Trichoderma Harzianum Strain Research Articles

Novel culture chamber to evaluate in vitro plant-microbe volatile interactions: Effects of Trichoderma harzianum volatiles on wheat plantlets

The field of plant-microbe interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) still faces several limitations due to the lack of reliable equipment. We present a novel device designed to evaluate in vitro plant-microbe volatile interactions, the plant-microbe VOC Chamber. It was tested by evaluating the effects exerted on wheat development by volatiles from three Trichoderma harzianum strains, a wild type and two genetically modified strains; one expressing the tri5 gene, which leads to the synthesis and emission of the volatile trichodiene, and the other by silencing the erg1 gene, impairing ergosterol production. The wild type and the erg1-silenced strain enhanced fresh weight and length of the aerial part, but reduced root dry weight. Interestingly, no differences were found between them. Conversely, the tri5-transformant strain reduced root and aerial growth compared to the control and the other strains. No differences were observed regarding chlorophyll fluorescence quantum yield and leaf chlorophyll content, suggesting that the released BVOCs do not interfere with photosynthesis. The plant-microbe VOC Chamber proved to be a simple and reliable method to evaluate the in vitro effects of microbial BVOCs on plant development, perfect for the screening of microorganisms with interesting volatile traits.

Volatile-mediated interactions between Trichoderma harzianum and Acanthoscelides obtectus: A novel in vitro methodology to evaluate the impact of microbial volatile compounds on dry grain storage pests

Biological interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) is a well-established field that has been researched for decades. Although extensive focus is currently given to the control of insect pests using natural molecules, the study of volatile interactions between microorganisms and insects has been largely neglected and has only begun to attract more attention in recent years. In this work, we developed a novel protocol to assess the effects of microbial BVOCs directly produced by growing microbial strains on dry grain insect pests and the seed damage they cause, using VOC Chambers to evaluate both sealed and unsealed conditions. Four Trichoderma harzianum strains were tested against Acanthoscelides obtectus, a wild type and three of its transformants. These had been previously obtained by introducing the tri5 gene and thus overproducing the volatile trichodiene, or by silencing the erg1 gene, which encodes for a squalene epoxidase, therefore reducing ergosterol levels and increasing squalene ones in the fungus. Results demonstrated that ventilation plays a key role in these interactions. All fungal strains significantly increased adult mortality in sealed conditions, while this effect was barely noticeable in unsealed ones. Nevertheless, subsequent insect emergence from bean seeds and bean damage were still significantly reduced in both conditions. The erg1 silenced strains caused significantly higher levels of adult mortality than the rest in sealed conditions and lower insect emergence in both sealed and unsealed ones. Bean damage produced by insects was lower also when exposed to BVOCs from these strains in sealed conditions. Conversely, trichodiene overproduction did not show enhanced toxicity or significant reduction of insect emergence and bean damage in the tested conditions. Therefore, T. harzianum BVOCs, especially those from erg1 silenced strains, should be further researched for their potential use in the biological control of A. obtectus infestation in dry grain storing facilities. VOC Chambers have shown themselves to be a reliable method in the screening of in vitro volatile mediated interactions between growing microbial strains and insect pests.

Mitochondrial Genome Resource of a Grapevine Strain of Trichoderma harzianum, a Potential Biological Control Agent for Fungal Canker Diseases

Trichoderma spp. are commonly used as bioremediation agents, biological controls, and for making biofuels. Herein, a Trichoderma harzianum strain PAR3 was isolated from grapevine roots in central California, USA. As part of a larger whole genome sequencing effort, the mitochondrial genome (mitogenome) sequence was obtained for the PAR3 strain. The mitogenome is 27,631 bp containing genes of 14 core mitochondrial proteins, 25 tRNA, and two rRNAs and a GC% content of 27.55%. BLAST search using the PAR3 mitogenome as query against GenBank sequence database showed mitogenome MUT3171 (29,791 bp) of Trichoderma lixii was the most similar (Query Coverage = 99%; Percentage Identity=100.00%) with two major deletions, 1,339 bp and 821 bp. The PAR3 mitogenome sequence will provide a useful reference for comparing different Trichoderma strains from US and around the world.

