,3-glucanase produced by Pseudomonas cepacia. ...
... Chitinolytic enzymes together with -1, 3-glucanases or cellulases are most frequently considered to play a vital role in degrading the pathogen cell wall in bio-control (Chet, 1987). -1, 3-glucanase produced by P. stutzeri suppressed F. solani (Lim et al., 1991), whereas P. cepacia controlled R. solani, S. rolfsii and P. ultimum (Fridlender et al., 1993). Exploitation of lytic enzyme production trait for suppression of fungal disease has been reported by Inbar and Chet (1991) and Chernin et al. (1995). ...
... The fungal pathogen is also degraded by the destruction of cell wall component by the enzymes, for example, chitinase, β-1,3-glucanase, proteases, and produced by PGPR [155]. Extracellular chitinase and laminarinase enzymes synthesized by Pseudomonas stutzeri are known to degrade mycelia of Fusarium solani [156]. Frankowski et al. [157] studied that the production of chitinase enzyme through Serratia plymuthica C48 restricted the germ tube elongation and spore germination in B. cinerea. ...
Plant growth‐promoting rhizobacteria (PGPR) are diverse groups of plant‐associated microorganisms, which can reduce the severity or incidence of disease during antagonism among bacteria and soil‐borne pathogens, as well as by influencing a systemic resistance to elicit defense response in host plants. An amalgamation of various strains of PGPR has improved the efficacy by enhancing the systemic resistance opposed to various pathogens affecting the crop. Many PGPR used with seed treatment causes structural improvement of the cell wall and physiological/biochemical changes leading to the synthesis of proteins, peptides, and chemicals occupied in plant defense mechanisms. The major determinants of PGPR‐mediated induced systemic resistance (ISR) are lipopolysaccharides, lipopeptides, siderophores, pyocyanin, antibiotics 2,4‐diacetylphoroglucinol, the volatile 2,3‐butanediol, N‐alkylated benzylamine, and iron‐regulated compounds. Many PGPR inoculants have been commercialized and these inoculants consequently aid in the improvement of crop growth yield and provide effective reinforcement to the crop from disease, whereas other inoculants are used as biofertilizers for native as well as crops growing at diverse extreme habitat and exhibit multifunctional plant growth‐promoting attributes. A number of applications of PGPR formulation are needed to maintain the resistance levels in crop plants. Several microarray‐based studies have been done to identify the genes, which are associated with PGPR‐induced systemic resistance. Identification of these genes associated with ISR‐mediating disease suppression and biochemical changes in the crop plant is one of the essential steps in understanding the disease resistance mechanisms in crops. Therefore, in this review, we discuss the PGPR‐mediated innovative methods, focusing on the mode of action of compounds authorized that may be significant in the development contributing to enhance plant growth, disease resistance, and serve as an efficient bioinoculants for sustainable agriculture. The review also highlights current research progress in this field with a special emphasis on challenges, limitations, and their environmental and economic advantages.
Fusarium wilt of watermelon [Citrullus lanatus (Thunb.) Matsumura & Nakai] is caused by Fusarium oxysporum f. sp. niveum (FON), is the most severe soil-borne disease under the prevailing conditions of Punjab. Present study was carried out during 2018–19 and 2019–20 at the pot house and research farm of Punjab Agricultural University, Ludhiana, Punjab to evaluate the antagonistic activity of 15 isolates of Bacillus spp. against FON. These isolates were screened by using dual culture assay and among them, Bacillus subtilus (B1) and Bacillus cereus (B3) showed maximum inhibition percentage i.e. 57.00 and 58.22, respectively. Similarly, these isolates (B1 and B3) showed maximum activity of chitinase (2.31 and 2.16 unit/ml) and glucanase (1.72 and 1.79 unit/ml). Further, Talc-based bio- formulations of B1 and B3 isolates were evaluated in pot house and research farm of Punjab Agricultural University, Ludhiana, Punjab. Amongst all the treatments, Bacillus subtilis (B1) and Bacillus cereus (B3) as seed + soil @15 g treatment showed maximum inhibition of FON. Therefore, based on our findings, Bacillus subtilis (B1) and Bacillus cereus (B3) used as seed + soil treatment was able to effectively manage the fusarium wilt in watermelon under pothouse and field conditions.
