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Biological Control of Fusarium Wilt of Cucumber by Chitinolytic Bacteria
Abstract
ABSTRACT Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp. 385, suppressed Fusarium wilt of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum in nonsterile, soilless potting medium. A mixture of the two strains in a ratio of 1:1 or 4:1 gave significantly (P < 0.05) better control of the disease than each of the strains used individually or than mixtures in other ratios. Several formulations were tested, and a zeolite-based, chitosan-amended formulation (ZAC) provided the best protection against the disease. Dose-response studies indicated that the threshold dose of 6 g of formulation per kilogram of potting medium was required for significant (P < 0.001) suppression of the disease. This dose was optimum for maintaining high rhizosphere population densities of chitinolytic bacteria (log 8.1 to log 9.3 CFU/g dry weight of potting medium), which were required for the control of Fusarium wilt. The ZAC formulation was suppressive when added to pathogen-infested medium 15 days before planting cucumber seeds. The formulation also provided good control when stored for 6 months at room temperature or at 4 degrees C. Chitinase and beta-1,3-glucanase enzymes were produced when the strains were grown in the presence of colloidal chitin as the sole carbon source. Partial purification of the chitinases, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and activity staining, revealed the presence of five bands with molecular masses of 65, 62, 59, 55, and 52 kDa in the case of Paenibacillus sp. 300; and three bands with molecular masses of 52, 38, and 33 kDa in the case of Streptomyces sp. 385. Incubation of cell walls of F. oxysporum f. sp. cucumerinum with partially purified enzyme fractions led to the release of N-acetyl-D-glucosamine (NAGA). NAGA content was considerably greater when pooled enzyme fractions (64 to 67) from Paenibacillus sp. were used, because they contained high beta-1,3-glucanase activity in addition to chitinase activity. Suppression of Fusarium wilt of cucumber by a combination of these two bacteria may involve the action of these hydrolytic enzymes.
... Chitinolytic enzymes have been considered important in the biological control of soil borne pathogens because of their ability to degrade fungal cell walls, of which a major component is chitin. In recent years, the use of chitinolytic enzymes in application fields such as biological pest control, the degradation of chitin rich wastes and the production of chitin hydrolysates for pharmaceutical or chemical purposes and for the food/feed industry has become of increasing interest (Di Pietro et al., 1993;Chen & Chang, 1994;Lorito et al., 1994Lorito et al., & 1996Fenice et al., 1998;El-Sawah, 1999 andSingh et al., 1999). The sensitivity of the fungal cell wall to chitinolytic enzymes has been exploited by releasing chitinolytic bacteria to control plant pathogens in the rhizosphere (Flach et al., 1992 andEl-Sawah, 1999). ...
... Enzymatic preparation obtained from the culture fluid of B. licheniformis displayed a high antifungal activities toward all substrates tested and the maximum antifungal activity was observed against the cell wall preparation. Preliminary studies also indicate that other metabolites are also produced (Singh et al., 1999). Similar results were obtained by Abdel-Fatah & Khella (1995) and Singh et al. (1999) ...
... Preliminary studies also indicate that other metabolites are also produced (Singh et al., 1999). Similar results were obtained by Abdel-Fatah & Khella (1995) and Singh et al. (1999) ...
... 300 and Streptomyces sp. 385 by production of chitinase and b-1,3-glucanase was also reported (Singh et al. 1999). Similarly, Pseudomonas cepacia produced b-1,3 glucanase which lysed the cell wall of Rhizoctonia solani, Sclerotium rolfsii and Pythium ultimum and decreased the occurrence of the disease caused by these pathogens (Fridlender et al. 1993). ...
... strain 300 and Streptomyces sp. strain 385 against F. oxysporum (Singh et al. 1999) Cellulase produced by Bacillus subtilis YJ1 (Li- Jung et al. 2010) Cellulase produced by Cellulomonas spp. ASN2 (Muhammad et al. 2012) Carboxymethyl cellulase and polygalacturonase produced by Bacillus coagulans (Odeniyi et al. 2009) Cellulase produced by Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis (Basavaraj et al. 2014) Protease produced by Pseudomonas aeruginosa PGPR2 against Macrophomina spp., Rhizoctonia spp., and Fusarium spp. ...
... Similarly, F. oxysporum was inhibited by the β-1,3-glucanases produced by Paenibacillus and Streptomyces sp. (Singh et al. 1999). One more study reported seven β-1,3glucanases synthesized from T. harzianum under various growth conditions (Vazquez et al. 1998). ...
Fungal and oomycete pathogens are the significant causes of many plant diseases leading to major annual economic loss. This results in today’s shortfall of food supply due to which many millions of lives are insufficiently fed. One way to combat the loss is use of fungicides, but repeated use of fungicides resulted in evolved resistance in these pathogens leading to severe loss of yield. Hence, biological methods are proving to be more beneficial as compared to the chemical ones. Plant growth-promoting rhizobacteria (PGPR) employ a variety of mechanisms to promote plant growth and development, of which production of various hydrolytic enzymes against the fungal pathogens plays an important role. PGPR as biocontrol agents have been tried in many plants. Recently vast studies referring to the isolation and characterization of these hydrolytic enzymes have determined their ability to control plant pathogens. Enzymes like protease, chitinase, cellulose, and glucanase are known to act on the fungal cell wall leading to its degradation and finally the lysis of the fungal cell. Many bacterial species like B. subtilis, B. cereus, B. subtilis, B. thuringiensis, S. marcescens, R. solani, F. oxysporum, S. rolfsii, P. ultimum, etc. are shown to synthesize such enzymes that can affect the cell wall integrity of the pathogens and inhibit them. These hydrolytic enzymes produced by PGPR play an important role in the control of various plant pathogens and thereby improve the plant growth, making an efficacious biocontrol agent. These PGPR release their antifungal metabolites in a sustained manner due to which it is difficult for the target organism to develop resistance against them as it is experienced with the chemical fungicides. PGPR can be used individually or in combinations in the sector of plant growth and protection.KeywordsBiocontrol agentsFungiHydrolytic enzymesOomycetesPhytopathogens
... India is the second largest producer of vegetables in the world next to china. A growing demand for vegetables in India could be met by increased area under production, using hybrids, and adoption of improved agro-techniques including protected cultivation (Singh et al., 1999). Protected cultivation of vegetables against major biotic stresses may increase crop productivity and quality. ...
... Chitinase and β1,3-glucanase produced by Streptomyces sp. 385 lysed cell walls of Fusarium oxysporum (Singh et al.,1999). ...
Cancer is a deadly disease characterized by uncontrolled cell growth that causes physiological harm.
Asia is reported to be the top in both cancer incidence (57.3 percent) and cancer death (48.4 percent). Patients
have substantial side effects from treatment methods such as chemotherapy and radiation, which include nausea,
allergic reactions, decreased immune response, haemorrhage and toxicity. Humanity urgently has to explore
newer resources in order to discover medications to battle such a horrible illness. Plants hold promise as a
consistent supply of anticancer compounds such as polyphenols, brassinosteroids and taxols that unlike
manufactured medications have no side effects. The current study looked at the antioxidant and in vitro
cytotoxicity of Prunus armeniaca (apricot) kernel against two human cancer cell lines from different organs,
SHSY-5Y (CNS) and SW-620 (colon). Methanolic extracts were employed as test material and anticancer
activity was evaluated using the SRB assay at a concentration of 100 μg/mL. The antioxidant activity of
methanolic extract was determined using DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay. The kernel exhibited
antioxidant property with IC50 of 137.31 μg/mL and possess in vitro cytotoxic efficacy against one human cancer
cell line, SHSY-5Y from CNS tissue, with a growth suppression of 73%, but no cytotoxicity against colon
cancer. To summarize, kernel contains cytotoxic components that will be useful to those suffering from CNS
cance
... They consist of number of sites that can easily undergo hydrolysis as they are esters. The principle reactions associated with the microbial degradation are oxidation, hydrolysis, dealkylation and alkylation (Singh et al., 1999). Notable step in decontamination of organophosphate is the microbial degradation causing hydrolysing of the bonds P-O-alkyl and P-O-aryl. ...
... In the similar way genes were engineered to enhance the production of 2, 3-butanediol dehydrogenase. Genetic engineering using Cre/Lox site combination with PCR is also used (Borriss 2015;Erdogan and Benlioglu 2010;Singh et al., 1999;Wu et al., 2015). ...
... They consist of number of sites that can easily undergo hydrolysis as they are esters. The principle reactions associated with the microbial degradation are oxidation, hydrolysis, dealkylation and alkylation (Singh et al., 1999). Notable step in decontamination of organophosphate is the microbial degradation causing hydrolysing of the bonds P-O-alkyl and P-O-aryl. ...
... In the similar way genes were engineered to enhance the production of 2, 3-butanediol dehydrogenase. Genetic engineering using Cre/Lox site combination with PCR is also used (Borriss 2015;Erdogan and Benlioglu 2010;Singh et al., 1999;Wu et al., 2015). ...
... Enzymes such as chitinases, cellulases, -1,3 glucanases, proteases, and lipases are produced by some biocontrol bacteria and have the ability to partially break down the cell walls of certain dangerous fungus. It has been discovered that PGPB that produce one or more of these enzymes have biocontrol action against a variety of pathogenic fungus, such as Sclerotium rolfsii, Botrytis cinerea, Fusarium oxysporum, Phytophthora spp., Rhizoctonia solani, and Pythium ultimum [116][117][118][119]. ...
The ability of agricultural systems to provide food globally may be limited by growing environmental concerns. The biggest issue the world is now dealing with is climate change. By 2050, food production will need to quadruple to fulfill the world's food demand. In the field of sustainable agriculture, plant growth-promoting bacteria (PGPB) have been shown to have a significant impact. Increasing agricultural yield while using less synthetic chemical fertilizers and pesticides is a major issue in today's world. It has been demonstrated that using PGPB to promote plant development through direct or indirect means is a sustainable method of raising agricultural yields. Among PGPB's processes are the control of hormonal and nutritional balance, the development of resistance against plant diseases, and the solubilization of minerals to facilitate their simple absorption by plants. Moreover, PGPB interact both antagonistically and synergistically with microorganisms in bulk soil and the rhizosphere, which indirectly accelerates plant development. Numerous bacterial species have been reported in the literature to function as PGPR and to effectively enhance plant development. There is a difference, nevertheless, between the PGPR's function as a biofertilizer and its method of action (mechanism) for plant development. Therefore, this analysis fills in the aforementioned void and provides an overview of PGPR's mechanism as a biofertilizer for sustainable agriculture.
... Endophytic microorganisms, virtually ubiquitous in nearly all plant species (Owen and Hundley, 2004;Ryan et al., 2008;Khan et al., 2015), establish residence within the living tissues of their host plants without causing any discernible harm (Reinhold-Hurek and Hurek, 2011;Brader et al., 2014;Türkölmez et al., 2023). These endophytic bacteria possess a diverse array of functional traits, encompassing the capacity for phosphate solubilization (Wakelin et al., 2004), phytohormone production (Shi et al., 2009), hydrolytic enzymatic activities, including lytic hydrolases (Chernin and Chet, 2002), glucanases (Singh et al., 1999), chitinases (Frankowski et al., 2001), antibiotic synthesis (Demain, 1981;Ezra et al., 2004), siderophore secretion (Lodewyckx et al., 2002), and nitrogen fixation (Watanabe et al., 1979). These multifaceted abilities contribute significantly to nutrient acquisition by the plant and serve to augment overall plant growth. ...
