Bioassays using detached tobacco leaves to determine the sensitivity of Peronospora tabacina to fungicides

Two bioassay methods are described which use detached tobacco leaves to measure the sensitivity of Peronospora tabacina to systemic fungicides. Tobacco leaves (13–15 cm²), treated with fungicides before or after detachment from the plant, were inoculated with sporangia in water drops and, after incubation in beakers and Petri plates, the disease severity and/or production of sporangia was determined 4–7 days after treatment with the fungicides. Of 15 systemic fungicides applied to detached leaves, eight N-phenylamides at 0.066?1.0 μg ml^?1 controlled blue mould; metalaxyl was the most effective fungicide. Isolates of P. tabacina, collected in the field from tobacco plants grown in soil treated with metalaxyl, were not resistant to the fungicide applied to detached leaves prior to inoculation. The fungicide, applied to leaves before detachment, was used to measure the efficacy of five systemic N-phenylamide fungicides sprayed on the basal and unsprayed distal portions of the leaves. Blue mould was controlled on the basal portion of the leaf by all the fungicides at 0.66?1.0 μg ml^?1, but it required the application of 3–30 times more chemical on the basal portion to achieve comparable blue mould control on the distal part of the leaf.     

Cocultures of Peronospora tabacina and Nicotiana Species to Study Host-Pathogen Interactions

ABSTRACT Long-term cocultures of the tobacco blue mold pathogen, Peronospora tabacina, with Nicotiana tabacum and N. repanda callus were derived from infected host plant tissue. In this apparently contaminant-free system, sporulation occurred under similar conditions as in intact plants. Sporangia were collected from cocultures and used to complete Koch's postulates. The cocultures were grown under two light regimes. One consisted of 23 h of light followed by 1 h of darkness and the second comprised total darkness. Sporulation occurred frequently in the 23 h light-grown cocultures but resulted in production of abnormal sporangiophores and sporangia. Production of normal sporangiophores and sporangia was achieved by transferring light-grown cocultures to overnight darkness and resulted in necrosis of the callus. Cocultures of Peronospora tabacina with either host species, grown in total darkness, frequently sporulated with minimal necrosis over the course of 1 year. Thus, cocultures should prove useful as a source of Peronospora tabacina over extended periods of time at low risk of pathogen release, for studying the physiology of Peronospora tabacina- Nicotiana interactions, maintaining Peronospora tabacina lines for genetic studies, and providing a reliable source of axenic inoculum for research.     

Interactions of Peronospora tabacina with Roots of Nicotiana spp. in Gnotobiotic Associations

ABSTRACT Peronospora tabacina is an obligate plant pathogen that causes downy mildew disease on several species of Nicotiana, including N. tabacum (tobacco). The primary objective of this study was to use gnotobiotic associations to describe interactions between the pathogen and roots of either N. tabacum (cv. KY14) or N. repanda. We found that the pathogen was capable of moving systemically from shoots to roots of both host species and emerged from the root tissues as hyphae. We also demonstrated that root-associated hyphae were infectious on roots of nearby plants and resulted in new systemic infections. Following overnight darkness, sporulation of the pathogen was observed on infected roots exposed to air on both host species. We also found that within 2 months in culture, structures resembling resting stages of Peronospora tabacina were produced on hyphae emerging from roots of N. repanda but not N. tabacum. These findings appear relevant to both the epidemiology of the disease and to future studies of this and other downy mildew pathosystems.     

Genetic Uniformity Among Isolates of Peronospora tabacina, the Tobacco Blue Mold Pathogen

ABSTRACT As a first step toward analysis of genetic variation and population structure in Peronospora tabacina, we used a collection of random genomic DNA fragments to survey for restriction fragment length polymorphisms (RFLPs) in DNA from a collection of isolates from Kentucky and other tobacco-growing regions of the United States. Also included in the study were isolates from the wild tobacco species, Nicotiana repanda, and from ornamental tobacco, N. alata. In a preliminary survey using DNA from 10 pathogen isolates, no polymorphisms were detected at six single-copy DNA loci using 22 probe-enzyme combinations. Moderately repetitive and highly repetitive regions of the genome were also remarkably similar between isolates, with only 6 of 15 different probes identifying genetic differences. Some of the polymorphic probes were then used to analyze a larger collection of isolates, most of which were from Kentucky. This resulted in the identification of very few additional polymorphisms, indicating that the population of P. tabacina that infects the Kentucky tobacco crop is genetically very homogeneous. The low level of polymorphism detected in this study overall, suggests that genetic variability may be lacking in P. tabacina populations throughout the United States. Two of the RFLP markers gave hybridization patterns that were consistent with P. tabacina being diploid. Frequencies of alleles at these loci and linkage disequilibrium between different marker loci indicated that genetic recombination does not occur frequently in the pathogen population. DNA polymorphisms that were identified in this study enabled us to differentiate the pathogen population into at least 10 haplotypes. One isolate was analyzed in detail and was shown to be genetically stable through several rounds of single-spore isolation and through several pathogenic cycles.     