Two-component systems (TCSs) are widespread regulatory systems in bacteria, which control cellular functions and play an important role in sensing various external stimuli and regulating gene expression in response to environmental changes. Among the nineteen genes for the two-component system found in the whole genome of Pseudomonas stutzeri LH-42, one of the TCS coded by the HK-1 gene, has a structural domain similar to the HAMP domain, which plays an important role in regulating bacterial virulence in other bacteria. In this study, the deletion mutant LH-42△HK-1 was successfully constructed using the lambda Red recombinase system. Compared with the wild-type strain, the mutant strain LH-42△HK-1 showed a significantly slower growth time and a longer stationary phase time. In addition, in the plate bacteriostatic experiment with Escherichia coli DH5α as an indicator strain, the inhibition zone size of the mutant strain showed significantly less than the wild-type strain(P<0.05), indicating that the virulence of the mutant strain was significantly reduced compared with the wild-type strain. Overall, the results indicate that the deletion of the gene HK-1 decreased bacterial virulence in Pseudomonas stutzeri LH-42.
Antibiotics are becoming less effective as drug resistance spreads
throughout the world, making it ever more difficult to treat the disease. The
effect of biotic stress on antimicrobial activity of Lawsonia inermis L.
(Henna) was studied as an alternate to antimicrobial agents against
multidrug resistant microorganisms. The study was carried out in Molecular
Biochemistry Lab, Department of Biochemistry, University of Agriculture
Faisalabad. The plant seedlings were given biotic stress with Fusarium
solani. The induced extracts were harvested at different time intervals and
the antimicrobial activity was investigated by disc diffusion method. The
induced plant extracts revealed highest susceptibility against S. aureus
(30±0.02) and Pasteurella multocida (30±0.01) at 12 hpi. The least activity
was shown against all the strains at 0 hpi. The highest antifungal activity
was found against Ganoderma lucidum (55±0.05) at 24 hpi. The results
demonstrated a significant difference in the antimicrobial activities of all
the strains with and without fungal stress (P<0.05). The fungal induced
extracts of Lawsonia inermis L (Henna) with enhanced antimicrobial activity
may have the potential of being alternative and cost- effective agents
against antimicrobial resistance.
Microbes are organisms that are partner inhabitants of humans and other animals. They have been known to exist in various habitats including water, soil, air, and the interiors of animals as well as plants. These wonders have been known to perform several key roles such as decomposition and nutrient cycling. Their roles in the environment have got the limelight and many sectors are recognized where microbes can be used. Industries, agriculture, and environment are the three major sectors in which microbes can be utilized for various purposes. Agriculture is an important sector that feeds the worldwide population with several types of cereals and fruits. In the agriculture sector, different types of chemical inputs are applied to increase the yield and feed the growing population. Microbial-based inputs are a new trend that has been applied for quite a while now. Microbial-based inputs have been considered sustainable inputs that help in crop productivity. Microbes improve crop production by fulfilling the requirement of plants, along with protecting them from various biotic and abiotic stresses. This is an introductory chapter for the book Trends of Agricultural Microbiology for Sustainable Crops Production and Economy, which deals with the role of beneficial microbiomes for plant growth and plant protection for agricultural sustainability.
Bacillus bacteria are advantageous antagonistic organisms that can be used as bio-control agents. This study is aimed at screening the antagonistic activity of different strains of isolated Bacillus bacteria and molecular identification of the superior chitinase producer strain against dermatophytes fungi. Soil samples were collected from different places of Kotoor city, Gharbia Governorate, Egypt and Al Madina Al Munawwarah, Kingdom of Saudi Arabia. A collection of Bacillus isolated from soil was tested in vitro against the dermatophytes: Microsporum sp. and Trichophyton sp. The bacterial strains Kh-B1 and Kh-B2 showed the highest antagonistic activity against dermatophytes pathogenic fungi. The highest amount of chitinase productivity (13.6 units/ml) was obtained from the original Bacillus strain (Kh-B1) at 3 days of incubation. BLAST analysis of the amplified 16S ribosomal RNA gene sequence identified the Bacillus strain (Kh-B1) as Paenibacillus macerans. Upon the mutation induction by UV light, the highest chitinase-producing mutant was Kh-UVB-4 as it showed 305.88 percent production higher than the wild-type strain. While, upon the mutation induction by EMS, the highest amount of chitinase produced was 54.8 units/ml by mutant Kh-ESB-20, and it has produced 402.94% more than the original untreated strain. The application of RAPD-PCR protocol using three 15-mer random primers was used to determine the genetic effects of mutagenic treatments on the wild type strain (Kh-B1) as well as to demonstrate the genetic variability between the five most chitinase producing mutants and the wild type (Paenibacillus macerans).