This study aimed to evaluate the physiological and biochemical properties and enzyme activities of endophytic bacteria obtained from different wheat genotypes, as well as their effectiveness against Fusarium culmorum, which causes root and crown rot in wheat. The results obtained from double culture tests of isolates against F. culmorum showed that the inhibition rate varied between 80.56% and 13.90%. The inhibition rate against F. culmorum was 80.59% for Bacillus subtilis (MM11), 69.41% for Stenotrophomonas maltophilia (EY5), and 61.10% for Enterobacter sp. (MY3) under in vitro conditions, the most effective isolates. Pseudomonas putida (EM9) and Pseudomonas orientalis (MM21) isolates gave positive results in all tests in the production of amylase, cellulase, phosphatase, ACC deaminase, and siderophore. To identify six promising isolates, 16S rRNA gene-based sequence analysis was utilized. The efficacy of bacterial strains against F. culmorum, pot experiments were conducted in a growth room (in vivo). The results demonstrated that the combination of S. maltophilia, Enterobacter sp., and B. subtilis (MY3+EY5+MM11) yielded the most favorable outcomes in terms of disease severity, plant height, wet weight, dry weight, root wet weight, and root dry weight. The combination of Stenotrophomonas rhizophila, P. putida, and P. orientalis (EY1+EM9+MM21) exhibited promising results. Utilizing effective bacterial strains is anticipated to reduce the dependence on and costs associated with chemical fertilizers and pesticides while minimizing their environmental impact. Furthermore, these strains show potential for commercial applications pending further validation procedures. The findings from this study significantly contribute to the field of biological control strategies against F. culmorum by leveraging the diverse capabilities of endophytic bacteria.
... Formation of clearing zones surrounding inoculated sites indicated chitinase enzyme production. Quantitative estimation of b-1, 3-glucanase was carried out as per the methodology outlined by Singh et al. (1999). Laminarin, a glucan polymer is cleaved by b-glucanase resulting in the release of glucose molecules. ...
The main objectives of the present investigation was to characterize and recognize potential indigenous Rhi-zobium spp. associated with red clover nodules, as well as to evaluate the influence of prospective rhizobia inoculation (individual and consortia) on common bean plant growth. Total of 8 rhizobia like bacteria were isolated and were investigated for their specific PGP traits, production of hydrolytic enzymes. Results revealed, four isolates synthesize IAA (22.3 § 1.45 to 88.6 § 1.45 mg mL À1), three isolates solubilized phosphate (40.69 § 1.25 to 216.3 § 1.31 mg mL À1), three isolates solubilized potassium, produced siderophore, and showed chitinase production, two isolates possess ACC deaminase activity, one isolate was able to solu-bilize zinc, 5 isolates produced b-1, 3-glucanase (0.38 § 0.005 to 2.82 § 0.011 % units) and four isolates produced cellulase. Two bacterial isolates found to exhibit most of the PGP activities, were selected and identified on the basis of phenotypic, biochemical tests and 16S rRNA sequencing, as Rhizobium tropici IHTF-1 and Rhizobium mayense IHTF-2. Interaction of these potential rhizobium strains individually and consortia with common bean plants under greenhouse conditions boosted root and shoot length, fresh and dry bio-mass compared to un-inoculated control plants. SEM study revealed that both the strains, IHTF-1 and IHTF-2 colonized common bean roots. These results imply that Rhizobium spp., identified in this work, can be used as bio-inoculants to increase common bean plant production in a sustainable way.
... Its presence in the ecosystem, in various forms, is important in preventing and treating soil-borne illnesses from Sclerotium rolfsii and Rhizoctonia solani in beans and cotton, respectively (Ordentlich et al. 1988;Shapira et al. 1989). One approach that is recommended is the use of lytic enzymes such as chitinase, which results in the breakdown of pathogenic fungal cell walls (Ordentlich et al. 1988;Chet et al. 1990;Singh et al. 1999). In addition to its role in biological pest management, this enzyme has a wide variety of uses because of its ecofriendly nature, which helps to sustain maintain healthy soil and, as consequences, enhance plant growth and development and yields. ...
Soil enzymes `activate various key reactions necessary for the life processes of soil-based microorganisms in agriculture. Soil enzymes are important for the energy transfer that occurs through the breakdown of organic matter in soil and cycling of nutrients. Moreover, it is also beneficial in the maintenance of structure of soil. Soil enzymes are primarily produced by microbes. All changes in environmental factors and soil management techniques are quickly reacted to by soil enzymes. Their effects are directly impacted by the soil’s physiochemical and biological properties. Soil enzymes are used as detectors to assess the microbiological state and physio-chemical parameters of the soil. Understanding the possible functions of various soil enzymes in maintaining the health of soil can help with soil health management and fertility control. Enzymes serve as essential activating agents of biological processes, and they are acknowledged to have a significant function in maintaining the ecology as well as the health of the soil. The bulk of enzymatic activity found in soil is produced from free enzymes or within the cell, the majority of which are microbial in origin. A detailed understanding of the functions of enzymes found in soil is essential to ensure that soil remains healthy because soil is an essential component for the terrestrial environment and it nourishes every aspect of life on earth. A greater knowledge of the significance of the activity of soil enzymes in sustaining the health of soil could give an excellent chance for a combined biological evaluation of soils because of their essential function in numerous soil biochemical processes.
... These demonstrated that pH was an important factor in microbial community transfor mation and was strongly related to microbial richness and diversity (52). Fusarium wilt, caused by Fusarium oxysporum, is among the most dangerous soilborne diseases and can affect plants of a wide range (53). According to numerous studies, RSD practices can effectively decrease soilborne pathogens (54,55), which is consistent with our study that Fusarium abundance was significantly decreased in all RSD treatments. ...
Reductive soil disinfestation (RSD) is an effective method to inhibit soilborne pathogens. However, it remains unclear how RSD combined with different types of organic materials affects the soil ecosystems of perennial plants. Pot experiments were conducted to investigate the effects of RSD incorporated with perilla (PF), alfalfa (MS), ethanol, and acetic acid on soil properties, enzyme activities, microbial communities and functions, and seedling growth. Results showed that RSD-related treatments improved soil properties and enzyme activities, changed microbial community composition and structure, enhanced microbial interactions and functions, and facilitated seedling growth. Compared with CK, RSD-related treatments increased soil pH, available nitrogen, and available potassium contents, sucrase and catalase activities, and decreased soil electric conductivity values. Meanwhile, RSD-related treatment also significantly reduced the relative abundance of Fusarium while increasing the relative abundance of Arthrobacter , Terrabacter , and Gemmatimonas . The reduction was more evident in PF and MS treatment, suggesting the potential for RSD combined with solid agricultural wastes to suppress pathogens. Furthermore, the microbial network of RSD-related treatment was more complex and interconnected, and the functions related to carbon, nitrogen, sulfur, and hydrogen cycling were significantly increased, while the functions of bacterial and fungal plant pathogens were decreased. Importantly, RSD-related treatments also significantly promoted seed germination and seedling growth. In summary, RSD combined with solid agricultural wastes is better than liquid easily degradable compounds by regulating the composition and function of microbial communities to improve soil quality and promote plant growth.
IMPORTANCE
Reductive soil disinfestation (RSD) is an effective agricultural practice. We found that RSD combined with solid agricultural wastes is better than that of liquid easily degradable compounds, may improve soil quality and microbial community structure, inhibit the proliferation of pathogenic bacteria, and contribute to the growth of replanted crops. Thus, RSD combined with solid agricultural wastes is more effective than liquid easily degradable compounds.
... Combinations of Paenibacillus sp. and a Streptomyces sp. suppressed Fusarium wilt of cucumber better than when either was used alone [12] and a combination of Pseudomonas fluorescens and Stenotrophomonas maltophila improved protection of sugar beet against Pythium damping off in comparison with either applied individually [13]. Combinations of fungi and bacteria have also been shown to provide enhanced biocontrol. ...
Groundnut (Arachis hypogaea L.) is an important oil seed crop in the world belonging to the Leguminosae family. It is one of the essential food and cash crops of our country. In India, a large number of diseases attack groundnut [1]. Among soil borne diseases, stem rot or white mold caused by Sclerotium rolfsii Sacc. [2] is an important disease causing significant yield losses in several groundnut growing countries (Mehan et al., 1994). Sclerotium rolfsii Sacc. is a destructive soil borne fungal pathogen of oil seed crop in India. Different practices are recommended for management of groundnut stem rot such as deep summer ploughing, destruction of plant debris, crop rotation with jowar and bajra, seed treatment with carbendazim or captan or mancozeb or tebuconazole, soil drenching with hexaconazole, application of ammonium sulphate or calcium ammonium nitrate instead of urea and application of gypsum at flowering stage. Further, no single treatment is full proof and disease continues to cause losses in farmers’ fields. Biological control offers an interesting alternative to fungicides for sustainable management of soil borne diseases. Biocontrol is a non-chemical measure, could be effective as chemical control by various techniques. Among the techniques, the mixture of antagonists was studied to enchance the control efficacy. In most studies, the involvement of only mechanism of biological control is demonstrated. Involvement of more than one mechanism has been reported in only a few systems. Use of several biocontrol agents with several mechanisms of control fits in well with the concepts of integrated disease management, in which several means of disease suppression are applied concurrently. When single or more means of mechanisms are not effective, the others may compensate for the former absence. The present study, involved three major bacterial antagonists viz., Streptomyces violaceusniger, Streptomyces exfoliatus and Pseudomonas fluorescens to find out the effective dose of mixtures. The different doses from 10-1 to 10-5 were studied, the dose 10-1 of Streptomyces violaceusniger have more efficiency of 86.70, than S. exfoliatus. The efficacy of Streptomyces violaceusniger with combination of other antagonists were tested and found that, the mixture of Streptomyces violaceusniger and P. fluorescens have synergistic activity than any other combinations which have synergistic factor greater than one.
... Stenotrophomonas sp., Serratia spp., Streptomyces spp., Agrobacterium radiobacter, Lysobacter spp., and Paenibacillus spp. are examples of genera studied for antibiotic production potential (Fridlender et al., 1993;Singh et al., 1999;Islam et al., 2005). ...
Turmeric (Curcuma longa L.), a significant commercial crop of the Indian subcontinent is widely used as a condiment, natural dye, and as a cure for different ailments. Various bioactive compounds such as turmerones and curcuminoids have been isolated from C. longa that have shown remarkable medicinal activity against various ailments. However, reduced soil fertility, climatic variations, rapid urbanization, and enhanced food demand, pose a multifaceted challenge to the current agricultural practices of C. longa. Plant growth-promoting microbes play a vital role in plant growth and development by regulating primary and secondary metabolite production. Rhizospheric associations are complex species-specific interconnections of different microbiota with a plant that sustain soil health and promote plant growth through nutrient acquisition, nitrogen fixation, phosphate availability, phytohormone production, and antimicrobial activities. An elaborative study of microbiota associated with the roots of C. longa is essential for rhizospheric engineering as there is a huge potential to develop novel products based on microbial consortium formulations and elicitors to improve plant health, stress tolerance, and the production of secondary metabolites such as curcumin. Primarily, the purpose of this review is to implicate the rhizospheric microbial flora as probiotics influencing overall C. longa health, development, and survival for an increase in biomass, enhanced yield of secondary metabolites, and sustainable crop production.
... Outro mecanismo adotado pelos microrganismos benéficos para inibir o crescimento de patógenos na planta hospedeira é a interação direta com o parasita liberando enzimas líticas, como quitinase, â-1, 3 glucanase, protease e lipase, que degradam a parede celular das células de fungos e bactérias patogênicos . Como exemplo de produção de enzimas líticas, podemos citar o estudo que observou bactérias do gênero Bacillus promovendo antagonismo direto e secreção de enzimas líticas que degradaram a parede celular de Sclerotium rolfsii, atualmente Athelia rolfsii (mofo cinzento) (Kumari, Bishnoi et al. 2021), entre outros patógenos como: Botrytis cinerea, Sclerotium rolfsii, Fusarium oxysporum, Phytophthora spp., Rhizoctonia solani e Pythium ultimum (Singh, Shin et al. 1999, Frankowski, Lorito et al. 2001, Czaban, Rasmussen et al. 2018, Agrofit 2019, Agrofit 2019). ...
