D. K. Berner

A land management based approach to integrated Striga hermonthica control in sub-Saharan Africa

Striga hermonthica, an obligate root parasite of grasses, Is one of the most severe constraints to cereal production in sub-Saharan Africa. In the recent past, prior to increased production pressure on land, S. hermonthica was controlled in African farming systems by prolonged crop rotations with bush fallow. Because of increasing need for food and concomitant changes in land management practices, however, these fallow rotations are no longer extensively used. Shorter crop rotations and fallow periods have also led to declines in soil fertility which present a very serious threat to African food production. A sustainable solution will be an integrated approach that simultaneously addresses both of these major problems. An integrated programme that replaces traditional bush fallow rotation with non-host nitrogen-fixing legume rotations, using cultivars selected for efficacy in germinating S. hermonthica seeds, is outlined. The programme includes use of S. hermonthlca-free planting material, biological control, cultural control to enhance biological suppressiveness, host-plant resistance, and host-seed treatments.

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.

Genetic variability of Striga asiatica (L.) Kuntz based on AFLP analysis and host-parasite interaction

AFLP analysis was used to estimate genetic variability within and among 14 populations of Striga asiaticaL. Kuntze collected from different locations within the Republic of Benin. The mean within-population genetic distances ranged from 0.028 to 0.038, while the mean among-population genetic distances ranged from 0.019to 0.088, with an assumed minimum genetic distance of0.01 in each case. Intra- and inter-population variation was reflected by a highly significant R2 of 0.61for the regression of geographic distance versus genetic distance. Interactions of the different Strigapopulations with susceptible host genotypes, 8338-1 (Zea mays) and CK60B (Sorghum bicolor),indicated a high degree of host-specialization, with CK60B failing to support growth of the parasite from any of the populations. The various Striga populations also exhibited different degrees of virulence on susceptible host plants. Our results support the hypothesis that different populations of this parasite may well be considered and treated as ecotypes in plant breeding programs developing resistance to S. asiatica.

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.

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.

Effects of Daily Temperature Highs on Development of Phakopsora pachyrhizi on Soybean

Although considerable information exists regarding the importance of moisture in the development of soybean rust, little is known about the influence of temperature. The purpose of our study was to determine whether temperature might be a significant limiting factor in the development of soybean rust in the southeastern United States. Soybean plants infected with Phakopsora pachyrhizi were incubated in temperature-controlled growth chambers simulating day and night diurnal temperature patterns representative of the southeastern United States during the growing season. At 3-day intervals beginning 12 days after inoculation, urediniospores were collected from each plant and counted. The highest numbers of urediniospores were produced when day temperatures peaked at 21 or 25°C and night temperatures dipped to 8 or 12°C. When day temperatures peaked at 29, 33, or 37°C for a minimum of 1 h/day, urediniospore production was reduced to 36, 19, and 0%, respectively, compared with urediniospore production at the optimum diurnal temperature conditions. Essentially, no lesions developed when the daily temperature high was 37°C or above. Temperature data obtained from the National Climatic Data Center showed that temperature highs during July and August in several southeastern states were too high for significant urediniospore production on 55 to 77% of days. The inhibition of temperature highs on soybean rust development in southeastern states not only limits disease locally but also has implications pertaining to spread of soybean rust into and development of disease in the major soybean-producing regions of the Midwestern and northern states. We concluded from our results that temperature highs common to southeastern states are a factor in the delay or absence of soybean rust in much of the United States.

Leaf Anthracnose, a New Disease of Swallow-Worts Caused by Colletotrichum lineola from Russia

