M. R. Bonde

Evaluation of virulence of Phakopsora pachyrhizi and P. meibomiae isolates.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi and recently discovered for the first time in continental United States, has been of concern to the U.S. agricultural industry for more than 30 years. Since little soybean rust resistance is known, and resistance is often difficult to detect or quantitate, we initiated a project to develop a better, more quantitative, method. The methodology determined the average numbers and diameters of uredinia in lesions that developed on leaves of inoculated plants 14 days after inoculation. It was used to compare virulence of P. pachyrhizi isolates from Asia and Australia and P. meibomiae from Puerto Rico and Brazil, collected as many as 30 years earlier, with isolates of P. pachyrhizi recently collected from Africa or South America. Susceptible reactions to P. pachyrhizi resulted in tan-colored lesions containing 1 to 14 uredinia varying greatly in size within individual lesions. In contrast, on these same genotypes at the same time of year, resistance to other P. pachyrhizi isolates was typified by 0 to 6 small uredinia in reddish-brown to dark-brown lesions. Using appropriate rust resistant and rust susceptible genotypes as standards, examination of uredinia 14 days after inoculation allowed quantitative comparisons of sporulation capacities, one measure of susceptibility or resistance to soybean rust. The study verified the presence and ability to detect all known major genes for resistance to soybean rust in the original sources of resistance. It demonstrated that soybean lines derived from the original PI sources, and presumed to possess the resistance genes, in actuality may lack the gene or express an intermediate reaction to the rust pathogen. We suggest that a determination of numbers and sizes of uredinia will detect both major gene and partial resistance to soybean rust.

Polymerase Chain Reaction Assays for the Detection and Discrimination of the Soybean Rust Pathogens Phakopsora pachyrhizi and P. meibomiae

ABSTRACT Soybean rust occurs in Australia and many countries throughout Africa, Asia, and South America. The causal agents of soybean rust are two closely related fungi, Phakopsora pachyrhizi and P. meibomiae, which are differentiated based upon morphological characteristics of the telia. Determination of the nucleotide sequence of the internal transcribed spacer (ITS) region revealed greater than 99% nucleotide sequence similarity among isolates of either P. pachyrhizi or P. meibomiae, but only 80% sequence similarity between the two species. Utilizing differences within the ITS region, four sets of polymerase chain reaction (PCR) primers were designed specifically for P. pachyrhizi and two sets for P. meibomiae. Classical and real-time fluorescent PCR assays were developed to identify and differentiate between P. pachyrhizi and P. meibomiae. Identification of P. pachyrhizi from infected soybean leaves using the real-time PCR assay will allow for more rapid diagnoses.

New Legume Hosts of Phakopsora pachyrhizi Based on Greenhouse Evaluations

Phakopsora pachyrhizi, the causal organism of soybean rust, was first found in the continental United States in 2004 and has been found on soybean, kudzu, Florida beggarweed, and three Phaseolus species in the field. The pathogen has been reported to occur on more than 90 legume species worldwide and it is likely to infect native and introduced legume species in the United States. The objective of this study was to determine if 176 species representing 57 genera of legumes, the majority of which are either native or naturalized to soybean-growing areas of the United States, could be hosts of P. pachyrhizi. Between one and three accessions of each species, a total of 264 accessions, were inoculated with a mixture of four isolates of P. pachyrhizi. Severity and sporulation were rated on a 1-to-5 scale at 14 and 28 days after inoculation. P. pachyrhizi was confirmed by the presence of sporulating uredinia and/or immunological assay on 65 new species in 25 genera; 12 of these genera have not been reported previously as hosts. Many of the newly identified hosts grow in the southern United States, and like kudzu, could serve as overwintering hosts for P. pachyrhizi.

ON THE CONSPECIFICITY OF ENDOTHIA EUGENIAE AND CRYPHONECTRIA CUBENSIS

Isolates of Endothia eugeniae, a pathogen of clove (Syzygium aromaticum), and Cryphonectria cubensis, a pathogen of eucalyptus (Eucalyptus spp.), were examined with chemotaxonomic techniques, including thin-layer chromatography of fungal pigments and isozyme analysis. The bisanthraquinone pigments skyrin and oxyskyrin were isolated from mycelial extracts of each species. Isolates of E. eugeniae and C. cubensis could not be differentiated by polyacrylamide gel electrophoresis of buffersoluble proteins. The organisms also shared alleles at 16 presumed genetic loci, as detected by isozyme analysis. This study confirms the conspecificity of E. eugeniae and C. cubensis.