A primeira edição online do Congresso de Ciências Agrárias da Unioeste foi bem-sucedida, cumprindo seus objetivos e envolvendo significativamente os participantes. Plataformas digitais como YouTube, StreamYard, Microsoft Teams e Bitly foram escolhidas para a realização de palestras, mesas redondas, apresentações de trabalhos e minicurso, com um custo total de US$60,00. O comprometimento das pessoas envolvidas foi fundamental para o sucesso do evento, apesar de não serem profissionais de organização de eventos online.
Algumas dificuldades, como a limitação do acesso ao chat do Microsoft Teams para os participantes que acessavam por links, e problemas relacionados ao pagamento de assinaturas em sites internacionais. Essa situação destaca a necessidade de adaptação das instituições universitárias e suas fundações diante dessa nova realidade.
O ambiente remoto para eventos, já em ascensão devido ao avanço tecnológico, foi acelerado pela pandemia. A capacitação e adaptação do evento a esse formato ressaltam a importância da resiliência e da continuidade da extensão universitária.
... CC was harvested and kept in the refrigerator until its utilization for liquid chitinase screening media (CSM) or agar plates. The recipe was adapted and modified from [81,82]: 20 g/L (2% w/v) CC, 0.7 g/L K 2 HPO 4 , 0.3 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 · 5H 2 O, 10 mg/L FeSO 4 · 7H 2 O, and 20 g/L agar (optional), adjusting to pH 6.5 for plates or 7 for liquid medium. After autoclaving, 1 mg/L ZnSO 4 and MnCl 2 were added from sterile filtrated stock solutions prior to the pouring of agar plates/inoculation of liquid media. ...
In nature, chitin, the most abundant marine biopolymer, does not accumulate due to the action of chitinolytic organisms, whose saccharification systems provide instructional blueprints for effective chitin conversion. Therefore, discovery and deconstruction of chitinolytic machineries and associated enzyme systems are essential for the advancement of biotechnological chitin valorization. Through combined investigation of the chitin-induced secretome with differential proteomic and transcriptomic analyses, a holistic system biology approach has been applied to unravel the chitin response mechanisms in the Gram-negative Jeongeupia wiesaeckerbachi. Hereby, the majority of the genome-encoded chitinolytic machinery, consisting of various glycoside hydrolases and a lytic polysaccharide monooxygenase, could be detected extracellularly. Intracellular proteomics revealed a distinct translation pattern with significant upregulation of glucosamine transport, metabolism, and chemotaxis-associated proteins. While the differential transcriptomic results suggested the overall recruitment of more genes during chitin metabolism compared to that of glucose, the detected protein-mRNA correlation was low. As one of the first studies of its kind, the involvement of over 350 unique enzymes and 570 unique genes in the catabolic chitin response of a Gram-negative bacterium could be identified through a three-way systems biology approach. Based on the cumulative data, a holistic model for the chitinolytic machinery of Jeongeupia spp. is proposed.
... Quantitative production of chitinase by the high chitinase-producing Streptomyces isolates Quantitative production of chitinase by all isolates showing hydrolysis zones > 4 mm were also determined as described previously (Singh et al., 1999) using the minimal synthetic medium (Tweddell et al., 1994) amended with 2 mg mL −1 of colloidal chitin. The reaction mixture containing 1 mL of culture supernatant from a chitin amended minimal synthetic medium and 1 mL of colloidal chitin (20 g L −1 ) in 50 mM sodium acetate buffer (pH 6.8) was incubated in a water bath for 1 h at 50 °C. ...
Actinobacteria are ubiquitous Gram-positive filamentous bacteria that are well-known for their ability to control plant diseases through mechanisms such as the production of antibacterial and antifungal antibiotics, induction of plant resistance , parasitism, and even simple competition for resources. For their potential as nematode biocontrol agents, streptomycete actinobacteria have received the greatest attention. The current study aimed to isolate chitinase-producing streptomycete actinobac-teria from eggplant rhizospheres with the potential to be used as biocontrol agents for the root-knot nematode Meloidogyne incognita. Twenty-four different strains of streptomycete actinobacteria were isolated and tested for chitinase production. Only five high chitinase-producing isolates were chosen for further experiments and were identified as ST01; Strepto-myces levis, ST07; S. enissocaesilis, ST10; S. plica-tus, ST15; S. rochei, and ST16; S. sparsus. The isolates were identified using cultural, morphological, Supplementary Information The online version contains supplementary material available at https:// doi. biochemical, physiological and chemotaxonomical characteristics as well as16S rRNA gene sequenc-ing. The phylogenetic analyses indicated that the actinobacteria isolates in this study belonged to the genus Streptomyces. The in vitro tests compared the culture filtrates of five isolates at three different concentrations (5, 10, and 20%) for their ability to reduce nematode numbers and the percentage of eggs hatching of M. incognita. When exposed to higher concentrations (20%) for longer periods of time (up to 72 h), second stage juveniles (J2) of M. incognita had a higher mortality rate. The Streptomyces strains, ST15, ST10, ST16, ST01, and ST07, significantly (P < 0.05) reduced egg hatching by 86.8, 85.4, 78.9, 77.8, and 68.1%, respectively. In addition, these five isolates were tested under greenhouse conditions to control M. incognita on eggplants. In both the presence and absence of M. incognita, the treatment of strain ST15 was the most effective and significantly (P < 0.05) increased the length, fresh weight, and dry weight of eggplant roots and shoots, total chlorophyll and carotenoids contents compared to the control and to the other strains (ST10, ST16, ST01, and ST07). Strain ST15 significantly (P < 0.05) reduced the number of galls, number of eggs, number of egg masses and the population of M. incognita in soil compared to the control and to the other strains. Malonalde-hyde, hydrogen peroxide concentration, and the anti-oxidant enzyme (superoxide dismutase, peroxidase, and catalase) activity were all significantly (P < 0.05) decreased in eggplant tissues after treatment with strain ST15, compared to infected control. Based on our research, we have concluded that chitinase-producing Streptomyces strains, and specifically St15, have the potential to be used as significant biocontrol agents against the root-knot nematode M. incognita and as promotor of eggplant performance.
... Because of their ability to breakdown fungal cell walls, microbial chitinolytic enzymes have been deemed crucial in the biological control of several plant diseases (Shapira et al. 1989). Serratia (Ordentlich et al. 1988), Aeromonas (Inbar andChet 1991), Chromobacterium (Park et al. 1995), Enterobacter (Chernin et al. 1997), Arthrobacter (Frändberg and Schnürer 1998), and Paenibacillus (Singh et al. 1999) have all been reported to be effective biological control agents of many soil-borne plant pathogens through the production of chitinase. All of the above examples corroborated the current study's findings that chitinase synthesis by the three extremophilic isolates can explain the pathogen and disease suppression attained in the current investigation. ...
Plant growth-promoting rhizobacteria are helpful soil microorganisms that are used extensively in the agricultural industry. In the current investigation, a total of 120 extremophilic bacterial isolates were successfully obtained from a variety of drastic soils. These bacterial isolates were tested to determine their effectiveness as plant growth promoters and biocontrol agents against the two most frequent soil-borne plant pathogenic fungi, Pythium ultimum, and Rhizocotnia solani. Results indicated that numerous isolates demonstrated antifungal activity against P. ultimum and R. solani, through the production of diffusible antifungal metabolites, cell-wall degrading enzymes (chitinase, cellulase), and siderophores. In addition, many of these extremophilic bacteria fixed atmospheric nitrogen, solubilized insoluble phosphate and zinc, and produced indole-3-acetic acid (IAA) and salicylic acid. The 16 S rRNA analysis revealed that the most potent isolates with multiple modes of action were identified as Bacillus halotolerans, Massilia alkalitolerans, and Bacillus aryabhattai. Under greenhouse conditions, these three isolates, either individually or in combination were used as microbial inoculants, and their effects on the development of tomato and maize were evaluated both with and without the presence of the two pathogens. Application of the three isolates as individual or combined treatments significantly (P < 0.05) reduced the growth of P. ultimum and R. solani in vivo, and significantly (P < 0.05) increased the percentage of survived seedlings as evidenced by the significant (P < 0.05) increase in plant resistance as measured by the in planta contents of total phenols, polyphenol oxidase, and peroxidase. Additionally, the treatment of any one of the three isolates, either individually or in combination, significantly (P < 0.05) improved the development of tomato and maize, as evidenced by a considerable increase in the amount of total nitrogen and dry weight yield. The utilization of these bacterial isolates presents a potentially fruitful alternative to the excessive use of potentially harmful agrochemicals.
... Fridlenderet al. (1993) reported the hydrolytic inhibition of Rhizoctonia solani, Sclerotium rolfsii, and Pythium ultimum by b-1,3-glucanases of Bacillus cepacia. Singh et al. (1999) reported two strains of Paenibacillus and Streptomyces sp., which produce b-1,3-glucanases that inhibited the growth of F. oxysporum. Palumbo et al. (2005) reported the inhibition of Bipolari s leaf spot of tall fescue and Pythium damping-off of sugar beet by Lysobacter enzymogenes due to production of enzyme beta-1,3-glucanases (Jose et al., 2014).Similarly, glucanolytic rhizobacteria Achromobacter mucicolens, Enterobacter cloacae, Serratia marcescens, and Bacillus spp. ...
... Biocontrol is an eco-friendly technique that effectively manages the soil borne pathogens (Saleem et al., 2000). Immense work has been done worldwide for using the antagonistic fungi (Trichoderma, Gliocladium, etc.) and bacteria (Pseudomonas, Bacillus, etc.) isolated from suppressive soils against the wilt diseases caused by Fusarium (Singh et al.,1999). Biological control of Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. ...
Bottle gourd (Lagenaria siceraria L.) is an important vegetable crop belonging to the gourd group in cucurbitaceae family. The production and productivity of this vegetable crop is affected by many diseases, of which wilt (Fusarium oxysporum f.sp. lagenariae) plays a significant role. Among different management strategies available, biocontrol seems to be promising as the use of chemical fungicides in disease management has many limitations including fungicide resistance and environment hazards. Biological control can play an effective role in combating this problem by effectively managing the disease. Considering the importance of disease, the present study was conducted with an objective to evaluate the potential biocontrol agents against Fusarial wilt of bottle gourd. In this study, several isolates of Trichoderma and Pseudomonas were isolated from wilt affected bottle gourd growing regions of Punjab and were screened and later tested under in vitro and in vivo conditions to determine their antagonistic effect on Fusarium oxysporum f.sp. lagenariae. Dual culture and volatile assay showed that two isolates of Trichoderma (T1 and T2) and one Pseudomonas isolate (P1) had superior antagonistic potential, when tested under in vitro conditions. Of these potential isolates, Trichoderma isolate T2 showed maximum mycelial growth inhibition of Fusarium oxysporum f.sp. lagenariae (76.33%) in dual culture. On the basis of molecular characterization, the potent isolates T1 and T2 were identified as Trichoderma asperellum and isolate P1 was identified as Pseudomonas aeruginosa. The potent bioagents (T1, T2 and P1) were then evaluated for their bioefficacy under in vivo conditions for two growing seasons in year 2018-19. Maximum disease inhibition 84.62 and 80.00% was reported by Trichoderma asperellum isolate T2 in March and June sown bottle gourd crop, respectively. Besides, Trichoderma asperellum (T2) exhibited highest plant growth promotion with total length (5.43m and 4.83m) alongwith enhanced yield i.e. 235.99 q/acre and 229.00 q/acre in March & June seasons, respectively. Based on the findings, the biocontrol agents viz. Trichoderma asperellum (T2 and T2) and Pseudomonas aeruginosa (P1) have greater potential in managing wilt of bottle gourd and their bioformulations can be used at commercial scale to manage the disease at farmer’s field.