Genetic resistance to Peronospora tabacina in Nicotiana langsdorffii, a South American wild tobacco

Several accessions of Nicotiana langsdorffii, a wild tobacco relative native to South America, express an incompatible interaction in response to infection by Peronospora tabacina, an o?mycete that causes blue mold disease of tobacco (N. tabacum) and many other species of Nicotiana. In resistant accessions of N. langsdorffii, such as S-4-4, incompatibility takes the form of necrotic lesions that appear 48 h after pathogen inoculation, restricting pathogen growth, and suppressing subsequent asexual sporulation. Significantly elevated levels of salicylic acid and expression of a defense-related gene (HSR203J) were observed in S-4-4 leaves following blue mold infection. Genetic segregation analysis in F(2) and modified backcross populations showed that blue mold resistance is determined by a single dominant gene (NlRPT) present in S-4-4. Further characterization of this unique host-pathogen interaction has revealed that (i) necrotic lesion resistance is due to the hypersensitive response (HR), (ii) HR-mediated resistance is present in 7 of 10 N. langsdorffii accessions examined, but not in closely related species, (iii) in some accessions of N. langsdorffii, resistance is expressed in cotyledon tissue and seedling leaves as well as in adult plants, and (iv) several resistant accessions including S-4-4 express an unregulated ("runaway") HR in response to P. tabacina infection.     

Development of Contamination-Free Restriction Fragment Length Polymorphism Probes for the Obligate Biotroph Peronospora tabacina, an Oomycete Causing Blue Mold of Tobacco

ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism.     

一种测定大白菜霜霉病菌对甲霜灵敏感性方法的建立及应用

建立了一种新方法—离体子叶浸泡法并用于大白菜霜霉病菌对甲霜灵敏感性的检测。离体子叶在含有0.005%吐温20和0.1%丙酮的甲霜灵中浸泡3h后,接种浓度为1×104孢子囊/mL的大白菜霜霉病菌孢子囊悬浮液,保湿培养6d后观察子叶发病状况。采用离体子叶浸泡法测定了50株大白菜霜霉病菌对甲霜灵的敏感性,结果显示部分大白菜霜霉病菌菌株对甲霜灵的敏感性降低,有抗药性亚群体出现。研究表明离体子叶浸泡法重复性好,具有较高的敏感性和精密度,符合生产实际,适用于大白菜霜霉病菌对杀菌剂的敏感性检测。     

Production of oospores by Peronospora viciae f.sp. fabae

Oospore production inVicia faba, cv. Metissa, was quantified in the field after plants had been inoculated with a sporangium suspension of a homothallic isolate ofPeronospora viciae f.sp.fabae. Oospores were produced abundantly during the hole growing season from 3 weeks after inoculation on. Oospores were found in all plant parts above soil level, except in the seeds. Most oospores were found in the leaves. Less oospores were formed in leaves inoculated in an older stage than in those inoculated in a younger stage. Towards the end of the season, in August, numbers of oospores in pods strongly increased.Oospore production in leaves of three cultivars, Metissa, Toret and Maris Bead, was studied in growth chambers at 5, 10, 15 and 20 °C at 16 h light. Oospores were formed earlier at higher temperatures than at lower temperatures. The ultimate numbers of oospores produced in leaves were highest at 10 and 15 °C. Similar numbers of oospores were formed in leaves of cultivars Metissa and Toret. In leaves of cv. Maris Bead significant less oospores were produced than in leaves of cv. Metissa and cv. Toret. Total numbers of oospores produced were not related to the level of host plant resistance to downy mildew. The percentage of asexually sporulating leaf area, assessed in a resistance test, was largest in cv. Metissa and smallest in cv. Toret.     

Production of conidia by Peronospora farinosa f. sp. spinaciae

Conidium production byPeronospora farinosa f. sp.spinaciae (pathotype 3) was measured daily on colonies induced on leaves of spinach cvs Breedblad Scherpzaad (BS) and Huro. Distinction was made between colonies grown at temperatures of 10 and 15°C. Spore production was expressed as number of spores produced per stoma. There was a significant difference between total spore production on lesions on BS at 10 °C and that on lesions on the three other cultivartemperature combinations tested. Also a significant difference in the average sporulation period was observed between lesions produced at 10 and those at 15 °C. The same significant difference in response to temperature was found in the sporulation per stoma in the course of time.Samenvatting De conidiënproduktie vanPeronospora farinosa f. sp. spinaciae (fysio 3) werd dagelijks bepaald aan kolonies, welke op bladeren van de spinaziecultivars Breedblad Scherpzaad (BS) en Huro werden geïnduceerd. Daarbij werd onderscheid gemaakt tussen kolonies welke zich bij 10 en bij 15 °C hadden ontwikkeld. De sporenproduktie werd omgerekend naar het aantal sporen per huidmondje. Er was een significant verschil tussen de totale sporenproduktie op lesies op BS bij 10 °C en die op lesies op de drie andere getoetste cultivar-temperatuur combinaties. Er kon ook een significant verschil in de gemiddelde sporulatieperiode worden waargenomen tussen de lesies ontstaan bij 10 en bij 15 °C. Een dergelimk significant verschil in temperatuurreactie kon ook worden gevonden in het verloop van de soorulatie per huidmondje in de tijd.