Maydis DNA Research Articles

Development of a qPCR Assay for Species-Specific Detection of the Tar Spot Pathogen Phyllachora maydis

Phyllachora maydis is a fungal plant pathogen that causes tar spot of corn ( Zea mays) in North and South America, causing devastating yield losses under favorable conditions. Although the causal agent is relatively easy to diagnose via macroscopic and microscopic observations, other diseases and conditions, such as insect frass, have been mistaken for tar spot of corn. Furthermore, conidia and ascospores in isolation can be difficult to visually distinguish from other fungi, and the development of signs and symptoms of the disease may not be observed until 12 to 20 days after infection. Therefore, we developed a TaqMan quantitative polymerase chain reaction (qPCR) assay for the detection and quantification of this pathogen to be used for diagnostics and airborne spore quantification. The assay was designed for the internal transcribed spacer region of P. maydis. The specificity of the assay was confirmed and tested against various nontarget Phyllachora species, corn pathogens, endophytes, and P. maydis samples from several states in the Midwest and from Mexico. The detection limit of this assay was determined to be 100 fg of genomic P. maydis DNA. To demonstrate the transferability of this technology, the assay was tested in different labs using various qPCR thermal cyclers. This assay can be used in downstream research involving latency period, disease prediction, and diagnostics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Cultivars Resistance Assay for Maize Late Wilt Disease.

Magnaporthiopsis maydis late wilt disease (LWD) in corn is considered to be the most severe in Israel and Egypt and poses a significant threat in other countries. Research efforts extending over a period of five decades led to the development of chemical, biological, agrotechnical, physical (solar disinfection) and other means for controlling late wilt disease. Today, some applications can reduce damage even in severe cases. However, cultivating disease-resistant maize varieties is the primary means for reducing the disease's impact. The current work uses a rapid (six days) laboratory seedling pathogenicity test and a full-season open encloser semi-field conditioned pots assay (101 days) to classify maize varieties according to their LWD resistance. To better evaluate differences between the cultivars, a real-time based molecular assay was applied to track the pathogen's presence in the plants' tissues, and visible light aerial imaging was used in parallel. The findings show that in cases of extreme sensitivity or tolerance (for example, in the highly susceptible Megaton cultivar (cv.) or the resistant Hatai cv.), a similarity in the results exists between the different methods. Thus, a reliable estimate of the varieties' sensitivity can be obtained in a seed assay without the need for a test carried out throughout an entire growing season. At the same time, in most situations of partial or reduced LWD sensitivity/resistance, there is no match between the various tests, and only the entire growing season can provide the most reliable results. Tracking the amount of M. maydis DNA in the plants' bodies is a precise, sensitive scientific tool of great importance for studying the development of the disease and the factors affecting it. Yet, no complete overlap exists between the fungal DNA amount and symptom severity. Such a correlation exists in high sensitivity or resistance cases but not in intermediate situations. Still, the valuation of the pathogen's establishment in asymptomatic corn hybrids can indicate the degree of LWD immunity and the chance of susceptibility development.

Trichoderma longibrachiatum and Trichoderma asperellum Confer Growth Promotion and Protection against Late Wilt Disease in the Field.