Kinetic Study of Fungal Growth of Several Tanninolytic Strains Using Coffee Pulp Procyanidins

Procyanidins are bioactive molecules with industrial and pharmaceutical relevance, they are present in recalcitrant agro-industrial wastes that are difficult to degrade. In this study, we evaluated the potential consumption of procyanidins from Aspergillus niger and Trichoderma harzianum strains in submerged fermentations. For this purpose, a culture medium containing salts, glucose, and procyanidins was formulated, where procyanidins were added to the medium after the near-total consumption of glucose. The submerged cultures were carried out in amber flasks at 30 °C and 120 rpm. The addition of procyanidins to the culture medium increased the formation of micellar biomass for all the strains used. The use of glucose affected the growth of A. niger GH1 and A. niger HS1, however, in these assays, a total consumption of procyanidins was obtained. These results show that the consumption of procyanidins by fungal strains in submerged fermentations was influenced by the pH, the use of glucose as the first source of carbon, and the delayed addition of procyanidins to the medium. The study showed that A. niger and T. harzianum strains can be used as a natural strategy for the consumption or removal of procyanidins present in recalcitrant residues of risk to the environment and human health.

Correction to: A native Trichoderma harzianum strain Th62 displays antagonistic activities against phytopathogenic fungi and promotes the growth of Celosia cristata

Correction to: A native Trichoderma harzianum strain Th62 displays antagonistic activities against phytopathogenic fungi and promotes the growth of Celosia cristata

Fungi Associated with Postharvest Diseases of Sweet Potato Storage Roots and In Vitro Antagonistic Assay of Trichodermaharzianum against the Diseases.

Sweet potato is the 11th most important food crop in the world and an excellent source of nutrition. Postharvest diseases were monitored in sweet potato storage roots collected from the local markets in Korea during 2021. Several diseases including Fusarium surface and root rot, charcoal rot, dry rot, and soft rot were observed in the postharvest sweet potatoes. A total of 68 fungal isolates were obtained from the diseased samples, and the isolates were grouped into 8 different fungal colony types. Based on multilocus phylogeny and morphological analysis of 17 representative isolates, the isolates were identified as Fusarium oxysporum, F. ipomoeae, F. solani, Penicillium citrinum, P. rotoruae, Aspergillus wentii, Mucor variicolumellatus (Mu. circinelloides species complex), and Macrophomina phaseolina. F. oxysporum was the predominant pathogen as this is the most common pathogen of sweet potato storage roots causing the surface rot disease, and M. phaseolina caused the most severe disease among the pathogens. Dual culture antagonistic assays were evaluated using Trichoderma harzianum strains CMML20–26 and CMML20–27. The results revealed that the two strains showed strong antifungal activity in different ranges against all tested pathogens. This study provides an understanding of diverse postharvest diseases in sweet potatoes and suggests potential biocontrol agents to manage the diseases. In addition, this is the first report of sweet potato storage root rot diseases caused by A. wentii, and P. rotoruae worldwide.

Molecular characteristics of a novel hypovirus from Trichoderma harzianum.

We report a novel mycovirus with a positive-sense single-stranded (+)ss RNA genome, belonging to the family Hypoviridae, infecting Trichoderma harzianum strain M6. The complete genome sequence is 13,813 nucleotides long, excluding the poly(A) tail at the 3' end. Sequence analysis revealed that the genome has a single large open reading frame (ORF) encoding a 4,118-amino-acid polyprotein harboring five conserved motifs of a protease, two conserved domains of a protein of unknown function, an RNA-dependent RNA polymerase, and a helicase. Sequence comparisons revealed that the deduced amino acid sequence of the polyprotein is similar to those of other hypoviruses and is most similar to that of Bipolaris oryzae hypovirus 1 (35.1% identity). Phylogenetic analysis using full-length RdRp and helicase sequences showed that this virus clustered closely with known members of the proposed genus "Alphahypovirus" of the family Hypoviridae. We accordingly designated this novel mycovirus "Trichoderma harzianum hypovirus 2" (ThHV2).