Rhizomicrobiome is of utmost significance in agricultural sciences on account of highly diverse, distinct rhizospheric microflora present in direct or indirect association with plants. It is vital for plant growth promotion due to its involvement in processes such as nutrient uptake and incorporation; enhancement of soil characteristics; regulation of the production of plant growth hormones, secondary metabolites and antibiotics; regulation of stress tolerance; and rhizoremediation. The diverse rhizomicrobiomes are significant for maintaining sustainability in agricultural practices. Fulfilment of increasing food demand with employment of minimal chemical fertilizers has become a great challenge for both researchers and farmers. PGPR and their secretions play a pivotal role in efficiently stimulating plant growth and enhance stress tolerance for the biotic and abiotic stresses. The versatile PGPR-based biofertilizers have been formulated for use in agricultural practices, thus minimizing chemical fertilizers and agrochemical input. Therefore, a deep and comprehensive insight into the plant rhizomicrobiome and their mechanism is critical for exploring the aforesaid sustainability of agricultural practices.
Nitrogen bubbles that is generated by microbial denitrification process is a new pollution-free clean material for mitigation of sand liquefaction. The current study aims to assess sustainability as well as distribution character of biogas bubbles in sand column under the condition of hydrostatics along with its performance of mitigating sand liquefaction under static loading. A series of laboratory experiments were conducted, and test results indicate that biogas bubbles has excellent sustainability in sand pores, and after 92 weeks, an increase of saturation from 84.5 to 85.1% marking only 0.6% rise. The volume of biogas generated by bacteria increases linearly with decrease of depth. Under ndrained condition, if saturation of sample decreased from 100% to around 92.4%, strain softening behavior will transfer to strain hardening, and undrained shear strength can be increased by approximately two times in both of compression and extension tests. The excess pore water pressure ratio and liquefaction potential index have significant reduction with the decrease of saturation, and the magnitude of impact on compression is comparatively bigger than the extension tests. This study validates that as a new material, biogas bubbles are very stable in soil, desaturation using nitrogen bubbles is an effective method for mitigating the liquefaction of sand under static loading conditions. Moreover, the study provides support for the desaturation mitigating static liquefaction of sand to prevent geological disasters and reveals its potential engineering practical value.
In a context of a changing environment, it is crucial to maximize our knowledge on all the mechanisms involved in plant microbiome interactions at the genetic, physiological, and ecological levels. This chapter reviews some recent advances in plant microbiome investigations and describes potential applications of such associations for the mitigation of both abiotic and biotic stresses to improve crop health and productivity. Understanding the full potential of microbes in the ecosystem functioning in general and their complex beneficial interactions in improving agriculture productivity in particular requires the development and improvement of compatible tools that can be verified in biological assays, always bearing in mind their reproducibility in situ on different scales. Progress in the engineering microbiome have made it possible to show how meta‐omics (metataxonomic, metagenomics, metatranscriptomics, and metaproteomics) can be potentially powerful tools to gain deeper knowledge of the functional capabilities of the microbiome and how they can shape ecosystems.
The term “endophyte” was first introduced by De Bay in 1886 to explore the symbiotic microbiota to contain the plant diseases like probiotic microbiota in human being. The modern intensive farming system enhanced the use of chemical synthetic pesticides to increase the crop production in agri-horti ecosystems for many decades. Due to the hazardous effect of pesticides on soil health, human health, and the environment, this is likely to be limited in future. As an alternative strategy, the secondary metabolites produced by agriculturally important microorganisms are explored, which are residing in the host plants as epiphytes and endophytes and play an important role in improving the quality of the crops. Microorganisms synthesize enzymes, compounds either alone or in association with the host plants, which are eco-friendly, toxic to plant pathogens, and do not harm human beings and other living organisms. Inoculation with endophytes modulates the synthesis of bioactive compounds with high pharmaceutical properties in addition to plant growth promotion. In this review, we have focused on the need for exploration of alternative microbial habitats for isolating novel microbes with chemical and functional diversity.
A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.