Late wilt disease (LWD) of maize, caused by Magnaporthiopsis maydis, is considered a major threat to commercial fields in Israel, Egypt, Spain, and India. Today’s control methods include chemical and agronomical intervention but rely almost solely on resistant maize cultivars. In recent years, LWD research focused on eco-friendly biological approaches to restrain the pathogen. The current study conducted during two growing seasons explores the potential of three Trichoderma species as bioprotective treatments against LWD. These species excelled in preliminary assays performed previously under controlled conditions and were applied here in the field by directly adding them to each seed with the sowing. In the first field experiment, Trichoderma longibrachiatum successfully rescued the plants’ growth indices (weight and height) compared to T. asperelloides and the non-treated control. However, it had no positive effect on yield and disease progression. In the subsequent season, this Trichoderma species was tested against T. asperellum, an endophyte isolated from susceptible maize cultivar. This experiment was conducted during a rainy autumn season, which probably led to a weak disease burst. Under these conditions, the plants in all treatment groups were vivid and had similar growth progression and yields. Nevertheless, a close symptoms inspection revealed that the T. longibrachiatum treatment resulted in a two-fold reduction in the lower stem symptoms and a 1.4-fold reduction in the cob symptoms at the end of the seasons. T. asperellum achieved 1.6- and 1.3-fold improvement in these parameters, respectively. Quantitative Real-time PCR tracking of the pathogen in the host plants’ first internode supported the symptoms’ evaluation, with 3.1- and 4.9-fold lower M. maydis DNA levels in the two Trichoderma treatments. In order to induce LWD under the autumn’s less favorable conditions, some of the plots in each treatment were inoculated additionally, 20 days after sowing, by stabbing the lower stem section near the ground with a wooden toothpick dipped in M. maydis mycelia. This infection method overrides the Trichoderma roots protection and almost abolishes the biocontrol treatments’ protective achievements. This study suggests a biological Trichoderma-based protective layer that may have significant value in mild cases of LWD.

The Microflora of Maize Grains as a Biological Barrier against the Late Wilt Causal Agent, Magnaporthiopsis maydis

The maize pathogen Magnaporthiopsis maydis causes severe damage to commercial fields in the late growth stages. This late wilt disease has spread since its discovery (the 1980s) and is now common in most corn-growing areas in Israel. In some fields and sensitive plant species, the disease can affect 100% of the plants. The M. maydis pathogen has a hidden endophytic lifecycle (developed inside the plants with no visible symptoms) in resistant corn plants and secondary hosts, such as green foxtail and cotton. As such, it may also be opportunist and attack the host in exceptional cases when conditions encourage it. This work aims to study the pathogen’s interactions with maize endophytes (which may play a part in the plant’s resistance factors). For this purpose, 11 fungal and bacterial endophytes were isolated from six sweet and fodder corn cultivars with varying susceptibility to late wilt disease. Of these, five endophytes (four species of fungi and one species of bacteria) were selected based on their ability to repress the pathogen in a plate confrontation test. The selected isolates were applied in seed inoculation and tested in pots in a growth room with the Prelude maize cultivar (a late wilt-sensitive maize hybrid) infected with the M. maydis pathogen. This assay was accompanied by real-time qPCR that enables tracking the pathogen DNA inside the host roots. After 42 days, two of the endophytes, the Trichoderma asperellum, and Chaetomium subaffine fungi, significantly (p < 0.05) improved the infected plants’ growth indices. The fungal species T. asperellum, Chaetomium cochliodes, Penicillium citrinum, and the bacteria Bacillus subtilis treatments were able to reduce the M. maydis DNA in the host plant’s roots. Studying the maize endophytes’ role in restricting the invasion and devastating impact of M. maydis is an essential initial step towards developing new measures to control the disease. Such an environmentally friendly control interface will be based on strengthening the plants’ microbiome.

Soil Bioassay for Detecting Magnaporthiopsis maydis Infestation Using a Hyper Susceptible Maize Hybrid.

Magnaporthiopsis maydis is the causal agent of severe maize late wilt disease. Disease outbreak occurs at the maize flowering and fruit development stage, leading to the plugging of the plant’s water vascular system, resulting in dehydration and collapse of the infected host plant. The pathogen is borne by alternative hosts, infected seeds, soil, and plant residues and gradually spreads to new areas and new countries. However, no soil assay is available today that can detect M. maydis infestation and study its prevalence. We recently developed a molecular quantitative Real-Time PCR (qPCR) method enabling the detection of the M. maydis DNA in plant tissues. Despite the technique’s high sensitivity, the direct examination of soil samples can be inconsistent. To face this challenge, the current work demonstrates the use of a soil bioassay involving the cultivation of a hyper-susceptible maize genotype (Megaton cultivar, Hazera Seeds Ltd., Berurim MP Shikmim, Israel) on inspected soils. The use of Megaton cv. may facilitate pathogen establishment and spread inside the plant’s tissues, and ease the isolation and enrichment of the pathogen from the soil. Indeed, this cultivar suffers from severe dehydration sudden death when grown in an infested field. The qPCR method was able to accurately and consistently identify and quantify the pathogen’s DNA in an in vitro seed assay after seven days, and in growth-chamber potted plants at as early as three weeks. These results now enable the use of this highly susceptible testing plant to validate the presence of the maize late wilt pathogen in infested soils and to evaluate the degree of its prevalence.