Effects of Temperature on Urediniospore Germination, Germ Tube Growth, and Initiation of Infection in Soybean by Phakopsora Isolates

ABSTRACT Temperature is a critical factor in plant disease development. As part of a research program to determine how specific environmental variables affect soybean rust, we determined temperature effects on urediniospore germination and germ tube growth of four isolates of Phakopsora pachyrhizi, one each from Brazil, Hawaii, Taiwan, and Zimbabwe, and an isolate of P. meibomiae from Puerto Rico, collected over a 25-year period. Also compared were the effects of temperature during a night dew period on initiation of disease by the P. pachyrhizi isolates. All variables were fit to a nonlinear beta function with temperature as the independent variable. Minimum, maximum, and optimum temperatures, along with shape parameters of the beta function for each variable, were statistically analyzed. All Phakopsora isolates behaved similarly as to how temperature affected urediniospore germination, germ tube growth, and initiation of disease. The results suggest that P. pachyrhizi has changed little in the past few decades with respect to how it responds to temperature and that previously collected research data continues to be valid, simplifying the development of soybean rust disease models.

Survival of Teliospores of Tilletia indica in Soil

This study was conducted to assess survival of Tilletia indica teliospores in a location in the northern United States. Soils differing in texture and other characteristics were collected from four locations, equilibrated to -0.3 MPa, and infested with teliospores of T. indica to give a density of 103 teliospores per gram of dry soil. Samples (22 g) of the infested soil were placed in 20-μm mesh polyester bags, which were sealed and placed at 2-, 10-, and 25-cm depths in polyvinyl chloride tubes containing the same field soil as the infested bags. Tubes were buried vertically in the ground at Bozeman, MT, in October 1997. Soil samples were assayed for recovery and germination of T. indica teliospores 1 day and 8, 20, and 32 months after incorporation of teliospores into soil. The rates of teliospores recovered from soil samples were 90.2, 18.7, 16.1, and 13.3% after 1 day and 8, 20, and 32 months after incorporation of teliospores into soil, respectively, and was significantly (P < 0.01) affected by soil source. The percentage of teliospore recovery from soil was the greatest in loam soil and lowest from a silt loam soil. The rate of teliospores recovered from soil was not significantly affected by depth of burial and the soil source-depth interaction during the 32-month period. The percentage of germination of teliospores was significantly (P < 0.01) affected by soil source and depth of burial over the 32-month period. The mean percentage of teliospore germination at 1 day, and 8, 20, and 32 months after incorporation into soils was 51.3, 15.1, 16.4, and 16.5%, respectively. In another experiment, samples of silty clay loam soil with 5 × 103 teliospores of T. indica per gram of soil were stored at different temperatures in the laboratory. After 37 months of incubation at 22, 4, -5, and -18°C, the rates of teliospore recovered from soil were 1.6, 2.0, 5.7, and 11.3%, respectively. The percentage of spore germination from soil samples was highest at -5°C. Microscopy studies revealed that disintegration of teliospores begin after breakdown of the sheath-covering teliospore. The results of this study showed that teliospores of T. indica can survive in Montana for more than 32 months and remain viable.

Survival of Tilletia indica Teliospores in Different Soils

To determine the potential for Tilletia indica, cause of Karnal bunt of wheat, to survive and become established in new areas, a teliospore longevity study was initiated in Kansas, Maryland, Georgia, and Arizona. Soil from each location was infested with T. indica teliospores and placed in polyester mesh bags. The bags were placed within soil from the same location within polyvinyl chloride pipes. Pipes were buried in the respective plots such that the bags were at 5-, 10-, and 25-cm depths. Each pipe was open at the ends to allow interaction with the outside environment, however fitted with screens preventing possibility of teliospore escape. In the Karnal bunt-quarantine area of Arizona, bags of infested soil also were placed outside the pipes. Teliospore-infested soil from each location was maintained dry in a laboratory. During the first 2 years, viability declined more rapidly in pipes than outside pipes, and more rapidly in fields in Kansas and Maryland than in Georgia or Arizona. After 2 years, viability declined nearly equally. In the laboratory over 3 years, viability decreased significantly more rapidly in dry soil from Kansas or Maryland than in dry soil from Georgia or Arizona, while pure teliospores remained unchanged. We hypothesized that soils, irrespective of weather, affect teliospore longevity.