With a growing population, the demand for crop production is an increasing trend. The main hindrance in this regard is the biotic stresses like diseases and pests attacking crop causing huge losses every year. Mainly the soil-borne pathogens are devastatingly affecting the crop production. It is challenging to predict, detect and diagnose a variety of soil-borne pathogens that are causing plant diseases. The soil-borne plant pathogens can be categorized under divisions of bacterium, virus, fungus or plant parasitic nematode. These pathogens are highly effective due to extensive surviving periods even in absence of host plant and also congenial environmental condition. The survivability of soil-borne pathogens in the soil varies for each of them. Most soil-borne pathogens are difficult to control by conventional procedures like the use of resistant cultivars. Extensive use of fungicides is expensive and affects nontarget microflora. The promising strategies used are crop rotation and cover cropping and using organic amendments (manures and composts) and biological control are alternative methods which can replace the use of chemical pesticides in the control of soil-borne pathogens. In recent years, biological control has become very popular and included in plant disease management, and it is considered as a practical and safe approach in many crops. Among these antagonistic microorganisms, actinobacteria are well-known and classified among the most active rhizobacteria. In recent decade actinobacteria have gained importance due to its extensive presence and active colonization ability in rhizosphere of plants, its ability to produce a wide variety of agro-active compounds effective against many pathogens. Actinobacteria, especially Streptomyces spp., have biocontrol action against a range of phytopathogens. The actinobacterial mechanisms imparting soil-borne disease control majorly involve antibiosis, hyperparasitism, production of cell wall-degrading enzymes, stimulation of nodulation, etc. Actinobacteria-treated plants showed ameliorated plant health. The role of actinobacteria, as the probable stimulator of ISR (induced systemic resistance), is a major aspect in disease control. It induces signalling pathway involved in plant disease resistance and produces many defence-related enzymes protecting plants against pathogen attack and also preventing its further spread. Hence actinobacteria are an effective biocontrol agent and can be used for the control of soil-borne pathogens.
A total of 120 rhizobacteria were isolated from seven different tea estates of Darjeeling, West Bengal, India. Based on a functional screening of in vitro plant growth-promoting (PGP) activities, thirty potential rhizobacterial isolates were selected for in-planta evaluation of PGP activities in rice and maize crops. All the thirty rhizobacterial isolates were identified using partial 16S rRNA gene sequencing. Out of thirty rhizobacteria, sixteen (53.3%) isolates belong to genus Bacillus, five (16.6%) represent genus Staphylococcus, three (10%) represent genus Ochrobactrum, and one (3.3%) isolate each belongs to genera Pseudomonas, Lysinibacillus, Micrococcus, Leifsonia, Exiguobacterium, and Arthrobacter. Treatment of rice and maize seedlings with these thirty rhizobacterial isolates resulted in growth promotion. Besides, rhizobacterial treatment in rice triggered enzymatic [ascorbate peroxidase (APX), catalase (CAT), chitinase, and phenylalanine ammonia-lyase (PAL)], and non-enzymatic [proline and polyphenolics] antioxidative defense reactions indicating their possible role in the reduction of reactive oxygen species (ROS) burden and thereby priming of plants towards stress mitigation. To understand such a possibility, we tested the effect of rhizobacterial consortia on biotic stress tolerance of rice against necrotrophic fungi, Rhizoctonia solani AG1-IA. Our results indicated that the pretreatment with rhizobacterial consortia increased resistance of the rice plants towards the common foliar pathogen like R. solani AG1-IA. This study supports the idea of the application of plant growth-promoting rhizobacterial consortia in sustainable crop practice through the management of biotic stress under field conditions.
Human efforts to develop agriculture have been known for a long time. Since becoming aware of the importance of this field, farmers and researchers have not ceased looking for methods and products to improve crop productivity and quality and to protect it from various aggressions and stress that it might undergo. Mechanisms using microbes as biofertilizers and biocontrol agents have been adopted recently as an alternative to agrochemicals. The use of beneficial microbes is an environment-friendly strategy, which play a major role in the stimulation of plant growth and in the biocontrol of plant pathogens. A better understanding of the use of these bacterial populations could allow a reduction of chemical inputs and pollutant pesticides in agricultural soils. The present review is limited to plant growth-promoting bacteria (PGPB); it summarizes the role of PGPB in soil fertilization and plant protection with a special emphasis on their mechanisms of action. Several examples of PGPB chosen from the literature are cited in this chapter. This review includes also examples of the agricultural application of these microbes.