... The supernatants were recovered and placed in the 2 ml tubes. Further extractions were repeated with 1 ml of solution and the two supernatants were mixed into a single extract according to the method of Singh et al. (1999). The total amino acids were then determined according to the ninhydrin modified method of Yemm and Cocking (1955). ...
Fungal diseases are one of the major causes of losses on wheat yield in the world. Recent studies on
plant defense mechanisms have highlighted the role of amino acids and total polyphenols in disease
tolerance. Thus, with the objective of identifying on the basis of morphological and physiological
variables the high-performance wheat accessions in high and low altitude, we characterized sixteen
wheat accessions via quantification of polyphenols (TPP) and amino acids (AA) and identified fungal
diseases affecting wheat in Centre Cameroon. Using the set of the three hexaploid wheat cultivars
where the 11 microsatellite markers data was available, a total of 29 alleles were detected among
cultivars and the number of alleles per locus ranged from 2 to 3 with an average of 2.64. Gene diversity
ranged from 0.44 to 0.67 with an average of 0.59, increasing with the number of alleles. Microsatellites
markers used had an average Polymorphic Information Content (PIC) value of 0.50, indicating that these
markers are useful and will make a contribution to the studies in hexaploid wheat. The assessment of
wheat plant to disease tolerance permitted to identify Septoria, Fusarium wilt; tanned spot and powdery
mildew in high and low altitude. Analyzes of TPP and AA have made it possible to discriminate
accessions that are tolerant to diseases. Hence, accession Atilla 1 was highly tolerant and had high
levels of TPP (3.5 ± 0.36 μg/mg) and AA (40.1 ± 1.21 μg/mg) while the accession Sup 152 highly
susceptible to disease had low levels (2.3 ± 0.1 and 2.1 ± 0.36 μg/mg, respectively). This study will
contribute to the extension of wheat in areas where conditions similar to the study sites will be
localized.
Key words: Amino acids, bread wheat, high and low altitude, fungal diseases, polyphenols.
... β-1,3-glucanase synthesized by Paenibacillus and Streptomyces spp. strains can easily disrupt pathogenic fungal cell walls (Singh & et al., 1999). ...
Variety of Microbial Organic Compounds That Induce Plant Responses
... The production of chitinase and β-1,3-glucanase by the BCAs (Singh et al., 1999) was quantitatively determined using the minimal synthetic medium (Tweddell et al., 1994) amended with 2 mg ml −1 of either colloidal chitin or laminarin (Sigma-Aldrich), respectively. Chitinase-specific activity was calculated by measuring the release of N-acetyl-D-glucosamine (NAGA) from colloidal chitin. ...
In the United Arab Emirates (UAE), sudden decline syndrome (SDS) is one of the major fungal diseases caused by Fusarium solani affecting date palm plantations. To minimize the impact of the causal agent of SDS on date palm, native actinobacterial strains isolated from rhizosphere soils of healthy date palm plants were characterized according to their antifungal activities against F. solani DSM 106836 (Fs). Based on their in vitro abilities, two promising biocontrol agents (BCAs), namely Streptomyces tendae UAE1 (St) andStreptomyces violaceoruber UAE1 (Sv), were selected for the production of antifungal compounds and cell wall degrading enzymes (CWDEs), albeit their variations in synthesizing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD). Although both isolates showed antagonism when applied 7 days before the pathogen in the greenhouse experiments, the ACCD-producing Sv was relatively superior in its efficacy against SDS over the non-ACCD-producing St. This was evident from the symptoms of SDS in diseased date palm seedlings which were greatly reduced by Sv compared to St. On a scale of 5.0, the estimated disease severity indices in Fs-diseased seedlings were significantly (P < 0.05) reduced from 4.8 to 1.5 and 0.5 by St and Sv, respectively. Thus, the number of conidia of Fs recovered from plants pre-treated with both BCAs was comparable, but significantly (P < 0.05) reduced compared to plants without any BCA treatment. In addition, a significant (P < 0.05) decrease in ACC levels of both the root and shoot tissues was detected inSv + Fs seedlings to almost similar levels of healthy seedlings. However, in planta ACC levels highly increased in seedlings grown in soils infested with the pathogen alone or amended with St prior to F. solani infestation (St + Fs). This suggests a major role of ACCD production in relieving the stress of date palm seedlings infected with F. solani, thus supporting the integrated preventive disease management programs against this pathogen. This is the first report of effective rhizosphere actinobacterial BCAs to provide protection against SDS on date palm, and to help increase agricultural productivity in a more sustainable manner in the UAE and the other arid regions.
... For the estimation of mycolytic enzyme production culture supernatants of the screened actinobacterial isolates were determined as a source of enzymes. Chitinase (Reisslig et al. 1955), protease (Meyers and Ahearn 1977) and β-1, 3 glucanase (Singh et al. 1999) activities were measured by determining the release of N-acetyl-Dglucosamine, tyrosine and glucose per hour, respectively. Hydrogen cyanide (HCN) production was determined by the method of Bakker and Schippers (1987). ...
The present study is aimed to evaluate the integrated effects of the chemical fungicide Bavistin (carbendazim 50% WP) on adaptive variants of rhizospheric actinobacteria in providing protection to the chickpea from wilt caused by Fusarium oxsysporum. Actinobacterial isolates recovered from the rhizospheric region were screened for their antibiosis against the fungal pathogen F. oxysporum. Among these actinobacteria two potent antagonistic isolates, Nocardiopsis sp. KWC01 and Streptomyces sp. KBR01, showed a significant profile by producing extracellular lytic enzymes, hydrogen cyanide, siderophore, indole acetic acid (IAA) and solubilizing phosphate. Both of them caused hyphal deformation in F. oxysporum as observed through scanning electron microscopy (SEM). Before applying a blended form of biological agents and the chemical in the field, bavistin adaptive variants of both the actinobacterial isolates were obtained. Thereafter EC50 of bavistin to kill F. oxysporum was determined and applied with actinobacterial isolates during field trials. The effect of EC50 of Bavistin on the specific growth rate of isolates was also examined, which showed enhanced growth of isolates at concentrations close to EC50 of bavistin. During field trials, unsurpassed results were obtained using blends of the actinobacterial consortium with a low dose of chemical fungicide. This combination led to an increase in wilt protection by 2.66% and grain yield by 8.69% over full dose of chemical fungicide. These results advocate the efficiency of integrated formulation containing Nocardiopsis sp. KWC01, Streptomyces sp. KBR01 and low dose of bavistin in wilt management and productivity enhancement of chickpea plants.
... strain 385 can lyse the cell walls of Fusarium oxysporum f. sp. cucumerinum (Singh et al., 1999). The regulatory systems involved in modulating the production of lytic enzymes like proteases and chitinases is GacA/GacS, GrrA/GrrS, and the colony phase variation (Corbell and Loper, 1995;Lugtenberg et al., 2001;Ovadis et al., 2004). ...
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.
... strain 385 can lyse the cell walls of Fusarium oxysporum f. sp. cucumerinum (Singh et al., 1999). The regulatory systems involved in modulating the production of lytic enzymes like proteases and chitinases is GacA/GacS, GrrA/GrrS, and the colony phase variation (Corbell and Loper, 1995;Lugtenberg et al., 2001;Ovadis et al., 2004). ...
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.
... It can be detected in a variety of ecosystems and this has proved its efficacy in regulating soil-borne diseases in beans and cotton, such as southern blight of crops and plant pathogenic fungus (Shapira et al. 1989). One of the hypothesized methods includes the chitinase, which degrades harmful fungi's cell walls (Nayak et al. 2020;Singh et al. 1999). In addition to its role as biological pest managing enzyme, there are other numerous directions for using this enzyme to maintain soil health and, as a result, boost plant development and ultimate harvests, which is possible due to its eco-friendliness (Das and Varma 2010). ...
Soil is the most vital part of terrestrial biota. Since all the related ecological communities thrive on it, the protection, maintenance and improvement of soil is of high importance. The quality of soil to function in a dynamic equilibrium with the thriving biosphere to sustain plant, animal and human life is now being considered as soil health. Assessment of soil quality solely depends on biological, chemical, physical indicators, as all of these contribute in maintaining soil health. The quality of soil can be evaluated directly by reviewing the soil enzymes. Soil enzymes are the fundamental factors of the soil system that are critical for the maintenance of ecosystem functioning and nutrient recycling. They have the capability of efficiently catalysing the decomposition of organic components in the soil system, which helps in maintaining the life processes of soil microorganisms and structural stabilization of soil. Enzymes present in soil are mostly of microbial origin, but can also be of plant or animal origin. These enzymes can be extracellular or intracellular depending on their location, and depending on their origin of derivation, they can be intracellular, free enzymes or cell-associated. There are vast number of enzymes that have great efficiency in soil improvement and maintenance of soil health. This chapter provides a detailed review of all the major soil enzymes and their activities in maintaining the health of soil and their prospective role in soil improvement.KeywordsBiological indicatorsNutrient recyclingSoil enzymesSoil healthSoil quality
... It can be detected in a variety of ecosystems and this has proved its efficacy in regulating soil-borne diseases in beans and cotton, such as southern blight of crops and plant pathogenic fungus (Shapira et al. 1989). One of the hypothesized methods includes the chitinase, which degrades harmful fungi's cell walls Singh et al. 1999). In addition to its role as biological pest managing enzyme, there are other numerous directions for using this enzyme to maintain soil health and, as a result, boost plant development and ultimate harvests, which is possible due to its eco-friendliness (Das and Varma 2010). ...
Antibiotics are bioactive compounds that selectively kill or mitigate the growth of microorganisms. The increasing human population, development, and industrialization resulted in an increased demand of antibiotics. The annual consumption of antibiotics round the globe has reached over 200,000 tons. Hence, alternative and cost-effective sources for the production of antibiotics are inevitable. Agricultural wastes, i.e., corn cobs, sawdust, rice hulls, and groundnut shell, are rich source of bioactive compounds. Therefore, the agro-waste can be utilized for industrial production of various value-added products including antibiotics. The composition, quantity, and quality of antibiotics produced from agro-waste depend on both starting material/substrate (raw waste) and the processing steps. By applying appropriate fermentation techniques, agro-waste can be used in cost-effective production of antibiotics. Recent studies reported the production of neomycin, oxytetracycline, and rifamycin using agro-wastes as substrate by solid state fermentation (SoSF). Several microorganisms were used for the production of these valuable products. In addition, the external energy sources were supplied to enhance the production of antibiotic.KeywordsAgro-wasteAntibioticsSolid state fermentation (SoSF)Mechanism of actionProcess optimization
... Combined application of Trichoderma + Pseudomonas (1%) of talc formulation as foliar spray was found next best treatment in reducing the twister disease severity. This reduction in disease is mainly due to their different modes of action viz., competition for nutrients (O'Sullivan and O'Gara, 1992), production of cell wall lytic enzymes (Singh et al. 1999) and induced systemic resistance (Nandakumar et al. 2001) [16] , the salicylic acid and lipopolysaccharide produced by P. fluorescens may act as local and systemic signal molecules in inducing resistance in plants (Van Peer and Schippers, 1992) [25] . The diversity of mechanisms available to Trichoderma spp. ...