Improvement of Nutraceutical Value of Parsley Leaves (Petroselinum crispum) upon Field Applications of Beneficial Microorganisms

Parsley (Petroselinum crispum) is an important aromatic herb that has gained importance in food and cosmetic industry, and it is used as medicinal plant due to the presence of compounds with biological activity. Several studies have demonstrated antioxidant, antimicrobial or cancer chemopreventive activity of different parts of parsley plants. We showed that the nutritional value of parsley leaves can be improved by treatments with beneficial microorganisms on the field crop. Streptomyces fulvissimus strain AtB-42 and Trichoderma harzianum strain T22 were applied, as singly or in combination (microbial consortium), at transplanting and two weeks later. After harvesting, plants were subjected to metabolomic analysis by LC and GC-MS. Spectrometric analysis resulted in the identification of seven polar metabolites. Results showed a significant difference in relative abundance of these metabolites among treatments. The AtB-42 application, alone or in combination with T22, induced the accumulation of petroselinic acid, while T22, alone or in combination, induced the accumulation of xanthotoxol/bergaptol and its derivative xanthotoxin/bergapten. The microbial consortium increased the accumulation of capsanthone compared to single treatments. No statistically relevant differences were found for the volatile fraction. It can be concluded that S. fulvissimus and T. harzianum significantly induced metabolic profile change of parsley and the accumulation of metabolites with nutraceutical value.

A microbial consortium-based product promotes potato yield by recruiting rhizosphere bacteria involved in nitrogen and carbon metabolisms.

SummaryThe effect of a microbial consortium‐based (MCB) biocontrol product, composed of Bacillus subtilis, Trichoderma harzianum strain and diatomaceous earth as a carrier, on potato yield, and potential modes of action for its effect were investigated. The MCB product (300 kg ha−1) was added to furrows in which the potato seed tubers each year for 3 years (2016, 2017 and 2018), while potato planting without the MCB product treatment served as the control. A metagenomic analysis indicated that bacterial phylotypes dominated the microbial community, with a relatively small contribution of archaea and fungal taxa. The relative abundance of beneficial bacterial taxa increased significantly in response to the MCB product treatment. Notably, a higher relative abundance of bacterial taxa with carbon fixation, carbon‐degrading and nitrogen metabolism properties were observed in the MCB product‐treated potato rhizosphere. This was also reflected in the identification of a greater abundance of genes encoding enzymes involved in nitrogen metabolism, carbon fixation and carbon degradation pathways in the conducted metagenomic analysis. The greater relative abundance of these beneficial bacterial taxa in the rhizosphere of MCB product‐treated plots, as well as the higher abundance of genes associated with the indicated cellular processes, were associated with an increase in tuber yield. The observed changes in microbial community structure at an early stage of tuber development appears to have a beneficial impact on tuber yield.

Bioremoval and tolerance study of sulfamethoxazole using whole cell Trichoderma harzianum isolated from rotten tree bark

Antibiotic contamination raises concerns over antibiotic resistance genes (ARGs), which can severely impact the human health and environment. Sulfamethoxazole (SMX) is a widely used antibiotic that is incompletely metabolized in the body. In this study, the research objectives were (1) to isolate the native strain of Trichoderma sp. from the environment and analyze the tolerance toward SMX concentration by evaluating fungal growth, and (2) to investigate the potential of SMX removal by fungi. The potential fungi isolated from rotten tree bark showed 97% similarity to Trichoderma harzianum (Accession no. MH707098.1). The whole cell of fungi was examined in vitro; the strain Trichoderma harzianum BGP115 eliminated 71% of SMX after 7 days, while the white rot fungi Trametes versicolor, demonstrated 90% removal after 10 days. Furthermore, the tolerance of fungal growth toward SMX concentration at 10 mg L−1 was analyzed, which indicated that Trichoderma harzianum BGP115 (the screened strain) exhibited more tolerance toward SMX than Trametes versicolor (the reference strain). The screened fungi isolated from rotted tree bark demonstrated the ability of SMX bioremoval and the potential to be tolerant to high concentrations of SMX.