Antimicrobial property against phytopathogens
In in vitro tests, two strains of Trichoderma harzianum failed to parasitize colonies of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis. However, these strains were strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum. When grown in liquid cultures containing laminarin, chitin or fungal cell walls as sole carbon sources, both strains of T. harzianum released, 3-β-glucanase and chitinase into the medium. Higher levels of these enzymes were induced in strain T-203 than in T-35 by hyphal cell walls of F. oxysporum. When the lytic enzymes produced by T-35 were incubated with hyphal cell walls of the test fungi, more glucose and N-acetyl-d-glucosamine was released from cell walls of R. solani and Sclerotium rolfsii than from those of F. oxysporum. Treatment of F. oxysporum cell walls with 2 m-NaOH, protease or trypsin prior to their incubation with the lytic enzymes of T. harzianum significantly increased the release of glucose and N-acetyl-d-glucosamine. The effect of these treatments on R. solani and S. rolfsii cell walls was much lower. These results suggest that proteins in the cell walls of F. oxysporum may make these walls more resistant than those of R. solani or S. rolfsii to degradation by extracellular enzymes of T. harzianum.
Elad, Y., Chet, 1., Boyle, P., and Henis, Y. 1983. Parasitism of Trichoderma spp. on Rhizoctonia so/ani and Sclerotium rolfsii- Scanning e lectron microscopy and nuorescence microscopy. Phytopathology 73:85-88. Hypha! interactions between either Trichoderma harzianum or T. hamawm, and Sclerotium rolfsii or Rhizoctonia so/ani were observed by scanning electron microscopy. Trichoderma spp. auached to the host either by hypha! coils, hooks, or appressoria. Lysed sites and penetration holes were found in hyphae of the plant pathogenic fungi, following reinoval of parasitic hyphae. High 13-(1 ,3) glucanase and chitinase activities were detected in dual agar cultures when 1: harzianum parasitized S. rolfsii,
Trichoderma harzianum excreted β-1, 3-glucanase and chitinase into the medium when grown on laminarin and chitin, respectively, or on cell walls of the pathogen Sclerotium rolfsii, as sole carbon source. Trichoderma harzianum also showed high activity of both enzymes when grown on homogenized S. rolfsii sclerotia. Glucanase activity increased by 67% when the fungus was grown on a mixture of laminarin and glucose (3:1, v/v). Similarly, high lytic activity was detected in wheat bran culture of the fungus and in soil inoculated with this culture. Protease and lipase activity were detected in the medium when the antagonist attacked mycelium of S. rolfsii.Isolates of T. harzianum were found to differ in the levels of hydrolytic enzymes produced when mycelium of S. rolfsii, Rhizoctonia solani, and Pythium aphanidermatum in soil was attacked. This phenomenon was correlated with the ability of each of the Trichoderma isolates to control the respective soilborne pathogens.
Specific strains of the Pseudomonas fluorescens-putida group have recently been used as seed inoculants on crop plants to promote growth and increase yields. These pseudomonads, termed plant growth-promoting rhizobacteria (PGPR), rapidly colonize plant roots of potato, sugar beet and radish, and cause statistically significant yield increases up to 144% in field tests1-5. These results prompted us to investigate the mechanism by which plant growth was enhanced. A previous study indicated that PGPR increase plant growth by antagonism to potentially deleterious rhizoplane fungi and bacteria, but the nature of this antagonism was not determined6. We now present evidence that PGPR exert their plant growth-promoting activity by depriving native microflora of iron. PGPR produce extracellular siderophores (microbial iron transport agents)7 which efficiently complex environmental iron, making it less available to certain native microflora.
Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.
A bacterium R–4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.
Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.
A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested, A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.
There are three roles for chitinases in fungi:
(a)
Most spectacularly, they are involved in the gross autolysis associated with the release of spores in some basidiomycete fruit bodies. Examples include the maturation of puffballs, Lycoperdon species1, and the autodigestion of gill tissue following spore release in the ink-caps, Coprinus species2.