Interactions between Magnaporthiopsis maydis and Macrophomina phaseolina, the Causes of Wilt Diseases in Maize and Cotton.

Fungal pathogens are a significant threat to crops worldwide. The soil fungus, Magnaporthiopsis maydis, severely affects sensitive maize hybrids by causing the rapid wilting of plants at the maturity stage. Similarly, the soil fungus, Macrophomina phaseolina, develops in a variety of host plants, which leads to rot and plant mortality. The presence of both pathogens together in diseased cotton plants in Israel suggests possible interactions between them. Here, these relationships were tested in a series of experiments accompanied by real-time PCR tracking in maize and cotton. Despite the fact that neither of the pathogens was superior in a growth plate confrontation assay, their co-inoculum had a significant influence under field conditions. In maize sprouts and fully matured plants, infection by both pathogens (compared to inoculation with each of them alone) led to lesser amounts of M. maydis DNA but to increased amounts of M. phaseolina DNA levels. These results were obtained under a restricted water regime, while optimal water irrigation led to less pronounced differences. In water-stressed cotton sprouts, infection with both pathogens led to an increase in DNA amounts of each of the pathogens. Whereas the M. maydis DNA levels in the double infection remain high at the end of the season, a reduction in the amount of M. phaseolina DNA was observed. The double infection caused an increase in growth parameters in maize and cotton and decreased levels of dehydration in maize plants accompanied by an increase in yield production. Dehydration symptoms were minor in cotton under an optimal water supply. However, under a restricted water regime, the double infection abolished the harmful effect of M. phaseolina on the plants’ development and yield. These findings are the first report of interactions between these two pathogens in maize and cotton, and they encourage expanding the study to additional plant hosts and examining the potential involvement of other pathogens.

Uncovering the Host Range for Maize Pathogen Magnaporthiopsis maydis.

The fungus Magnaporthiopsis maydis is a soil-borne, seed-borne vascular wilt pathogen that causes severe damage to sensitive Zea mays L. (maize) hybrids throughout Egypt, Israel, India, Spain, and other countries. It can undergo virulence variations and survive as spores, sclerotia, or mycelia on plant residues. Maize, Lupinus termis L. (lupine) and Gossypium hirsutum L. (cotton) are the only known hosts of M. maydis. Identification of new plant hosts that can assist in the survival of the pathogen is an essential step in restricting disease outbreak and spread. Here, by field survey and growth chamber pathogenicity test, accompanied by real-time PCR analysis, the presence of the fungal DNA inside the roots of cotton (Pima cv.) plants was confirmed in infested soil. Moreover, we identified M. maydis in Setaria viridis (green foxtail) and Citrullus lanatus (watermelon, Malali cv.). Infected watermelon sprouts had delayed emergence and development, were shorter, and had reduced root and shoot biomass. M. maydis infection also affected root biomass and phenological development of cotton plants but caused only mild symptoms in green foxtail. No M. maydis DNA was detected in Hordeum vulgare (barley, Noga cv.) and the plants showed no disease symptoms except for reduced shoot weight. These findings are an important step towards uncovering the host range and endophytic behavior of M. maydis, encouraging expanding this evaluation to other plant species.

Evaluating Azoxystrobin Seed Coating Against Maize Late Wilt Disease Using a Sensitive qPCR-Based Method.