Survival of Teliospores of Tilletia indica in Arizona Field Soils

The survival of teliospores of the Karnal bunt of wheat pathogen, Tilletia indica, was determined in field plots in Tucson, AZ. Two methods were used to test viability during a 48-month period in which 21-μm-pore-size polyester mesh bags of teliospore-infested soil were buried in irrigated and nonirrigated field plots at two sites. One method determined the total number of viable teliospores in a soil sample, regardless of whether or not they could be extracted from the soil using a sucrose centrifugation technique. The total number of viable teliospores declined over time in both irrigated and nonirrigated field plots and in the same soils in the laboratory. Based on nonlinear regressions, total number of viable teliospores decreased from 55.7% at time zero to 9.7 and 6.7% for nonirrigated and irrigated field soils, respectively, in 48 months. Total number of viable teliospores in soil in the laboratory decreased from 55.7 to 34.0% after 48 months. The second method determined germination percentages of teliospores extracted from the soil samples by means of a sucrose centrifugation technique. Based on linear regressions of transformed data, germination of teliospores extracted from irrigated and nonirrigated field soils, and control (laboratory) soil, significantly decreased over time. The rate of decrease in germination was significantly greater for teliospores from irrigated field plots than from nonirrigated plots and the laboratory soil. At time zero, 55.7% of teliospores germinated, and by 48 months, average germination of teliospores extracted from soil in nonirrigated plots had decreased to 13.6% compared with 4.4% in irrigated plots and 36.8% for teliospores in the laboratory control. Regression over time of total number of viable teliospores accounted for more of the overall variability than did regression over time of germination percentages of extracted teliospores. Neither field site nor soil depth had any effect on total number of viable teliospores or on teliospore germination percentages.

Myclobutanil as a curative agent for chrysanthemum white rust

In response to a recent outbreak of chrysanthemum white rust, caused by Puccinia horiana, in California, research was conducted to test the fungicide myclobutanil for its effectiveness as a foliar chemical control for the disease. Myclobutanil applied 5 days after exposure of susceptible plants to sporidial inoculum had extremely strong curative properties and usually prevented disease development in either inoculated plants or cuttings obtained from presymptomatic infected mother plants. Even though lesions with pustules developed on inoculated plants when they were sprayed with the fungicide at 10, 15, or 20 days after sporidial inoculation, the pathogen produced few sporidia in an otherwise conducive environment. Myclobutanil at 100 mg a.i./liter, however, was not highly effective for preventing infection when sprayed onto plants 5 days before inoculation, although it did reduce disease incidence. The data indicate that myclobutanil (100 mg a.i./liter) used as a dip treatment for chrysanthemum cuttings prior to planting is suitable as a regulatory treatment for exclusion and eradication of chrysanthemum white rust

Comparative Susceptibilities of Legume Species to Infection by Phakopsora pachyrhizi

Knowledge of the host range of Phakopsora pachyrhizi is important to agriculture in the United States because of the distinct possibility that economic losses could occur to crops other than soybean. Furthermore, it is possible that alternative hosts could provide a means of overwintering of the pathogen, providing inoculum to initiate epidemics in future years. To clarify the potential importance of soybean rust on nonsoybean legumes and their role in overwintering of the disease, multiple accessions of clover, cowpea, pea, kudzu, lima bean, snap bean, and single accessions of coffee senna, Florida beggarweed, hemp sesbania, hyacinth bean, partridge pea, and showy crotalaria were inoculated under greenhouse conditions with urediniospores of P. pachyrhizi; infected soybean plants served as a control. The four criteria used to assess susceptibility were lesion density, proportion of lesions with sporulating uredinia, average number of uredinia per lesion, and average uredinia diameter, each determined 2 weeks following inoculation. Based on lesion densities, percentage of lesions with sporulation, and average numbers of uredinia per lesion, soybean, kudzu, and pea were the most susceptible species, followed by snap bean. However, because infected pea plants defoliated rapidly, urediniospore production presumably was limited, lessening the potential for epidemics on pea. Cultivars of snap bean produced numerous brown to reddish-brown lesions, many of which sporulated, but numbers of uredinia per lesion were lower than on soybean, kudzu, or pea. The presence of both tan (susceptible) and reddish-brown (resistant) lesions on kudzu demonstrated physiological differentiation on that host. Some kudzu plants appeared to be potentially excellent hosts for overwintering of the disease. The average number of uredinia per lesion appeared to be a valid measurement with which to compare host susceptibilities, and may have epidemiological significance. High susceptibility of a host was characterized by numerous uredinia with a wide range of sizes within individual lesions. In contrast, low susceptibility to rust was characterized by no or a few small uredinia.