ABSTRACT Infection of Nicotiana obtusifolia plant introduction (PI) #555573 by the downy mildew pathogen Peronospora tabacina resulted in a compatible interaction, in which P. tabacina penetrated and freely colonized host leaf tissue. This interaction became incompatible 5 to 6 days later, with the appearance of necrotic lesions (NLs) and inhibition of pathogen growth and subsequent sporulation. NL development depended upon the presence of P. tabacina in host tissue, was not due to the effects of other microbes, and occurred co-incident in time with the pathogen's ability to produce asexual sporangia on a susceptible N. obtusifolia genotype. Inhibition of the necrotic response by CoCl(2) (a calcium channel blocker) and pathogen-induced transcription of a defense-related gene (PR-1a) suggested that necrosis was due to hypersensitive cell death in the host. In contrast, N. obtusifolia PI#555543 did not exhibit hypersensitivity upon infection by P. tabacina, but rather developed characteristic symptoms of tobacco blue mold disease: chlorotic lesions accompanied by abundant pathogen sporulation. Disease reactions scored on PI#555573 x PI#555543 F(2) progeny inoculated with P. tabacina sporangia indicated that the resistance phenotype was due to the action of a single gene from N. obtusifolia PI#555573, which we have named Rpt1. To date, Rpt1 is the only gene known to confer a hypersensitive response (HR) to P. tabacina infection in any species of Nicotiana. A survey of wild N. obtusifolia revealed that the HR to P. tabacina was expressed in the progeny of 7 of 21 (33%) plants collected in southern Arizona, but not in the progeny of plants originating from Death Valley National Park in California and the Big Bend National Park in west Texas.

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.

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.

A 232-bp DNA sequence was obtained from random amplified polymorphic DNA of Peronospora tabacina, the blue mold pathogen of tobacco. This sequence had homology to P. tabacina DNA but not to DNA of other downy mildew or oomycetous fungi tested. The fragment was determined to be part of a repetitive DNA sequence that was ubiquitous in a worldwide collection of P. tabacina. Oligonucleotides were designed for amplification of this sequence by polymerase chain reaction. The fragment was reliably amplified with amounts of DNA (1-10 fg) that are less than that contained in a single P. tabacina sporangiospore. Use of this technique enabled the detection of P. tabacina DNA directly in local lesions, systemic vascular infections, and other infected parts of tobacco plants. The use of spore traps followed by amplification of this fragment facilitated the prediction of a local disease epidemic. Use of this highly specific and reliable detection method could prove valuable for regulatory agencies and for epidemiological and etiological studies.

A laboratory bioassay to distinguish isolates of Peronospora tabacina D.B. Adam was developed utilizing 7-mm acetone-dipped disks cut from tobacco leaf panels from several cultivars of Nicotiana tabacum, sporangiospores of P. tabacina, a germination indicator (Calcofluor), and fluorescence microscopy. Sporangiospore germination percentage of each isolate X cultivar pair was determined. Mean germination of sporangiospores differed between domestic and international collection locations (except between the two Mexican isolates). These results suggest the presence of different pathotypes of P. tabacina.

Aphanomyces euteiches was detected in soil or root samples from fields cropped to alfalfa in 47 counties from most major alfalfa-producing regions in Kentucky. Soil samples from 121 fields in 30 counties were tested for the presence of A. euteiches and Phytophthora medicaginis using a baiting technique. A. euteiches and P. medicaginis were detected in 57 and 9.9%, respectively, of samples collected. Given the prevalence of alfalfa-infecting strains of A. euteiches, studies are needed to determine whether the use of Aphanomyces-resistant cultivars will result in enhanced performance of alfalfa in Kentucky

Pathogenic mycorrhizal fungi may be a major reason crops must be rotated to maintain soil productivity. We studied the role such fungi may have in the maintenance of productivity of soil for tobacco (Nicotiana tabacum L.) by rotation with tall fescue (Festuca arundinacea Schreb). Tall fescue 'Kentucky 31' and continuous tobacco plots were established on a central Kentucky field previously shown to be infested with the mycorrhizal fungus Glomus macrocarpum Tul and Tul., the cause of a stunt disease of tobacco. Fumigation with 67% methyl bromide-33% chloropicrin (MBC, trichloronitromethane) covered with plastic was used as a control. Rotation with fescue for 2 yr and fumigation both reduced disease incidence when tobacco was planted on all plots the third year. Effects of rotation and fumigation were not additive, indicating that both control the same disease agent. Only G. macrocarpum, of three mycorrhizal fungi present in high populations, was associated with the disease. Rotation and fumigation controlled mycorrhizal colonization of roots and the build-up of populations of G. macrocarpum in the root zones of plants. Mycorrhizal fungi should be considered in research on the effects of crop rotation on productivity.