Harpophora maydis, a phytopathogenic fungus, causes late wilt, a severe vascular maize disease characterized by relatively rapid wilting of maize plants near fertilization. The disease is currently controlled using resistant varieties. Here, we evaluated seed coating efficiency with azoxystrobin against H. maydis in a series of in vitro and in vivo trials. A real-time polymerase chain reaction (qPCR)-based method was developed and proved to be a sensitive, accurate tool for monitoring H. maydis DNA inside infected seeds, sprouts, and tissues of mature plants. In the early growth stages, the chemical coating drastically reduced the pathogen DNA prevalence in host tissues and minimized the suppressing effect on the plants' biomass and development. In an infested field, the qPCR assay identified the pathogen 20 days after seeding, up to a month before conventional PCR detection. In the resistant fodder maize cultivar 32D99, which showed only minor disease symptoms, the seed coating blocked fungal progression and increased cob and plant weight by 39 and 60%, respectively. Nevertheless, this treatment was unable to protect a sensitive maize hybrid, cultivar Prelude, at the disease wilting breakout (60 days after sowing). These results encourage further examination of azoxystrobin and other fungicides in the field using the qPCR detection method to evaluate their efficiency.

Diagnosis and Control of <i>Harpophora maydis</i>, the Cause of Late Wilt in Maize

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by relatively rapid wilting of maize plants, before tasseling and until shortly before maturity. In Israel, the disease becomes a major problem in recent years. The pathogen currently controlled using varieties of maize has reduced sensitivity. In earlier work, we modified a molecular method for use as a diagnostic tool to evaluate the disease progress in field infested plants. Several fungicides suppressed H. maydis in vitro and in a detached root pathogenicity assay. Seedling pathogenicity assay enables us to identified H. maydis DNA in the host root and stem tissues 18 days after sowing in both susceptible and tolerant maize plants. Although the infested plants exhibited no wilt symptoms, their roots were significantly shorter in length. This seedling assay was used to demonstrate the suppressive effect of the fungicide Flutriafol on H. maydis virulence. The method of assaying the pathogen in a series of trials starting in a plate assay, followed by a detached root and resulting in a seedlings pathogenicity assay, using molecular and morphological approaches could be generalized to other plant pathogens.

Use of PCR to detect infection of differentially susceptible maize cultivars using Ustilago maydis strains of variable virulence.

Ustilago maydis was specifically detected in infected maize plants by means of the polymerase chain reaction (PCR) using oligonucleotides corresponding to a specific region downstream of the homeodomain of the bE genes of the pathogen. The reaction gave rise to amplification of a ca. 500-bp product when tested with U. maydis DNA, but no amplification was detected with DNA from fungi not related to U. maydis. Using these primers, U. maydis was detected in infected maize plants from differentially susceptible cultivars as early as 4 days after inoculation with strains of variable degrees of virulence. Detection of U. maydis at early stages of infection, or in asymptomatic infected plants should assist in studies on plant-pathogen interactions.

Cloning and disruption of the ornithine decarboxylase gene of Ustilago maydis: evidence for a role of polyamines in its dimorphic transition.

The gene encoding ornithine decarboxylase (ODC) from Ustilago maydis was cloned. A conserved PCR product amplified from U. maydis DNA was synthesized and used to screen a genomic library of the fungus. Alignment of its deduced protein sequence with those of other cloned ODCs showed a high degree of homology. Gene replacement was obtained by removal of a central part of the gene and insertion of the hygromycin resistance cassette. The null mutant thus obtained displayed no ODC activity and behaved as a polyamine auxotroph. This result is evidence that a single ODC gene exists in the fungus, and that U. maydis utilizes the ODC pathway as the only mechanism for polyamine biosynthesis. When grown in polyamine-containing media, the null mutant accumulated a polyamine pool which further sustained its normal rate of growth in polyamine-free media for approximately 12-16 h. When putrescine concentrations lower than 0.5 mM were employed, the mutant grew at a normal rate but was unable to engage in the dimorphic transition. Under conditions favourable for mycelial growth, the mutant grew with a yeast-like morphology in liquid media, and formed smooth colonies consisting of yeast cells on solid media. Reversion to normal dimorphic phenotype required high concentrations of putrescine or spermidine. These results are evidence that concentrations of polyamines higher than those necessary to sustain vegetative growth are required for the dimorphic transition in U. maydis.

Characterization of telomeric regions from Ustilago maydis.