(b)
They have a nutritional role. In the case of soil saprophytes such as Aspergillus species3,4 and Trichoderma species5, chitinase enables them to utilise chitinous debris from dead invertebrates and fungi as food sources. In the case of pathogens of crustacea, insects and fungi, chitinases also enable them to penetrate their hosts. Examples include the crayfish pathogen Aphanomyces astaci
6, insect pathogens such as Beauvaria and Cordyceps species7,8. There is however no convincing evidence that there is any appreciable re-cycling of the chitin in cell walls9,10 even though chitinase accumulates to a marked extent in old cultures11,12,i.e. there does not seem to be any appreciable autophagic utilisation of chitin in starving cultures
(c)
They have a morphogenetic role in the growth and differentiation of all chitin-containing fungi. This, the most fundamental of the three roles, has however proved the most difficult to obtain evidence for. Indeed some authors question whether chitinase and other lytic enzymes are involved in hyphal apical growth. Burnett13 suggests that “teleologically speaking, the apex would seem to be a most dangerous location for a lytic entity!”, and Wessels14 proposes a model for hyphal growth that does not require wall lytic enzymes. The unitary model for hyphal growth proposed by Bartnicki-Garcia15 does however see the control of apical wall growth as being the result of a “delicate balance between wall synthesis and wall lysis”, with some of the vesicles being transported to the apex containing lytic enzymes, to keep the wall at the apex in a plastic and extensible condition. Certainly in every case investigated chitinase activities can be detected in actively growing chitinous fungi. Examples include Mucor and Phycomyces species16–20,Neurospora crassa
21,22Aspergillus nidulans
9,23, Saccharomyces cerevisiae
24 and Candida albicans
25.
A bacterium R-4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus. Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production. A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested. A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.
Bacteria associated with the plant rhizosphere may have beneficial effects on plant growth by providing nutrients and growth factors, or by producing antibiotics and siderophores, which antagonize phytopathogenic fungi and bacteria. There is considerable experimental support for the idea that plant growth promoting bacteria may be used as bio–fertilizers or biological disease control agents to increase agricultural yields. Recent advances in our understanding of the molecular biology of the systems responsible for plant growth stimulation are opening the way to strain improvement by genetic engineering.
An Arthrobacter sp. which actively lysed Fusarium roseum was found to liberate chitinase (E.C. 3.2-1.14, chitin glycanohydrolase), an enzyme essential for the hydrolysis of chitin, a major component of fusarial hyphal walls. Factors involved in the production of chitinase were investigated by modifying culture conditions and assaying for enzyme activity. Production occurred on colloidal chitin as well as on native chitin supplemented with yeast extract or peptone. Enzyme production paralleled growth; liberation of enzyme took place during the log phase with the maximum yield being obtained at the stationary phase. Addition of the non-ionic surfactant, polyoxyethylene sorbitan monooleate (Tween 80) increased enzyme yield. An inverse relationship was found between the amount of enzyme produced and the quantity of n-acetyl-glucosamine liberated. The enzyme was generally not produced when grown on various other carbohydrates. These findings suggest that chitinase is inducible and that chitin breakdown is regulated by a repressor-inducer mechanism.Initial hydrolysis rates of colloidal chitin were proportional to the concentration of chitinase used. Optimal pH and temperature for enzyme activity were 4.9 and 50°C, respectively. Purification of the chitinase was obtained by (NH4)2SO4 precipitation followed by DEAE-cellulose and Sephadex chromatography, achieving a 12-fold increase in specific activity.
When Pseudomonas stutzeri was grown in iron-deficient conditions it excreted a colourless compound which was identified as nocardamine, a trihydroxamate, which is produced in similar conditions by actinomycetes. The production of this compound (which is related to the ferric iron content of the culture medium), as well as its ability to form very stable Fe(III) complexes, suggest that it is a desferri-siderophore.
This chapter discusses the cell wall synthesis in apical hyphal growth. It appears that the hyphal apex is best viewed as a highly polarized system of exocytosis. Wall materials, extracellular enzymes, and probably other substances are excreted at the growing end of a tubular cell. The most obvious cellular features that accompany this polarized system are (1) the unidirectional flow of vesicles in the cytoplasm fusing with the plasma membrane at the apex, (2) the gradients in wall synthesis at the apex, and (3) the cytoplasmic gradients in ion distribution that are maintained at the apex. New microscopic techniques reveal a cytoskeletal organization of the cytoplasm at the apex, which may be crucial to its polarized activity. Growth of the wall at the hyphal apex requires that the wall in this region has plastic properties, which contrast with the requirement of rigidity elsewhere in the hypha. A widely held view involves the participation of wall-lytic enzymes in plasticizing the wall at the apex and in allowing new wall material to be inserted. A critical evaluation of the evidence presented to support this view makes this hypothesis less attractive. As an alternative a steady-state model is discussed based on recent observations in the author's laboratory. This model holds that the assemblage of polymers synthesized at the apex is inherently plastic. However, this assemblage develops rigidity by interactions, in the wall, between and among the various individual polymers present while the wall segment moves in subapical directions during elongation. This model seems to fit many of the original observations made on living hyphae.