Field experiments were conducted in San Andres Tuxtla, Veracruz, Mexico, during 1986 and 1989 to test the effectiveness of induced systemic resistance (immunization) against metalaxyl-tolerant strains of Peronospora tabacina, causal agent of tobacco blue mold. Plants of Nicotiana tabacum cultivar Jaltepec were stem-injected with sporangiospores of metalaxyl-tolerant strains of P. tabacina. Half of the plants were treated with metalaxyl. The number and size of blue mold lesions that developed because of infections caused by ambient inoculum were significantly fewer and smaller on plants injected with P. tabacina as compared with noninjected stem controls, regardless of metalaxyl applications. Some vigorously growing, naturally infected plants with necrotic stem lesions but markedly reduced foliar lesions of blue mold were observed in commercial fields surrounding the experimental plots. Stem necrosis was consistently associated with these plants. Stem necrosis was not detected in plants infected heavily with foliar lesions of blue mold. Several attempts to isolate the causal agent of necrosis associated with acquired resistance to blue mold failed. Only live sporangiospores of P. tabacina produced necrosis typical of that found in the naturally protected plants. Natural immunization apparently occurs at scattered locations on the gulf coast of Mexico and is associated with vigorous growth. Furthermore, the endemic nature of the disease, presence of sexual stages of the fungus, long-term moisture in the field, and high constant disease pressure created conditions very favorable for the development of tolerance to any protective agent, such as metalaxyl, with a single site of action; however, development of tolerance to immunization has not yet been observed.

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.

Fumigation of soil with 67% methyl bromide-33% chloropicrin (MBC) was more effective than fumigation with 98% methyl bromide-2% chloropicrin (MB) in increasing growth and accelerating maturity of tobacco in three fields in Central Kentucky infested with the tobacco stunt pathogen. However, both fumigants had similar effects on yields. Two fields had more species of endogonaceous fungi and higher populations of endogonaceous spores than the third, in which the disease was more severe. In all three fields, Glomus macrocarpum was prevalent, reproduced on tobacco, and was controlled by fumigation. For plants growing in nonfumigated soil, height at harvest time was correlated negatively with populations of spores of G. macrocarpum, and less strongly with G. microcarpum and Acaulospora scrobiculata. No such correlations occurred with plants growing in fumigated soil. Roots of plants growing in both fumigated and nonfumigated soil were colonized by mycorrhizal fungi throughout the growing season, but by harvest time, roots of plants growing in nonfumigated soil were colonized 10-fold more than those of plants growing in fumigated soil. However, stunting was not correlated with colonization. These experiments generally support inoculation experiments indicating that G. macrocarpum causes tobacco stunt disease.

AbstractWhen the petioles of detached tobacco leaves (10–17 cm2) were incubated in aqueous solutions containing [14C]metalaxyl, uptake of the fungicide was dependent on the temperature and photoperiod. Detached leaves took up 78% more [14C]metalaxyl at 26°C than at 16°C. The rate of uptake in the light at 21°C was linear, but after an additional 20h in the dark, there was only twice as much fungicide in the leaves. Different sized leaves contained the same amount of fungicide per cm2 area. Uptake by detached leaves of the 14C‐labelled anilide lactones ofurace and RE‐26940 [2‐methoxy‐N‐(tetrahydro‐2‐oxo‐3‐thienyl)acet‐2′,6′‐xylidide] was similar to that of metalaxyl. At the concentration of metalaxyl (66 ng ml−1) that controlled blue mould (Peronospora tabacina) on detached tobacco leaves, the amount of fungicide in the leaves was found to be 7.25 ng. Autoradiography showed that the distribution of [14C]metalaxyl in detached leaves after incubation for 23h was uniform, although higher concentrations of the label were present in the smaller veins of the leaves.