The isolation of telomeres from the phytopathogenic fungus Ustilago maydis is described. The telomeric repeat from the plant Arabidopsis thaliana, TTTAGGG, cross-hybridizes to Bal31-sensitive fragments of U. maydis DNA and detects many or all of the U. maydis chromosomes separated by pulsed-field gel electrophoresis (PFGE). This telomeric repeat was used to screen a library enriched for chromosome ends. Three clones were isolated which contained the tandemly repeated sequence TTAGGG. This sequence is identical to some known telomere repeats found in humans and other vertebrates as well as in some protozoa and moulds. In addition, the three telomeric clones had an almost identical 376 bp segment of middle-repeated telomere-associated sequences adjacent to the telomeric repeat. This segment hybridized to many or all U. maydis chromosomes separated by PFGE and showed a hybridization pattern in genomic digestions similar to that of the telomeric repeat. These results indicate that in U. maydis the same segment of telomere-associated sequences is located adjacent to the telomeric repeat in many or all chromosomes, which suggests that it may have a common role in chromosome function.

Characterization of Ustilago maydis DNA binding protein one (UBP1)

The DNA binding properties of a protein from the lower eukaryote Ustilago maydis have been characterized. Using both filter binding and gel retention assays, we demonstrate that this protein, termed UBP1 ( Ustilago binding protein one), binds preferentially to DNA molecules lacking chain interruptions. The introduction of DNA breaks by a restriction enzyme or a purified nuclease, from Ustilago maydis, causes the dissociation of protein-DNA complexes. UBP1 stimulates the relaxation of negatively supercoiled DNA, mediated by Ustilago type I topoisomerase, through a mechanism most likely involving the association of UBP1 with the DNA rather than with the topoisomerase. The prebinding of UBP1 to DNA templates, subsequently assembled into minichromosomes, results in the development of a disorganized nucleosomal array. Possible roles for UBP1 in processes that involve changes in DNA topology, such as chromatin assembly, are discussed.

Development of a GUS reporter gene system for the maize pathogen Ustilago maydis

A GUS reporter gene system has been developed for the maize smut pathogen, Ustilago maydis, by making a transcriptional fusion between the coding region of the Escherichia colt gusA gene and an U. maydis hsp70-like gene promoter. This construction was transferred into U. maydis in a plasmid (pGR3) which also carried the carboxin resistance ( Cbx r ) gene as a selectable marker. GUS activity in these transformants appeared to be the result of expression of the gusA gene under the control of the hsp70 promoter sequences. GUS activity was visualized in colonies of transformants grown on agar plates and was quantitatively assayed in cell extracts. These results demonstrated the versatility of the GUS reporter system for studying gene expression in U. maydis. A derived plasmid (pGR4) which contained a promoterless gusA gene was also constructed. Transformants containing pGR4 did not express GUS activity. This plasmid now forms the basis of a promoterprobe vector for isolating U. maydis DNA fragments with gene promoter activity.

Detection and Identification ofPeronosclerospora sacchariin Maize by DNA Hybridization

The causal organism of an incidence of maize downy mildew in Southern China proved difficult to classify by standard techniques. The pathogen, subsequently identified as Peronosclerospora sacchari, was detected by DNA hybridization in endosperm, pericarp, and pedicel tissues, but not in embryos of infected maize seeds. Plasmid pCLY83, which had been selected from a P. maydis DNA library, served as the probe (...)

Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.

Differential expression of Ustilago maydis DNA sequences during induction of nitrate reductase enzyme activity.

A differential hybridisation screen of an Ustilago maydis genomic DNA library was used to identify DNA sequences transcribed at higher levels under growth conditions which induce nitrate reductase activity. After two rounds of screening, four different sequences showed a strongly enhanced hybridisation signal with an induced cDNA probe relative to a repressed cDNA probe. The sequence in plasmid pMH3007 hybridised to a U. maydis RNA transcript of a 4.2 kb. This is identical in size to a transcript, also nitrate inducible, which hybridised to cloned Aspergillus nidulans nitrate reductase gene sequences at a reduced stringency.

Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.

Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.

A DNA polymerase from Ustilago maydis. Evidence of proof-reading by the associated 3' leads to 5' deoxyribonuclease activity.

The 3' leads to 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.

A DNA polymerase from Ustilago maydis. 2. Properties of the associated deoxyribonuclease activity.

The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3' leads to 5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.