Black swallow-wort, Vincetoxicum nigrum (L.) Moench (= Cynanchum louiseae Kartesz & Gandhi), and pale swallow-wort, V. rossicum (Kleopow) Borhidi (= Cynanchum rossicum (Kleopow) Borhidi), are invasive plants belonging to the family Apocynaceae and are the targets of biological control efforts to control their spread in the United States. In 2010, a disease on a related species, V. scandens Sommier & Levier, was observed in the Krasnodar area of Russia. Disease symptoms were many small, dark red-to-purple leaf spots, approximately 2 to 5 mm in diameter, with white centers. Leaf spots were found on the upper leaf surface. Leaf tips and margins of leaves bearing many of these spots were necrotic. Symptomatic leaves were collected and sent to the BSL-3 containment facility at the Foreign Disease-Weed Science Research Unit (FDWSRU) of the USDA, ARS in Frederick MD. Surface-disinfested symptomatic leaves were incubated at 20 to 25°C in sterile moist chambers. After several days, acervuli and brown setae were observed inside the leaf spots. Pure cultures, designated FDWSRU 10-002, were obtained by transferring spore masses with sterile glass needles onto 20% V8 juice agar. Seeds of V. scandens, collected in Russia, were placed in a freezer at -20°C for 6 weeks and then germinated in sterile petri plates on moist filter paper. The seedlings were then transplanted and grown in a 20°C greenhouse under 12 h of light. Kochs postulates were fulfilled as follows: 2-month-old plants each of V. scandens, V. nigrum, and V. rossicum were inoculated with spores from 2-week-old cultures of isolate 10-002. Plants were inoculated by spraying an aqueous suspension of 106 spores per ml onto each plant until all leaves were wet. Plants were placed in 20 to 24°C dew chambers for 18 h and then placed in a 20°C greenhouse. Two weeks later, diseased leaves with the same symptoms observed in the field were harvested from each species, and the fungus was reisolated from seven of seven inoculated V. scandens plants, one of two V. nigrum plants, and four of four V. rossicum plants. Measurements of fungus fruiting structures were taken from cultures grown on synthetic nutrient-poor agar (SNA) (1). Conidiophores were brown, septate, and branched. Conidia were one-celled, hyaline, smooth walled, ovoid to oblong, falcate, and 20.1 to 26.2 × 1.7 to 3.6 μm (mean ± s.d. = 23.5 ± 1.3 × 2.6 ± 0.4 μm). Lengths of the conidia conformed to the description of Colletotrichum lineola Corda (1), but the conidia were slightly narrower than described. To induce appressoria formation, approximately 104 conidia were placed on sterile dialysis membranes on top of SNA in petri dishes that were wrapped in foil and incubated at 24°C for 24 h. After this time, appressoria were observed with a microscope at ×400 magnification. The appressoria were dark brown, smooth walled, ellipsoidal, and 5.5 to 25.5 × 3.6 to 12.1 μm (mean ± s.d. = 13.4 ± 4.0 × 7.3 ± 2.1 μm), which conformed to the description of appressoria of C. lineola Corda (1). DNA sequences of ITS1, 5.8S, and ITS2 were submitted to GenBank (No. HQ731491), and after BLAST analysis, aligned 100% to 15 previously identified isolates of C. lineola in GenBank. Voucher specimens of the fungus have been deposited in the U.S. National Fungus Collection and were designated as BPI 881105 and BPI 881106. Host range and efficacy tests are planned to determine the suitability of C. lineola for biological control of swallow-worts in the United States. Reference: (1) U. Damm et al. Fungal Divers. 39:45, 2009.

First report of Silybum marianum as a host of Puccinia punctiformis.

Silybum marianum (L.) Gaertn. (milk thistle) is a problematic invasive weed in the western United States. The rust fungus, Puccinia punctiformis (F. Strauss) Rohl., is found throughout the world as a pathogen of Cirsium arvense (L.) Scop. (Canadian thistle). Recently, plants of S. marianum grown from surface-disinfested seeds in our quarantine greenhouse were parasitized by a rust. Apparently, an isolate of P. punctiformis collected from C. arvense in Turkey that was present in the greenhouse had spread to adjacent S. marianum plants and caused infection without applying any artificial dew period. Ribosomal internal transcribed spacer region sequences from fungal spore DNA isolated from the two hosts were identical. Initial signs on S. marianum were abundant, fragrant spermogonia on large leaves. These signs occur on secondary shoots of C. arvense and are indicative of systemic fungal infection (1). As the fungus infection developed on S. marianum, uredinia and urediniospores were produced. Sori on older leaves also produced teliospores. Urediniospores from infected leaves were harvested and sprayed uniformly on eight 17-day-old plants of S. marianum grown in isolation from P. punctiformis. The spore suspension consisted of 4 mg urediniospores suspended in 40 ml distilled water. Inoculated plants were incubated for 18 h in a dew chamber at 20°C in the dark and transferred to a greenhouse (20 to 25°C, 30 to 50% relative humidity, and natural light). After 13 days, uredia with urediniospores developed on four of the plants. Using the same procedure, inoculations were repeated on plants of S. marianum and S. eburneum Coss. & Durieu (the only other species described in the genus) with urediniospores of a domestic isolate of the fungus from C. arvense in Maryland. Of 51 inoculated plants of S. marianum, 23 became infected and produced uredinia. None of the 12 inoculated plants of S. eburneum showed symptoms of infection. In nature, C. arvense and S. marianum occupy different ecological areas. C. arvense is found predominately in humid temperate habitats, while S. marianum is found in habitats with a dry Mediterranean climate. Life cycles of each host are also different. C. arvense is a perennial that emerges in spring and dies back in winter, while S. marianum is a winter annual that emerges in fall and dies in late spring. Because of the differences in life cycles combined with the different geographical distribution, P. punctiformis from C. arvense may rarely encounter susceptible S. marianum plants in the field. Since fungal spores can be produced routinely on artificially inoculated plants, there might be potential to use P. punctiformis for biological control of S. marianum. To our knowledge, this is the first report of S. marianum as a host for P. punctiformis. Reference: (1) A. H. R. Buller. Puccinia sauveolens and its sexual process. Page 345 in: Researches on Fungi. Vol VII. The Sexual Process in the Uredinales, Toronto, Canada, 1950.