The biosynthesis of a yellow-green, fluorescent, water-soluble pigment by Pseudomonas jluorescens occurred only when the bacteria were iron-deficient and was not directly influenced by the nature of the organic carbon source. The pigment formed a very stable Fe3+ complex and was purified in this form. Pseudomonasfluorescens produced only one molecular species of fluorescent pigment; however, its lability under mild alkaline conditions led to the formation of several pigmented decomposition products. The spectral properties of the pure pigment, its molecular weight (1500 rf: 75) and its stability constant for Fe3+ (of the order of were determined. Both its biosynthesis and its chemical properties (formation of a stable Fe3+ complex) suggest that the fluorescent pigment is a desferrisiderophore.
A number of bacteria capable of lysing Fusarium oxysporum were isolated from soil. One of these, a strain of Bacillus cereus, was studied in detail. Living and dead fusarium mycelium as well as cell-wall preparations were digested by the bacterium. Chitin and the hyphae of a number of other fungi also served as carbon sources for the active organisms, but there was no lysis of species of Pythium, Streptomyces, Agrobacterium, or Pseudomonas. Lysis appeared to be associated with chitinase and laminarinase activity of B. cereus, but incubation of fungus mycelium with chitinase alone, or with chitinase in combination with laminarinase, proteases and cellulase, did not result in lysis. N-Acetylhexosamine appeared during the digestion. Evidence was obtained to suggest that lytic microorganisms could destroy fungal mycelium in sterile soil.
CONCERN about the widespread use of chemicals to control plant pathogens
has made it desirable that alternative methods, including biological
control1, should be examined. This study of natural fungal
parasites of a plant pathogenic fungus indicates the potential of such
organisms in controlling plant diseases.
THERE have been many attempts to make use of biological control measures for the suppression of plant pathogens, borne by soil. Frequently, these attempts involve the inoculation into soil of organisms which antagonize the pathogens in vitro ; such procedures try to disturb the natural microbiological equilibrium by the simple introduction into the environment of individual isolates. The frequent failure of the added micro-organisms to become established is not surprising because the biological associations and antagonisms within the ecosystem determine the composition of the microflora, the climax population being a reflexion of the physical and chemical characteristics of the habitat. By modifying the habitat, however, it is possible to alter the composition of the microflora, and such microbiological alterations may destroy plant pathogens borne by soil.
The addition of either fluorescentPseudomonas strain B10, isolated from a take-all suppressive soil, or its siderophore, pseudobactin, to bothFusarium-wilt and take-all conducive soils inoculated withFusarium oxysporum f. sp.lini orGaeumannomyces graminis var.tritici, respectively, rendered them disease suppressive. Our findings suggest that disease suppressiveness is caused in part by
microbial siderophores which efficiently complex iron(III) in soils, making it unavailable to pathogens, thus inhibiting their
growth. Amendment of exogenous iron(III) to disease-suppressive soils converted them to conductive soils presumably by repressing
siderophore production.
Fine structure observations of hyphal interactions between a recently described mycoparasite, Pythium nunn, and plant pathogenic fungal species suggested that enzymatic dissolution of cell walls of host fungi was involved. The fluorescence, localized about cell wall lysis at points of interaction when stained with Calcofluor White M2R New, also suggested enzymatic activity since this stain usually binds to oligomers in regions of incomplete cell wall polymers. Again, mycoparasitic activity of P. nunn releases 14CO2 from labelled cell walls of a plant pathogenic species of Pythium and Rhizoctonia solani. Culture filtrates of P. nunn had high cellulose and β-1,3-glucanase activity when the fungus was grown on cell walls of Pythium spp. Chitinase and β-1,3-glucanase were produced by P. nunn in cultures containing cell walls of R. solani and Sclerotium rolfsii but low activity was associated with cell walls of Fusarium oxysporum f. sp. cucumerinum. These hydrolytic enzymes were produced in dual cultures of P. nunn with several Pythium spp., Phytophthora spp., Mucor sp., Rhizopus sp., R. solani and S. rolfsii but were not detected with 10 non-host deuteromycete fungi. This difference may be due to the outer cell wall layer of mucilaginous material associated with these non-host fungi since trypsin- and or KOH-treated hyphae of non-host F. oxysporum f. sp. cucumerinum released -acetyl-d-glucosamine in the presence of crude enzymatic preparations from P. nunn. This suggests that the potential host range of P. nunn is limited by components on the outer layer of fungal cell walls. Pythium nunn also produced factors inhibiting growth and/or propagule germination of a plant pathogenic Pythium sp. in vitro and in soil.