Arabidopsis Restricts Sugar Loss to a Colonizing Trichoderma harzianum Strain by Downregulating SWEET11 and -12 and Upregulation of SUC1 and SWEET2 in the Roots.

Phosphate (Pi) availability has a strong influence on the symbiotic interaction between Arabidopsis and a recently described root-colonizing beneficial Trichoderma harzianum strain. When transferred to media with insoluble Ca3(PO4)2 as a sole Pi source, Arabidopsis seedlings died after 10 days. Trichoderma grew on the medium containing Ca3(PO4)2 and the fungus did colonize in roots, stems, and shoots of the host. The efficiency of the photosynthetic electron transport of the colonized seedlings grown on Ca3(PO4)2 medium was reduced and the seedlings died earlier, indicating that the fungus exerts an additional stress to the plant. Interestingly, the fungus initially alleviated the Pi starvation response and did not activate defense responses against the hyphal propagation. However, in colonized roots, the sucrose transporter genes SWEET11 and -12 were strongly down-regulated, restricting the unloading of sucrose from the phloem parenchyma cells to the apoplast. Simultaneously, up-regulation of SUC1 promoted sucrose uptake from the apoplast into the parenchyma cells and of SWEET2 sequestration of sucrose in the vacuole of the root cells. We propose that the fungus tries to escape from the Ca3(PO4)2 medium and colonizes the entire host. To prevent excessive sugar consumption by the propagating hyphae, the host restricts sugar availability in its apoplastic root space by downregulating sugar transporter genes for phloem unloading, and by upregulating transporter genes which maintain the sugar in the root cells.

SCREENING, ISOLATION AND CHARACTERIZATION OF HEAT STRESS TOLERANT TRICHODERMA ISOLATES: SUSTAINABLE ALTERNATIVE TO CLIMATE CHANGE

To provide food security with quality crops for exponentially growing population brought intense pressure on the limited land and natural resources among developing countries. Abiotic stresses such as continuously rising temperature as consequences of global warming is adding pressure to existing problems by adversely affecting crop productivity through physiological changes in plants. Hence there is need of qualitatively potential vegetable crops that can withstand changing environmental conditions such as Okra (Abelmoschus spp.) holding high level of nutrients along with economic importance. But requirement of high temperature and humidity for its cultivation make the plant prone to several phytopathogens that ultimately leads to severe qualitative and quantitative losses depending upon the plant growth stage getting affected. Due to the unenviable problems of chemical fertilizers, biocontrol agents were applied as auxiliary treatments either single or in combination that possess fewer consequences on the environment. But due to increasing environmental and soil temperature, activities of these formulations are getting hindered. Therefore, there is an urgent requirement to procure high temperature stress tolerant strains along with antagonistic and plant growth promoting abilities. In the current study, we mainly focused on isolation of high temperature tolerant Trichoderma harzianum (BHU P4) strain with antagonistic abilities against fungal pathogen Sclerotium rolfsii causing collar rot disease. The strain was also studied for plant growth promoting attributes in okra plant which resulted in increased fresh weight, dry weight, chlorophyll content and nutrient content in comparison to control and pathogen challenged plants. This study was associated with an improvement in the level of total phenol, SOD, PO and PAL enzymes in order to regulate the host defense mechanism against its confrontation with S. rolfsii.

Trichoderma harzianum Strain T22 Modulates Direct Defense of Tomato Plants in Response to Nezara viridula Feeding Activity

Plant growth-promoting fungi belonging to genus Trichoderma are known to help plants when dealing with biotic stressors by enhancing plant defenses. While beneficial effects of Trichoderma spp. against plant pathogens have long been documented, fewer studies have investigated their effect on insect pests. Here, we studied the impact of Trichoderma root colonization on the plant defense responses against stink bug feeding attack. For this purpose, a model system consisting of tomato plant, Solanum lycopersicum cv Dwarf San Marzano, Trichoderma harzianum strain T22 and the southern green stink bug, Nezara viridula, was used. We firstly determined stink bug performance in terms of relative growth rate and survival on tomato plants inoculated by T. harzianum T22. Then, we evaluated relative expression of plant defense-related genes on inoculated plants induced by stink bug feeding. We found evidence that T. harzianum T22 affects tomato defense responses against N. viridula nymphs leading to reduction of growth rate. Our results also showed that T. harzianum T22 enhances plant direct defenses by an early increase of transcript levels of jasmonic acid marker genes. Yet this effect was time-dependent and only detected 8 h after herbivore induction. Taken together, our findings provide better understanding on the mechanisms underlying tomato induced resistance against herbivorous stink bugs.