Finalizing host range determination of a weed biological control pathogen with best linear unbiased predictors and damage assessment

Colletotrichum gloeosporioides f. sp. salsolae (Penz.) Penz. & Sacc. in Penz. (CGS) is a facultative parasitic fungus being evaluated as a classical biological control agent of Russian thistle or tumbleweed (Salsola tragus L.). In initial host range determination tests, Henderson’s mixed model equations (MME) were used to generate best linear unbiased predictors (BLUPs) of disease severity reaction to CGS among 89 species of plants related to S. tragus. The MME provided: (1) disease assessments for rare and difficult or impossible to grow species, (2) environmentally independent measures of disease severity, (3) measures of disease severity for species versus a sample of material tested in a greenhouse, (4) objective indicators of susceptible and non-susceptible species, (5) a means to objectively compare disease on targets versus non-targets. Of the 89 species evaluated by the MME, eight native N. American species were predicted to be susceptible. As a result of these predictions, these eight species were further evaluated to determine the amount of actual damage caused by CGS. This was done by comparing root and shoot areas and weights between non-inoculated plants and plants inoculated with CGS. Results showed that several of the species exhibited some minor reduction in root weight and root area, but none of the species had any damage to above-ground plant parts. This supports the BLUP output in the initial host range determination tests. As a result of both analyses, there is no evidence that CGS would cause any non-target effects in nature.

Evidence for Systemic Infection by Puccinia horiana, Causal Agent of Chrysanthemum White Rust, in Chrysanthemum.

Puccinia horiana, causal agent of the disease commonly known as chrysanthemum white rust (CWR), is a quarantine-significant fungal pathogen of chrysanthemum in the United States and indigenous to Asia. The pathogen was believed to have been eradicated in the United States but recently reappeared on several occasions in northeastern United States. The objective of the study presented here was to determine whether P. horiana could systemically infect chrysanthemum plants, thus providing a means of survival through winters. Scanning and transmission electron microscopy revealed the development of P. horiana on the surface and within leaves, stems, or crowns of inoculated chrysanthemum plants artificially exposed to northeastern U.S. winter temperatures. P. horiana penetrated leaves directly through the cuticle and then colonized the mesophyll tissue both inter- and intracellularly. An electron-dense material formed at the interface between fungal and host mesophyll cells, suggesting that the pathogen adhered to the plant cells. P. horiana appeared to penetrate mesophyll cell walls by enzymatic digestion, as indicated by the absence of deformation lines in host cell walls at penetration sites. The fungus was common in vascular tissue within the infected crown, often nearly replacing the entire contents of tracheid cell walls. P. horiana frequently passed from one tracheid cell to an adjacent tracheid cell by penetration either through pit pairs or nonpitted areas of the cell walls. Individual, presumed, fungal cells in mature tracheid cells of the crown and stems arising from infected crowns suggested that the pathogen might have been moving at least partially by means of the transpiration stream. The demonstration that chrysanthemum plants can be systemically infected by P. horiana suggests that additional disease control measures are required to effectively control CWR.