This chapter discusses a few aspects of microbiological control of plant pathogens, including its occurrence in nature, the most common microbial groups and the mechanisms involved, with emphasis on its practical implications. Microbiological control of plant diseases can be achieved directly, through inoculation, or indirectly, by changing the conditions prevailing in the plant's environment, and thus the microbiological equilibrium of its ecosystem, or by a combination of both approaches. The chapter presents a few examples demonstrating the control of plant diseases by inoculation. Microorganisms parasitic to plants constitute a tiny fraction of the microflora and microfauna inhabiting the vicinity and surfaces of plant organs. Disease severity is greatly increased when the pathogen is reintroduced in its pre-sterilized infection site, indicating that the saprophytic microorganisms inhabiting the surfaces of plant organs may serve as a biological buffer zone, preventing the pathogen from infecting its host. Selective pathogen suppression can be achieved by the use of specific chemicals, such as fungicides, which are applied at relatively low dosages. Soil treatment with fumigants, such as chloropicrin, methyl bromide or formalin, results in eradication of both the pathogen and most of the saprophytic microflora.
A streptomycete enzyme system active in lysing Aspergillus oryzae and Fusarium solani hyphal walls contained chitinase and several β-(1 → 3) glucanase components. The extracellular β-(1 → 3) glucanase and chitinase were shown to be instrumental in the process of dissolution of hyphal walls. The β-(1 → 3) glucanase and chitinase components alone did not effect solubilization of the hyphal walls, but they were active when used in combination. The major hyphal wall components were chitin and a glucan with β-(1 → 3) linkages. No cellulose was found. The presence of a wall core containing chitin but no glucan was demonstrated by incomplete enzymic hydrolysis. Digestion of F. solani walls yielded 14% glucose, 47% N-acetylhexosamine, and 6% of an insoluble residue which contained mannose, galactose, and a uronic acid.
THE enzyme contained in a culture fluid of Bacillus circulans has been shown to exert lytic activity towards Aspergillus oryzae and that a polymer of melibiose was liberated from the cell wall. However, the cell wall was not completely lysed by the enzyme owing to the large amount of chitin present. During the search for enzymes causing lysis of fungal cells, it was found that a mixed preparation of chitinase and lytic enzyme exerted strong lytic activity on the cell wall of A. oryzae.
Soils in many areas suppress certain plant diseases. Understanding the basis for this disease suppressiveness could lead to
improved plant health in less favorable areas. Some forms of disease suppression may be caused by bacteria in the genus Pseudomonas which aggressively colonize root surfaces. Increased plant growth and yield are closely associated with the capacity of some
of these bacteria to produce iron-binding compounds called siderophores. This article addresses the biological characteristics
of these soil-bome root epiphytes, their contribution to plant health, and their potential use in biotechnology.
Polysaccharide preparations: chitin
- J N Bemiller
- Bemiller J. N.
Bemiller, J. N. 1965. Polysaccharide preparations: chitin. Methods Carbohydr. Chem. 5:103.
Trichoderma-application, mode of action and potential as a biocontrol agent of soil-bome plant pathogenic fungi Innovative approaches to plant disease control End rot, surface rot, and stem lesions caused on sweet potato by Fusarium solani Biological control of pathogens: theory to application
- I Chet
Chet, I. 1987. Trichoderma-application, mode of action and potential as a biocontrol agent of soil-bome plant pathogenic fungi, p. 137-160. In I. Chet (ed.), Innovative approaches to plant disease control. John Wiley & Sons, Inc., New York. 7. Clark, C. A. 1980. End rot, surface rot, and stem lesions caused on sweet potato by Fusarium solani. Phytopathology 70:109-112. 8. Cook, R. J. 1985. Biological control of pathogens: theory to application. Phytopathology 75:25-29.
Fungicides for bedding plants
- R K Jones
- Jones R. K.
Jones, R. K. 1985. Fungicides for bedding plants. Bedding Plants Inc. News 16:3-4.