Onion (Allium cepa L) cultivation has been hampered by many pests and diseases. In recent times onion
twister has become most serious and threatening since two years. The severity of disease witnessed
drastic decline in production and shortage in domestic supply across the country. Its management was
studied using different fungicides along with bio-control agents Trichoderma and Pseudomonas in
combined mixture form during Rabi 2021. The experiment was conducted on a site previously cultivated
with onion and infested by sever twister disease. Among the different treatments, folair application of
carbendazim 12% + mancozeb 63% 75 WP at 2.5g/l (T9), Trichoderma + Pseudomonas mixture at 10 g/l
concentration (T13) and propiconazole 25 EC at 1 ml/l (T10) recorded less PDI of 16.59, 17.26 and 18.54
respectively. Maximum PDI of 72.11 was observed in control plot. The yield was also significantly
superior in T9 with yield of 28.28 t/ha and B:C value of 5.74 followed by T10 which had yield of 27.45
t/ha and B:C ratio of 5.59 and T13 with an yield of 26.89 t/ha and B:C ratio of 5.46. Lowest yield (14.70
t/ha) was recorded in control which had B:C ratio of 3.03. It was necessary that among these best
fungicides or bio-control agents’ mixture, any one shall be adopted but spraying of three times at an
interval of 15 days in essential once the symptoms are first observed for effective control of the disease.
Cucumber wilt is an important soil borne disease in cucumber production, which seriously affects the development of the cucumber industry. Cleome spinosa also has pharmacological effects such as antibacterial, analgesic, anti-inflammatory, and insect repellent. To study the control effect and mechanism of Cleome spinosa fumigation on cucumber wilt disease, different concentrations of Cleome spinosa fragments were applied on cucumber plants infected with Fusarium oxysporum. Cleome spinosa fumigation significantly reduced the incidence rate of cucumber Fusarium wilt. Under the fumigation treatment of 7.5 g kg-1 Cleome spinosa fragments, the preventive effects were 74.7%. Cleome spinosa fragments fumigation can promote cucumber growth and synthesis of photosynthetic pigments, thereby improving individual plant yield and fruit quality. At 7.5 g kg-1 Cleome spinosa fragments fumigation treatment, the plant height and individual plant yield of cucumber increased by 20.3% and 34.3%, respectively. Cleome spinosa fumigation can enhance the activity of antioxidant enzymes in cucumber, maintain a balance of reactive oxygen species metabolism, and enhance the plant disease resistance. Moreover, Cleome spinosa can also regulate the activities of Mg2+-ATPase and Ca2+-ATPase, enhancing its resistance to Fusarium oxysporum. Moreover, number of bacteria and fungi significantly decreased under Cleome spinosa fumigation. Those results suggested that Cleome spinosa could effectively restrain cucumber Fusarium wilt. This study will provide a new idea for the further use of biological fumigation to prevent soil-borne diseases.
Agriculture is facing major challenges affected by erosion, salinity and soil degradation. Chemical pesticides and fungicides are more used the agricultural lands. Chemical pesticides and fungicides are more used environment and human diseases causes. The better approaches in agriculture, biocontrol microorganisms, and Plant Growth Promoting (PGP) have emerged as safe for the environment, and also safe alternatives to chemical pesticides. Plant associated microorganisms have helped in soil nutrients enhancement, nitrogen fixation, phosphate solubilization, siderophore production, β-1,3glucanase, cellulase, protease, and lipases. These microbes have tolerances to the biotic and abiotic stress, pH, salinity drought, extremes temperature, heavy metals, and pesticide pollution. This summarized and discussed in this review evaluated actinomycetes related research and its benefits. These bacteria are biocontrol of plant pathogens and enhance the plant growth in agriculture.
Chitinase has a wide distribution of fungi, plants, bacteria, yeast, humans, protozoa, and insects, in the environment. Chitinases have different functions in these organisms. The mineral composition of chitin, nutrients, and bacterial parasitism is often involved in chitinases. Fungal chitinases however play a physiological role in cell division and differentiation. Plant chitinase is part of the plant's immune system against fungal pathogens. In insects, chitinases during their development have been identified as being involved in the molting process. In human gastric juice, chitinases have also been identified. Chitinase chitin degradable enzyme has control over phytopathogens, physiological functions and destruction of chitinous waste.In this review focusing on the various sources of natural chitinase production in Actinomycetes was discussed.
Three endophytic bacteria, namely BvV, BvP and BvL, were newly isolated from the root nodules of bean, pea and lentil plants respectively cultivated in Mascara the northwest of Algeria, and identified by 16S ribosomal RNA gene sequencing as Brevundimonas naejangsanensis. These strains were able to produce hydrolytic enzymes and hydrogen cyanide. All strains produced a growth-promoting hormone, indole acetic acid, varying in concentration from 83.2 to 171.7 µg/mL. The phosphate solubilizing activity of BvV, BvP and BvL varied from 25.5 to 42.02 µg/mL for tricalcium phosphate. The three antagonistic Brevundimonas spp. showed in vitro the most inhibitory effect on mycelial growth of Fusarium redolens FRC (from 78.33 to 85.55%). Strain BvV, BvP and BvL produced also volatile metabolites which inhibited mycelial FRC growth up to 39.2%. All strains showed significant disease reduction in pot experiments. Chickpea Fusarium yellows severity caused by FRC was reduced significantly from 89.3 to 96.6% in the susceptible cultivar ILC 482 treated with antagonistic B. naejangsanensis. The maximum stimulatory effect on chickpea plants growth was observed by inoculation of strain BvV. This treatment resulted in a 7.40–26.21% increase in shoot height as compared to the control plants. It is concluded that the endophytic bacterial strains of B. naejangsanensis having different plant growth promoting (PGP) activities can be considered as beneficial microbes for sustainable agriculture. To our knowledge, this is the first report to use B. naejangsanensis strains as a new biocontrol agent against F. redolens, a new pathogen of chickpea plants causing Fusarium yellows disease in Algeria.
Produktivitas bawang merah secara nasional menunjukkan penurunan pada beberapa tahun terakhir. Permasalahan utama yang menyebabkan hal tersebut adalah rendahnya ketersediaan benih berkualitas bebas penyakit serta sistem produksi bawang merah dengan input kimia yang tinggi. TSS merupakan alternatif benih sehat yang dapat digunakan melalui produksi umbi mini. Penggunaan PGPR dan input organik kompos dalam produksi umbi mini merupakan solusi teknologi ramah lingkungan untuk meningkatkan produktivitas bawang merah. Penelitian dilakukan dengan Rancangan Acak Lengkap dengan empat perlakuan dan lima kali ulangan. Perlakuan yang diberikan yaitu pemberian (A) kompos saja (B) kompos+larutan PGPR konsentrasi 5 g/L, (C) kompos+larutan PGPR konsentrasi 10 g/L, dan (D) kompos+larutan PGPR konsentrasi 15 g/L. Hasil penelitian menunjukkan penggunaaan kompos dengan konsentrasi PGPR (Plant Growth Promoting Rhizobacteria) yang berbeda tidak memberikan pengaruh terhadap pertumbuhan dan hasil umbi mini bawang merah asal benih TSS (True Shallot Seed) varietas Tuk Tuk. Namun, terdapat kecenderungan bahwa takaran kompos dan PGPR (Plant Growth Promoting Rhizobacteria) dengan konsentrasi 5 g/L berpotensi memberikan hasil umbi mini bawang merah yang terbaik.
Kata kunci : benih, kompos, PGPR, TSS, umbi mini
Endophytic bacteria is promised solution to suppress basal stem rot disease caused by fungus Ganoderma boninense . The antifungal activities of selected isolate endophytic bacteria from oil palm plantation in South Kalimantan was studied. Bacillus cereus was tested for its antifungal activities of crude chitinase and secondary metabolites against the growth of Ganoderma boninense mycelium with dual cultured method. The results were showed that chitinase had the largest inhibition zone (18.5%) against growth inhibition of fungi Ganoderma boninense’ s mycelium. A series of optimation assays of chitinase activity were conducted at pH 3 to 8 with 0.5 interval, and temperature at 30 to 70°C, with 5°C interval. Specific chitinase activities was measured using the colorimetric method. The highest specific chitinase activity significantly at 1.66393±0.04807 mU/μg (95% confidence level) at pH 5.5 and temperature 45°C.
Manipuri black aromatic rice, ‘Chakhao’, is a nutrient rich unique local rice cultivar of Manipur, India. Rhizosphere bacteria may have potential as antifungal agent and seedling growth promoter. Hence, the current investigation was aimed at screening, for the above traits, using bacteria, Bacillus spp., isolated from the soil of Rhizosphere of six Chakhao rice cultivars. Altogether, 323 bacterial isolates were obtained from the rhizospheric soils of 6 different cultivars of Chakhao, namely Chakhao Amubi (CA), Chakhao Poireiton (CP), Chakhao Sempak (CS), Chakhao Angoubi (CAng), Chakhao Angangbi (CR) and Chakhao Wairi (CW). All the rhizobacterial isolates were screened for antifungal activity against 5 rice fungal pathogens viz. Rhizoctonia solani (RS), Fusarium oxysporum (FO), Curvularia oryzae (CO), Pyricularia oryzae (PO) and Aspergillus niger (AN). The isolates were also subjected to PGP (plant growth promotion) assays such as ammonia, indole acetic acid (IAA), siderophore and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production, and phosphate solubilization. Of 323 isolates obtained, 64 were found to exhibit antifungal activity while 69 showed PGP traits. Twenty-five (25) isolates were found to be positive for both antifungal and PGP traits. Of 25 bioactive isolates, 4 (CR12, CW11, CA2 and CP2) potent isolates were shortlisted for further studies. The shortlisted potent isolates were subjected to quantitative estimation of PGP activities like phosphate solubilization, IAA and siderophore production. The strains could produce significant amount of IAA and siderophore and solubilize phosphate. Molecular characterization by 16S rDNA sequence analyses revealed the identity of the isolates: CR12: Bacillus subtilis (Accn. No.OM866257), CW11: Bacillus paralicheniformis (Accn. No.OM868047), CA2: Bacillus sp. CCMB1014 (Accn. No.OM868070) and CP2: Bacillus licheniformis (Accn. No.OM892495). Seedling vigor assays of the shortlisted potent strains were carried out on Chakhao Amubi rice. The isolates exhibited higher seedling vigor indices (CR12: 668.00, CA2: 618.66, CP2: 510.92 and CW11: 478.91) over the control (164.48). These rhizospheric strains have the potential to be developed as bioinoculants or biostimulants for enhancing Chakhao rice growth.
Chitin has substantial benefits in several sectors including agriculture. In the plant protection field, chitin can promote the growth of certain bacteria which has possibly controlled the spread of fungal pathogens. Thus, chitinolytic bacteria producing chitinase enzymes can inhibit the growth of fungi. The purpose of this study was to examine the morphological and physiological characteristics of chitinolytic bacteria which collected from Ipomea pes caprae in Makassar and its ability to produce chitinolytic enzymes on medial culture. The method started to grow the isolates on the nutrient agar which were added to colloidal chitin. Furthermore, the bacteria were identified by its color, form, elevation and edge as well as tested its physiological characteristics with KOH 3% test and catalase test. In addition, bacterial chitinolytic index was carried out by using the disc diffusion method. The results showed that each isolate had yellowish white and grayish white colors; morphological characters: round and irregular colony shapes, colony edges smooth and choppy as well as umbonature and convex elevations. The physiological test results indicate that all isolates had a positive response in the catalase test, one of the isolates reacted positively to the gram test and the others reacted negatively to the test. Each isolate had chitinolytic activity with indices of 0.10, 0.09, 0.70, and 0.11.