Biostimulant Activity of Azotobacter chroococcum and Trichoderma harzianum in Durum Wheat under Water and Nitrogen Deficiency

Biostimulants hold great potential for developing integrated sustainable agriculture systems. The rhizobacteria Azotobacter chroococcum strain 76A and the fungus Trichoderma harzianum strain T22, with demonstrated biostimulant activity in previous systems, were evaluated in Triticum durum cv Creso for their ability to enhance growth and tolerance to drought stress. Growth and drought tolerance were evaluated in conditions of low and high soil nitrogen, with two levels of water stress. T. harzianum increased plant growth (+16%) under control conditions and tolerance to moderate drought stress (+52%) under optimal fertilization, while A. chroococcum conferred a growth penalty (−28%) in well-watered conditions under suboptimal fertilization and increased tolerance only under extreme drought stress (+15%). This growth penalty was ameliorated by nitrogen fertilization. T. harzianum abundance was found to be positively correlated to extreme soil drying, whereas A. chroococcum-induced tolerance was dependent on soil nitrogen availability. These results indicate that while biostimulants may enhance growth and stress tolerance, nutrient availability soil and environmental conditions heavily influence these responses. These interactions should be considered when designing biostimulant products targeted to specific cultural conditions.

Mycoparasitic antagonism of a Trichoderma harzianum strain isolated from banana plants in Oaxaca, Mexico

Bananas are important crops in developing countries with tropical climate. In Mexico, the banana production has increased, and it must be guaranteed. The Panama disease, caused by the fungus Fusarium oxysporum f.sp. cubense threatens the current banana production, for what is necessary to implement methods to protect this crop. Fungi from genus Trichoderma are natural residents of the rhizosphere. This genus comprises mycoparasite species used to control diseases caused by phytopathogenic fungi, and also benefit plant development. In this report, we present data of the identification and characterization of the novel strain Trichoderma harzianum M110 that displays antagonism and biocontrol potential in laboratory conditions. Exploration of the rhizosphere and the endophytic microbial communities might help to identify microbes adapted to banana plants that can be incorporated in organic biological control formulations that ensure production of Fusarium-free plants and healthy fruits with export quality.

Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat.

Fusarium graminearum is the primary cause of Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. FHB reduces yield and contaminates grain with the trichothecene mycotoxin deoxynivalenol (DON), which poses a risk to plant, human and animal health. The first committed step in trichothecene biosynthesis is formation of trichodiene (TD). The volatile nature of TD suggests that it could be a useful intra or interspecies signalling molecule, but little is known about the potential signalling role of TD during F. graminearum-wheat interactions. Previous work using a transgenic Trichoderma harzianum strain engineered to emit TD (Th + TRI5) indicated that TD can function as a signal that can modulate pathogen virulence and host plant resistance. Herein, we demonstrate that Th + TRI5 has enhanced biocontrol activity against F. graminearum and reduced DON contamination by 66% and 70% in a moderately resistant and a susceptible cultivar, respectively. While Th + TRI5 volatiles significantly influenced the expression of the pathogenesis-related 1 (PR1) gene, the effect was dependent on cultivar. Th + TRI5 volatiles strongly reduced DON production in F. graminearum plate cultures and downregulated the expression of TRI genes. Finally, we confirm that TD fumigation reduced DON accumulation in a detached wheat head assay.