Millets are nutritionally rich crops, constituting an important part of the diverse food crops grown and consumed in most states of India and its surrounding regions. The different types of millets have different nutritional properties, and the nutrient profile varies depending on the soil conditions they are grown under. The millet rhizosphere harbours diverse and beneficial microbial communities playing myriad roles. The important roles of the microbes include supporting plant growth, pest and pathogen resistance, acclimatising the plants to harsh climatic conditions and anthropogenic pressures. The millet rhizobiome is unique since it may confer to drought resistance, participate in biogeochemical cycles of nitrogen, carbon, phosphorus, potassium, sulphur, etc., allowing the plants to adapt well in arid and nutrient-deficient soils. Furthermore, microbial diversity, abundance, and their multiple functions are essential for reductions in greenhouse gases (GHG) from agricultural soils, water retention, decomposition activities, increasing soil organic matter, weathering, etc. Advancing interdisciplinary tools for deciphering microbial communities including high throughput metagenome sequencing, advanced computational tools, etc. are increasingly being used to decipher and elucidate the microbial communities in the rhizosphere. Metagenomics has helped unravel many bacterial communities that are dominant in the millet rhizosphere, paving way for future characterization of their potential multifunctionalities. In this chapter, we have given an account of the metagenomics performed for different millets and the multifunctionalities associated with these future climate-adaptable crops.KeywordsMetagenomicsMultifunctional microbesRhizobiomeFinger milletHigh throughput sequencing
Chitin is the second most abundant polysaccharide worldwide as part of arthropods' exoskeletons and fungal cell walls. Low concentrations in soils and sediments indicate rapid decomposition through chitinolytic organisms in terrestrial and aquatic ecosystems. The enacting enzymes, so‐called chitinases, and their products, chitooligosaccharides, exhibit promising characteristics with applications ranging from crop protection to cosmetics, medical, textile, and wastewater industries. Exploring novel chitinolytic organisms is crucial to expand the enzymatical toolkit for biotechnological chitin utilization and to deepen our understanding of diverse catalytic mechanisms. In this study, we present two long‐read sequencing‐based genomes of highly similar Jeongeupia species, which have been screened, isolated, and biochemically characterized from chitin‐amended soil samples. Through metabolic characterization, whole‐genome alignments, and phylogenetic analysis, we could demonstrate how the investigated strains differ from the taxonomically closest strain Jeongeupia naejangsanensis BIO‐TAS4‐2T (DSM 24253). In silico analysis and sequence alignment revealed a multitude of highly conserved chitinolytic enzymes in the investigated Jeongeupia genomes. Based on these results, we suggest that the two strains represent a novel species within the genus of Jeongeupia, which may be useful for environmentally friendly N‐acetylglucosamine production from crustacean shell or fungal biomass waste or as a crop protection agent.
Crop infections with Fusarium spp. and associated mycotoxins have devastating economic implications and are a significant limitation to fifteen of the seventeen United Nations Sustainable Development Goals. Therefore, it is necessary to develop innovative strategies to reduce or eliminate diseases caused by Fusarium spp. in cultivated crops. Management of Fusarium-related diseases using beneficial microorganisms is a promising and eco-friendly solution. In this context, actinobacteria are considered one of the best candidates because of their multifunctional properties, which consist in the reduction of Fusarium spp. diseases and the promotion of plant growth. Actinobacteria represent a large Gram-positive bacterial phylum that is widespread in different environments, especially in soil. They are principally known for their versatile capacity to produce biologically active molecules with potential applications in different fields, including agriculture. As Fusarium species threaten diverse crops worldwide and cause global yield and quality losses that can negatively impact food security, the use of actinobacteria for plant diseases management is gaining interest as a safer and more potent alternative to ineffective synthetic-based pesticides. Thus, this work aims to review and highlight the importance of actinobacteria as microbial biocontrol agents and discuss the current research on Fusarium spp. diseases with a focus on species that threaten economically important crops, such as F. graminearum, F. oxysporum, F. solani, and F. culmorum. This review also presents actinobacteria-based products that have been patented for Fusarium disease control. We aim to provide valuable data, guidance and advancements for future research on microbial biocontrol against Fusarium-related diseases. In addition, this review emphasizes the need for further research about the impact of actinobacteria-Fusarium interactions on mycotoxin production. It also highlights the need of in-depth understanding microbial biocontrol mechanisms to ensure safe and sustainable crop production worldwide.
The area of soil exposed to root activity is called the rhizosphere which harbors diverse microbes that can aid in plant growth and resistance against biotic and abiotic stresses. Rhizosphere microbiome is defined as all the microbial species found in the rhizosphere, which have one of the most complex and diverse ecosystems on the planet. These rhizosphere microbial communities interact with the plants as beneficial or detrimental interactions. Beneficial rhizosphere microbes promote plant growth through abiotic stress tolerance, absorption of nutrients in plants and antagonism against several phytopathogens, while parasitic interaction causes diseases of plants which are economically important, leading to challenges in food security and reduction in productivity. In this chapter, we have discussed in detail the various interactions on microbe-plant and microbe-microbe interaction and also the role of rhizosphere microbiome in plant health and resistance.KeywordsBeneficialDetrimentalPlant healthMicrobesRhizosphere
Microbial consortium is a naturally occurring beneficial microbiome in the vicinity of plant root system. Artificially, bacterial mixture can be prepared after selecting them for a shared purpose of benefits such as nutrients, space, and other multifunctional benefit to the plants. The mixture of strains formulated in the form of bio-inoculants of microbial consortium can be seen in various ways including its use for biological control of plant diseases. Extensively, the applications of the microbial consortium have been proven more beneficial to the plants in comparison to the use of individual microorganisms, precisely contributing to sustainable development of overall ecosystem. The exploitation of microbial consortia is challenging to study synergism at eco-microbiological level thus offering a new scope of research towards rhizospheric bioengineering. Microbe–microbe interaction, etc., at theoretical and practical level may pay attention towards rediscovering. Their role as potential contender of boosting agricultural productivity is for the future benefits of the ever-increasing population. This review highlights the present state of knowledge and purpose of this scientific commentary to develop novel methods of using microbial mixtures that have relevance to developing microbial consortia for agriculture use. The success, impediments, and failure have also been presented to bring new scientific insight and technologies for wide dissemination of knowledge in scientific community. The research-based evidence clearly shows the advantages of microbial consortia whose function to plants or soil rhizosphere stimulate the natural process to overall benefits to plants in agro-ecosystems.KeywordsMicrobe–microbe interactionPhytopathogensDisease controlMicrobial consortiumBiocontrol agents
Fusarium cotton wilt is a devastating disease of the cotton crop throughout the world, caused by Fusarium oxysporum f.sp. vasinfectum (FOV). Chemical control has many side effects, so, biological controls have been widely used for the management of Fusarium wilt. This study aimed to investigate the possible use of an actinomycetes Saccharothrix algeriensis (SA) NRRL B-24137 to control FOV. To access in-vitro anti- Fusarium ability of SA NRRL B-24137, dual culture assay, spore germination and seed germination tests were carried out. Following in-vitro investigations, several pot tests in a greenhouse environment were used to evaluate the biological control potential of SA NRRL B-24137 against FOV. Dual culture assay and spore germination revealed that SA NRRL B-24137 showed significant anti- Fusarium activity.During spore germination 87.77% inhibition of spore germination were observed. In pot experiments, SA NRRL B-24137 primed cotton seeds resulted in a 74.0% reduction in disease incidence. In soil there was a significant reduction in FOV spores in the presence of SA NRRL B-24137. Positive correlation was also observed on different concentrations of SA NRRL B-24137 towards FOV reduction. The results of this study showed that SA NRRL B-24137 has the potential to be employed as a biocontrol agent against Fusarium cotton wilt, improving cotton growth characteristics and yield.
Plant growth promoting rhizobacteria (PGPR) residing in soil rhizosphere provide enormous beneficial effects to a plant host producing diverse secondary metabolites and enzymes useful for plant growth and protection. Siderophores, antibiotics, volatile compounds and hydrolytic enzymes are the major molecules secreted by the PGPRs, which have substantial antifungal properties and can provide plant protection. These compounds are responsible for the lysis and hyperparasitism of antagonists against deleterious fungal pathogens. Siderophore-producing PGPRs function by depriving the pathogen of iron nutrition. Antibiotics have been reported to be involved in the suppression of different fungal pathogens by inducing fungistasis, inhibition of spore germination, lysis of fungal mycelia. The PGPRs also secrete a wide range of low molecular weight volatile organic compounds (VOCs) that inhibit mycelial growth, sporulation, germination of phytophathogenic fungi, etc. Hydrolytic enzymes, mostly chitinase, protease and cellulose, lyse the cell wall of fungi. Therefore, plant growth-promoting rhizobacteria can be considered as an effective, eco-friendly, and sustainable replacement to the chemical fungicides. There are many PGPRs that perform very well in controlled conditions but not in field conditions, and hence the commercializing of hese products is not easy. Development of formulations with increased shelf life, a broad spectrum of action and consistent performance under field conditions can pave the way for commercializing the PGPRs at a faster rate. Journal of Bangladesh Academy of Sciences, Vol. 44, No. 2, 69-84, 2020
Chitin is an insoluble structural polysaccharide present in the exoskeleton of insects. One potential biopesticide is enzyme chitinase, which degrades chitin-soluble and insoluble oligosaccharides. Recently, chitinase enzymes as a biopesticide are developing to control insect and fungal pests—an alternative approach with enzyme-based biopesticides to avoid the chemical pesticides against pests and pathogens. The success in exploring chitinase from microbial sources, especially for agriculture, has a high volume low cost, and it also depends on the availability and active formulation of the product at a reasonable cost. The roles of chitinolytic enzymes as a biocontrol agent, different mechanisms to mass culture the chitinase and the formulations, and making the enzyme stable for the long term is the wide area to research. The chitin degradation, and identification of chitinase from different microbial sources with varying specificities, may make them more useful in commercial processes soon. In this chapter, Pseudomonas sp.-derived chitinase enzymes, essential in the agricultural field, benefit the plant by PGPR activity and mass multiplications of bacteria and the enzyme, production, and formulations in low cost, requirements to register the biopesticide and marketing were discussed in this chapter.KeywordsPseudomonas speciesMass productionFormulationShelf-lifeCommercializationBio-control agent
Chemical, biochemical, and physicochemical reactions are all involved in nutrient cycling in soil. Enzymes catalyze all biochemical processes in soil. Soil enzymes catalyze several biochemical processes that ensure the transformation of organic materials and the release of inorganic nutrients for plant growth and nutrient cycling. As a result of the significant role played by soil enzymes in improving the fertility of soil, an in-depth evaluation of the influence of soil microbial enzymes on the fertility of soil is essential for effective maintenance of soil fertility, utilization of soil resources, and enhancement of plant productivity. This chapter discusses (1) the detailed role of soil microbial enzymes in improving the fertility of soil, (2) the mechanisms of action of soil enzymes, and (3) the factors influencing the enzyme activity in soil.
Sustainable agriculture techniques to combat abiotic and biotic stresses in eco-friendly approaches are very essential to feed the increased population. Among the plant diseases, fungal pathogens are the biggest global threat causing huge losses in agriculture and food production. Plants and soil microbes interact to help each other for their growth and development. Plants can use the growth-promoting microbes as a weapon against various pathogens including fungi. The PGPR are capable to produce various classes/families of bioactive biomolecules, for example, non-ribosomal polyketide synthases (NRPS), polyketide antibiotics, siderophores, surfactants, hydrolytic enzymes (e.g., protease, lipase, etc.), volatile compounds, etc. By secreting one or more of these bioactive metabolites, the PGPR displays pathogen inhibition. Furthermore, the PGPR can consume nutrients from the soil by scavenging essential micronutrients that are required for the pathogen growth. Hence, these beneficial microbes have great potential application in agriculture, i.e., microbial solution is one of the most eco-friendly alternatives to the extensive use of pesticides and chemical fertilizer to combat fungal crop diseases.KeywordsAntagonistic propertyBiocontrolBiofungicideSoil microbes NRPSVolatile compounds
This work aimed to explore an antagonistic actinobacterial strain isolated from roots of Ziziphus lotus in bioformulation processes and the biocontrol of Rizoctonia solani damping-off of tomato seedlings. The strain Streptomyces lincolnensis SZ03 was investigated for the principal in vitro biocontrol mechanisms and then formulated in three different biofungicides Wettable Talcum Powder (WTP), Sodium-Alginate Propagules (SAP) and Clay Sodium-Alginate Propagules (CAP). Compared to a marketed control products (Serenade® and Acil 060FS®), the formulated biofungicides were investigated against the R . solani damping-off of tomato cv . Aïcha seedlings. The strain S . lincolnensis SZ03 produced chitinases, cellulases, β-1,3-glucanases, cyanhydric acid and siderophores and it showed strong antagonistic effect on the mycelial growth of R . solani . Bioautographic and HPLC analysis revealed the production of a single antifungal compound. The biofungicide WTP showed an attractive biocontrol effect by reducing significantly the disease severity index (DSI) compared to untreated seeds. No significant differences were obtained compared to the chemical treatment with Acil 060FS®. The viability of spores and biocontrol efficacy of the WTP were confirmed after one year storage. S . lincolnensis strain SZ03 has never been reported in the biocontrol of phytopathogenic fungal diseases and it open up very attractive prospects in the fields of biocontrol and crop improvement.