A native Trichoderma harzianum strain Th62 displays antagonistic activities against phytopathogenic fungi and promotes the growth of Celosia cristata

Trichoderma spp. are widely applied, eco-friendly mycofungicides and plant growth promoters. Native Trichoderma strains are likely to have more productive, stable biocontrol and biofertilizer activities since they are well adapted to the local environment. In this study, we isolated a native Trichoderma strain ‘Th62’ from the rhizosphere soil of wild Chelidonium majus plants in Harbin, China (126.6341°E, 45.7242°N). The isolated Trichoderma strain was identified as a T. harzianum strain via morphological observation and molecular methods based on the rDNA internal transcribed spacer region and elongation factor-1α gene sequences. Significant antagonistic activities of Th62 against five soil-borne fungal phytopathogens, Fusarium oxysporum, Sclerotinia sclerotiorum, Alternaria alternata, Cytospora chrysosperma, and Rhizoctonia solani, were confirmed by dual-culture assays. Furthermore, the crude fermentation products of Th62 also displayed antifungal activities against these five pathogens. To evaluate the function of Th62 as a biofertilizer, we subsequently applied Th62 on cockscomb (Celosia cristata L), a plant species with both ornamental and medicinal values, by inoculation with Th62 conidia at different concentrations, 1 × 1010 cfu mL−1, 1 × 1011 cfu mL−1, and 1 × 1012 cfu mL−1. The beneficial effects of Th62 were evaluated by measuring the growth and photosynthetic traits of the inoculated cockscomb plants, and the results demonstrated that Th62 significantly improved the photosynthetic efficiency, photosynthetic capacity, and the adaptability to intense light of the inoculated cockscomb plants compared to the controls. Consistently, Th62 inoculation significantly improved the growth and flower yield of cockscomb. We presented a positive case of isolating and applying native microbial resources on local plantation practices.

Isolation, screening, and molecular identification of lignin-degrading fungi isolates from Cibinong Science Centre, Indonesia Institute of Sciences, Cibinong, West Java

Lignin is a recalcitrant compound that has potential as fuels and chemicals on industries. Contamination of recalcitrant compound with the lignin-like structure on the ecosystem is being concerned worldwide. Fungi is the most targeted organisms with lignin-degrading ability, can secrets laccase and peroxidase to facilitate free radical chain reaction on lignin complex structure. Isolation, screening and identification for isolate with ligninolytic ability is the main purpose of this project. Phylum Ascomycota and Basidiomycota be the main target of this research. Isolation technics that being used are surface sterilization with distilled water and NaClO on PDA medium. Pure culture with ability to grow on lignin minimal medium with black liquor as the only carbon sources then are identified by molecular identification. Isolates that can grow on lignin minimal medium are identified as Fusarium verticillioides strain CBS 127178, Lasiodiplodia sp. LAS-2016 strain CBS 125262, Trichoderma harzianum strain 35814DRJ, and Lasiodiplodia theobromae strain CBS 127106. Four isolates obtained with lignin-degrading ability then preserved by cryopreservation technic for use in the next projects.

Mycosinthetized Ag, CuO and ZnO nanoparticles from a promising Trichoderma harzianum strain and their antifungal potential against important phytopathogens

Fungal green biosynthesis of nanoparticles (NPs) is a promising eco-friendly method for mass-scale production. In the present study Ag, CuO and ZnO nanoparticles were biogenically synthetized using a cell filtrate of a strain of Trichoderma harzianum as a reducer and stabilizer agent. The structure, morphology and physicochemical properties of the NPs were characterized through transmission electron microscopy, dynamic light scattering, wide angle X-ray scattering and thermogravimetric analysis. Since nanotechnology could offer promising applications in agricultural area, we evaluated the ability of the NPs to reduce the growth of important fungal phytopathogens as Alternaria alternata, Pyricularia oryzae and Sclerotinia sclerotiorum. Silver and CuO NPs reduced significantly the mycelial growth of A. alternata and P. oryzae in a dose dependent manner. This is the first report of a multiple extracellular biosynthesis of NPs from T. harzianum and the first time that CuO and ZnO NPs were obtained from this fungus. In addition, we highlighted the rapid production of NPs, as well as, the potential of Ag and CuO for the control of phytopathogens. On the other hand, the three types of NPs could be easily and sustainably produced on a large scale with the chance of having multiple applications in biotechnological processes.

Continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h−1. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.