The rhizomicrobiome comprises a wide variety of microorganisms that are essential for microbial colonization and root development in a wide variety of plants. A plant’s growth, development, and defense mechanisms would be impossible without the rhizomicrobiome’s microbes. In order to develop and operate properly, roots are essential to plants because they give structural support and aid in the intake of water and nutrients. This rhizobacteriome, a diverse bacterial population with particular roles that affect plant health, may be found in plant root exudates due to the complex variety of elements present. There are several metabolites produced by the plant-growth-promoting rhizobacteria (PGPR) in the rhizosphere near the plant roots that stimulate the plant’s development. Many PGPRs have the ability to solubilize phosphate, fix N2, produce biosynthesis of hydrolytic enzymes (hydrolase), produce phytohormones (phytoestrogens), produce siderophores (antibiotics), and more. Climate change, population growth, and the use of herbicides and insecticides have all had a significant influence on crop productivity in recent decades. Studies show that PGPR can boost plant growth and yield in a variety of species. As a result, PGPR dynamic microorganisms can be used as biofertilizers or biopesticides in agricultural techniques, which is critical to alleviating the urgent call for sustainable production. Rhizobacteriome, in particular PGPR found in the rhizosphere, and their many strategies for enhancing plant production are summarized in this chapter.KeywordsMicrobiomePlant–microbe interactionsRhizobacteriomeBiocontrolPGPRRhizobacteria
Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is one of the most disastrous fungal diseases. Biocontrol is a promising strategy for controlling fusarium wilt of banana. To explore endophytic actinomycetes as biocontrol resources against Foc TR4, antagonistic strains were isolated from different tissues of medicinal plants. Here, a total of 144 actinomycetes were isolated and belonged to Nonomuraea, Kitasatospora and Streptomyces. Forty-three isolates exhibited antifungal activities against Foc TR4. The strain labeled with 5-4 isolated from roots of Piper austrosinense had a broad-spectrum antifungal activity by the production of chitinase and β-1,3-glucanase and was identified as Streptomyces hygroscopicus subsp. hygroscopicus 5-4. Furthermore, disease index of banana wilt was significantly reduced by application of strain 5-4 in comparison with application of Foc TR4 alone. Exogenous application of strain 5-4 increased the expression levels of defense genes such as PAL, POD, PR-1, β-1,3-glucanase, LYK-1 and MPK-1. The antifungal mechanism assay demonstrated that extracts of strain 5-4 inhibited spore gemination and hyphal growth of Foc TR4, and caused abnormally swollen, deformity and rupture of Foc TR4 hypha. Thus, Streptomyces hygroscopicus subsp. hygroscopicus 5-4 could be used as a potential biological agent for controlling Fusarium wilt of banana.
Trichoderma sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus Trichoderma harzianum kj831197.1, and the cloning of xylanase genes in E. coli DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of T. harzianum kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The identified region was excised using restriction enzymes HindIII and EcoRI and cloned into a pUC19 plasmid vector. Optimization of fermentation conditions improved xylanase production about 23.9-fold.The antifungal efficacy of xylanase toward different phytopathogenic fungi was determined. The highest inhibition was against Corynespora cassiicola, Alternaria sp., Fusarium oxysporum, and Botrytis fabae. This study offered an economical, simple, and efficient method using Trichoderma harzianum kj831197.1 for the production of the xylanase enzyme via the submerged fermentation method.
Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.
Nearly 400 microorganism isolates, including bacteria, actinomycetes, and fungi, were collected from watermelon roots growing in soils sup pressive and nonsuppressive to Fusarium wilt of watermelon. These isolates were screened for their ability to restore suppressiveness to microwave treated suppressive soil and to reduce disease incidence in conducive field soil. Specific isolates of nonpathogenic Fusarium oxysporum from suppressive soil were the only organisms consistently effective in reducing disease (35 to 75% reduction) in both microwave-treated and natural field soils. Thus, we concluded that F. oxysporum was the primary antagonist responsible for suppression in this suppressive soil, although other organisms may contribute to suppressiveness. Selected isolates of F.oxysporum were effective in reducing disease when added to field soils at inoculum levels as low as 50 to 100 chlamydospores per g of soil, which was comparable to or below pathogen inoculum levels (100 to 200 CFU/g of soil). Root colonization data indicated that reduction of disease was not directly related to the ability of the antagonist to colonize roots extensively or to reduce colonization by the pathogen. Effective antagonists were not associated with specific vegetative compatibility groups, indicating antagonists represent diverse isolates. In split-root experiments, in which the antagonist and the pathogen were physically separated from each other, root colonization by selected isolates of F. oxysporum reduced disease incidence, verifying the mechanism of action as induced systemic resistance. Several isolates of F. oxysporum from this suppressive soil have potential for development as biocontrol agents.
Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.
Trichoderma koningii significantly reduced the severity of take-all of wheat caused by Gaeumannomyces graminis var. tritici in growth-chamber experiments and slightly enhanced the growth of wheat in the absence of G. graminis var. tritici. In field trials, T. koningii applied to the seed furrow increased the yield of spring wheat by 65% at Mt. Vernon, WA, and reduced crown root infection by G. graminis var. tritici on winter wheat by 40% at Pullman, WA. T. koningii was generally more suppressive of take-all than Pseudomonas fluorescens Q29z-80 or a mixture of P. fluorescens and P. putida strains at both sites. In field trials, the combination of T koningii and Q29z-80 increased yield compared to Q29z-80 alone but was not different from T. koningii alone. In growth-chamber experiments, combinations of T. koningii and any of six bacterial treatments provided substantially better disease control than the bacterial treatments applied alone. Combinations of T. koningii and certain bacterial treatments (e.g., P. chlororaphis 30-84, P. fluorescens Q2-87, and a four strain mixture) provided greater suppression of take-all than T. koningii alone. All combinations of T. koningii and fluorescent pseudomonads were compatible.
In an earlier study, treatment of radish seed with the bacteriumPseudomonas fluorescens WCS374 suppressed fusarium wilt of radish (Fusarium oxysporum f. sp.raphani) in a commercial greenhouse [Leemanet al., 1991b, 1995a]. In this greenhouse, the areas with fusarium wilt were localized or expanded very slowly, possibly due to disease suppressiveness of the soil. To study this phenomenon, fungi were isolated from radish roots collected from the greenhouse soil. Roots grown from seed treated with WCS374 were more abundantly colonized by fungi than were roots from nonbacterized plants. Among these were several species known for their antagonistic potential. Three of these fungi,Acremonium rutilum, Fusarium oxysporum andVerticillium lecanii, were evaluated further and found to suppress fusarium wilt of radish in a pot bioassay. In an induced resistance bioassay on rockwool,F. oxysporum andV. lecanii suppressed the disease by the apparent induction of systemic disease resistance. In pot bioassays with thePseudomonas spp. strains, the pseudobactin-minus mutant 358PSB- did not suppress fusarium wilt, whereas its wild type strain (WCS358) suppressed disease presumably by siderophore-mediated competition for iron. The wild type strains of WCS374 and WCS417, as well as their pseudobactin-minus mutants 374PSB- and 417PSB- suppressed fusarium wilt. The latter is best explained by the fact that these strains are able to induce systemic resistance in radish, which operates as an additional mode of action. Co-inoculation in pot bioassays, ofA. rutilum, F. oxysporum orV. lecanii with thePseudomonas spp. WCS358, WCS374 or WCS417, or their pseudobactin-minus mutants, significantly suppressed disease (except forA. rutilum/417PSB- and all combinations with 358PSB-), compared with the control treatment, if the microorganisms were applied in inoculum densities which were ineffective in suppressing disease as separate inocula. If one or both of the microorganism(s) of each combination were applied as separate inocula in a density which suppressed disease, no additional suppression of disease was observed by the combination. The advantage of the co-inoculation is that combined populations significantly suppressed disease even when their individual population density was too low to do so. This may provide more consistent biological control. The co-inoculation effect obtained in the pot bioassays suggests that co-operation ofP. fluorescens WCS374 and indigenous antagonists could have been involved in the suppression of fusarium wilt of radish in the commercial greenhouse trials.
Two models, based on the hyperbolic saturation relationship and probit function, that relate the densities of the biocontrol agent and pathogen with disease response were developed. The hyperbolic saturation (HS) and probit (PB) models and a modification of the negative exponential (MNE) model recently proposed by Johnson were evaluated. In the NE model, parameters c and k supply information on the efficiency of the pathogen and biocontrol agent, respectively, and provide endpoint values such as the maximum proportion of pathogen inoculum inactivated by the biocontrol agent (B). The HS model gives information on the 50% effective dose (ED(50)) for both the pathogen (K-x) and the biocontrol agent (K-z), asymptotic disease levels without biological control (Y-max), and the maximum proportion of pathogen the biocontrol agent can inactivate (I-max). The PB model provides information on the relative virulence of the pathogen (tau) and relative efficiency of the biocontrol agent (sigma) and on the ED(50) for the pathogen (lambda) and biocontrol agent (mu). Two patho-systems (an aerial and a root disease) and two types of biocontrol agent (antagonistic bacteria and nonpathogenic isolates of the pathogen) were compared. The data from Mandeel and Baker on biological control of Fusarium oxysporum f. sp. cucumerinum on cucumber with nonpathogenic isolates as well as our data on biological control of Stemphylium vesicarium on pear with selected isolates of Pseudomonas and Erwinia spp. were used. Data sets of the effect of several densities of the biocontrol agent and pathogen on disease levels were fitted to the models by nonlinear regression. Estimated parameters permitted quantitative comparisons among biocontrol-pathogen-host systems. The most valuable parameters obtained from the data sets that fitted adequately to the models were the proportion of pathogen potentially inactivated by the biocontrol agent, the relative efficiency and ED(50) of the biocontrol agent, and the ED(50) biocontrol/pathogen ratio. The values of B ranged from 0.79 to 0.98, and the values of I-max ranged from 0.96 to 1.04, indicating that a high proportion of the pathogen inoculum was susceptible to inactivation by the biocontrol agents. The values of sigma ranged from 0.7 to 1.4 and were consistent with the independent action of the biocontrol agents on the pathogen. The mean ED(50) for the biocontrol agent (HS and PB models) for the fungus-fungus pathogen-biocontrol system was 2 x 10(3) CFU/g of soil, and for the bacteria-fungus pathogen-biocontrol system, it was 6 x 10(6) CFU/ml. The ED(50) biocontrol/pathogen ratio ranged from 1 to 10 for the fungus-fungus biocontrol-pathogen system and from 77 to 435 for the antagonistic bacteria-fungus system, indicating the existence of a lower number of targets for fungus-fungus competition for common sites than with bacteria-fungus antagonism.
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.
Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.
The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.
The in vitro production of chitinases and beta-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO(3). The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for beta-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and beta-1,3-glucanases were detected. S. elegans culture filtrates, possessing beta-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.
Chitinase and beta-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and beta-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and beta-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and beta-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by beta-1,3-glucanase alone. However, combinations of purified chitinase and beta-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and beta-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.
The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4-beta-chitobiosidase, and glucan N-acetyl-beta-D-glucosaminidase, as well as glucan 1,3-beta-glucosidase activity. An endochitinase was purified to homogeneity. The enzyme had a molecular weight of approximately 41,000 Da and a pI of 7.8. The optimal range for enzyme activity was pH 4-6. The inhibitory effect of pure endochitinase and gliotoxin on the germination of conidia and germ tube elongation of Botrytis cinerea was tested in vitro. When applied alone at concentrations of 150 mug ml-1, the endochitinase inhibited spore germination of the test fungus and caused cell wall damage, resulting in bursting of hyphal tips. The ED50 value of gliotoxin was 1.25 mug ml-1. When gliotoxin and the endochitinase were applied together, a synergistic inhibitory effect was observed. Addition of 25 or 50 mug ml-1 of endochitinase reduced the ED50 of gliotoxin to 0.75 mug ml-1 and 0.5 mug ml-1, respectively. These enzyme concentrations applied alone showed no inhibitory effect. Furthermore, applied individually, 0.75 mug ml-1 of gliotoxin and 75 mug ml-1 of endochitinase caused no inhibition and 20% inhibition, respectively, whereas the combined application resulted in 95% inhibition. Synergistic antifungal activity of endochitinase and gliotoxin may play a role in biological control by G. virens.
An antagonistic rhizoplane bacterium (Serratia Marcescens) and a chitin-degrading microbe (Streptomyces anulatus) were used for establishing a binary microbe system for biological control of Fusarium wilt disease of tomato. For effectively tracing root-colonization, the rhizoplane bacterium was genetically marked with the lux genes and the tetracycline resistance gene through bacterial conjugation. Successful colonization by the bacterium on roots from bacterized seeds was visualized by bioluminescence of the transconjugant into which the lux gene operon had been integrated. Antifungal activity of the transconjugant was stable and effectire even when the bacterium was colonized on tomato roots. Multiplication of the transconjugant in soil was supported by the chitin-degrading microbe which had been previously added in chitin-supplemented soil. The chitin-degrading microbe produced extracellular endochitinases and hydrolyzed chitin which was added to soil. Chitohydrolytic products supported the multiplication and root-colonization of the rhizoplane bacterium in soil. The binary microbe system comprising of chitin-degrading microbe and gene-marked transconjugant was effective in controlling of tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici.
Fluorescent Pseudomonas strains were tested alone and in combinations for the ability to suppress take-all in greenhouse and growth chamber tests and to enhance the growth and yield of wheat in fields infested with Gaeumannomyces graminis var. tritici. In general, certain combinations of strains enhanced yield, whereas other mixtures and strains used individually did not. Overall, the best combinations were Q2-87 plus Q1c-80 plus Q8d-80 plus Q6Sc-80, Q2-87 plus Q1c-80 plus Q8d-80 plus Q69c-80, and Q1c-80 plus Q8d-80 plus Q6Sc-80. For example, in a field test on spring-sown wheat at Pullman, WA, during 1989, the treatment Q2-87 plus Q1c-80 plus Q8d-80 plus Q6Sc-80 significantly (P=0.05) increased yield 20.4% compared to a nontreated control, whereas each strain used individually had no significant effect on yield
Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-β-D-glucosaminidases, chitin 1,4-β-chitobiosidases, and an endochitinase. Chitabiosidases and an endochitinase were purified from dialyzed, concentrated culture filtrates using gel filtration, chromatofocusing, and isoelectric focusing. Three protein bands were evident in the purified chitobiosidase preparation, representing different levels of N-glycosylation of the same protein. The pI of all purified proteins was ∼3.9 []
Biological activity of chitosan on Pythium rot of cucumber was investigated. Growing cucumber plants in the presence of chitosan (100 or 400 μg/ml) controlled root rot caused by Pythium aphanidermatum and triggered several host defense responses, including the induction of structural barriers in root tissues and the stimulation of antifungal hydrolases (chitinase, chitosanase, and β-1,3-glucanase) in both the roots and leaves. Whereas chitosan did not cause any apparent phytotoxicity to cucumber plants, it adversely affected the growth of P. aplnanidermatum. A close examination of hyphal cells revealed that chitosan caused wall loosening, vacuolation, and, in some cases, protoplasm disintegration [...]
The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4β-chitobiosidase, and glucan N-acetyl-β-D-glucosaminidase, as well as glucan 1,3-β-glucosidase activity. An endochitinase was purified to homogeneity []
Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED 50 values for the endochitinase and the chitobiosidase were 35-135 μg ml -1 and 62-180 μg ml -1 , respectively. Complete inhibition occurred at 200-300 μg ml -1 []
A strain of Trichoderma, isolated from a sample of peat collected in New Brunswick, displays the ability to produce chitinases and inhibits growth of the pathogenic fungus, Fusarium oxysporum f. sp. radicis-lycopersici, in dual culture tests. Scanning electron microscope investigations of hyphal interactions show that growth inhibition of the host mycelium does not result from hyphal penetration by Trichoderma (...)
A chitinolytic isolate of Aeromonas caviae was isolated from roots of healthy bean plants grown in soil artificially infested with Sclerotium rolfsii. Under greenhouse conditions, the bacterium controlled Rhizoctonia solani and Fusarium oxysporum f.sp. vasinfectum in cotton (78 and 57% disease reduction, respectively) and S. rolfsii in beans (60% disease reduction). Seed coating was the most effective application method for controlling R. solani in cotton. There was no evidence of inhibition of the fungal pathogens by A. caviae. A caviae partially lysed live mycelium of R. solani, S. rolfsii and F. oxysporum f.sp. vasinfectum when their mycelium served as a sole carbon source in liquid medium. A high chitinolytic activity was found when colloidal chitin was used as a sole carbon source, with an optimum pH between 6.0–7.0. No β-3-glucanase was produced by the bacterium. After partial purification of the enzyme by affinity adsorption to colloidal chitin, three bands appear in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). One strong band with a molecular weight of ca. 80 kDa, and two weak bands with molecular weights of 48 and 59 kDa. Using the chromogenic substrate pNp-chitobiose, the partially purified chitinase from A. caviae was shown to act in an exo-splitting manner.
The chitinolytic system of the biocontrol agent Trichoderma harzianum is made up of two β-1,4-N-acetylglucosaminidases and four endochitinases. The expression of the various N-acetylglucosaminidases and endochitinases during mycoparasitism was found to be regulated in a very specific and finely tuned manner that was affected by the host. When T. harzianum was antagonizing Sclerotium rolfsii, an N-acetylglucosaminidase of 102 kDa (CHIT 102) was the first to be induced. As early as 12 h after contact, its activity diminished, and another N-acetylglucosaminidase of 73 kDa (CHIT 73) was expressed at high levels. However, when T. harzianum was antagonizing Rhizoctonia solani, the chitinase expression patterns differed considerably. Twelve hours after contact, CHIT 102 activity was elevated, and the activities of three additional endochitinases at 52 kDa (CHIT 52), 42 kDa (CHIT 42), and 33 kDa (CHIT 33) were detected. As the antagonistic interaction proceeded, CHIT 102 activity decreased, whereas the activity of the endochitinases gradually increased. The differential expression of T. harzianum chitinases may influence the overall antagonistic ability of the fungus against a specific host.
Two nonpathogenic strains of Fusarium oxysporum (C5 and C14) introduced into soil at 1.08 X 10(4) and 5 X 10(4) colony-forming units (cfu) per gram of soil significantly decreased the slope values of inoculum density-disease incidence curves generated for Fusarium wilt of cucumber induced by F. oxysporum f. sp. cucumerinum. Strain C14 reduced disease significantly more than C5. A three-phase mechanism was associated with biological control of Fusarium wilt by these agent. There was a significant reduction in germination of chlamydospores of F. o. cucumerinum in the rhizopheres of cucumbers infested with C5 or C14. Competition for infection sites with F.o. cucumerinum was demonstrated in soil infested with C14 but not C5. Enhanced systemic resistance of the host to inoculation with F. o. cucumerinum or Colletotrichum lagenarium occurred when wounded or nonwounded roots wer exposed to C14. Strain C5 significantly reduced disease induced by F. o. cucumerinum in similar experiments only if roots were wounded when the agents were introduced. When roots were exposed to either C5 or C14 and microconidia of F.o. cucumerinum applied to transversely severed stems of cucumber, significant reduction in germination of the pathogen occurred 72-96 hr after introduction of the biocontrol agents; however, wounding of roots was necessary for C5 to induce the response. Therefore, mechanisms of suppression of Fusarium wilt of cucumber by these nonpathogenic agents involved competition in the rhizosphere and infection sites, as well as induction of enhanced resistance in the host. Apparently, the ability of C14 to compete for infection sites, penetrate through intact root tissue, colonize the host, and induce resistance reactions in the host were attributes that contributed to its superior performance in biological control in comparison with C5.
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.
Rochelle salt, normally present in the dinitrosalicylic acid reagent for reducing sugar, interferes with the protective action of the sulfite, but is essential to color stability. The difficulty may be resolved either by eliminating Rochelle salt from the reagent and adding it to the mixture of reducing sugar and reagent after the color is developed, or by adding known amounts of glucose to the samples of reducing sugar to compensate for the losses sustained in the presence of the Rochelle salt. The optimal composition of a modified dinitrosalicylic acid reagent is given.
ABSTRACT Eighteen bacterial strains were individually assayed against Gibberella pulicaris (5 x 10(5) conidia per ml) by coinoculating antagonist and pathogen in wounds in cv. Russet Burbank potatoes. All antagonist concentrations (10(6), 10(7), and 10(8) CFU/ml) decreased disease (38 to 76% versus control, P < 0.05). When four strains were assayed at 11 concentrations (range 10(5) to 10(8) CFU/ml) against G. pulicaris, linear regression of the log-dose, log-response data was significant for all four strains (P < 0.001 to 0.01, R(2) = 0.50 to 0.74). Challenging G. pulicaris with all possible antagonist pairings within 2 sets of 10 antagonist strains (5 x 10(5) CFU of each strain per ml) resulted in 16 of 90 pairs controlling disease better than predicted based on averaging the performance of the individual strains making up the pair (P < 0.10). Successful pairs reduced disease by ~70% versus controls, a level of control comparable to that obtained with 100 times the inoculum dose of a single antagonist strain. Neither strain genus nor soil of origin were useful in predicting successful antagonist pairs. Factors potentially influencing dose-response relationships and the effectiveness of antagonist pairs in controlling disease are discussed.
ABSTRACT Ten wild-type strains and two benomyl-resistant mutants of Talaromyces flavus were examined for their ability to secrete the cell wall-degrading enzymes chitinase, beta-1,3-glucanase, and cellulase, to parasitize sclerotia of Sclerotium rolfsii, to reduce bean stem rot caused by S. rolfsii, and to secrete antifungal substance(s) active against Verticillium dahliae. The benomyl-resistant mutant Ben(R)TF1-R6 overproduced extracellular enzymes and exhibited enhanced antagonistic activity against S. rolfsii and V. dahliae compared to the wild-type strains and other mu tants. Correlation analyses between the extracellular enzymatic activities of different isolates of T. flavus and their ability to antagonize S. rolfsii indicated that mycoparasitism by T. flavus and biological control of S rolfsii were related to the chitinase activity of T. flavus. On the other hand, production of antifungal compounds and glucose-oxidase activity may play a role in antagonism of V. dahliae by retardation of germination and hyphal growth and melanization of newly formed microsclerotia.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.