Viable Urediniospores Research Articles

Effect of pathogen concentrations and period of leaf wetness on orange rust severity in Brazilian sugarcane cultivars

The most important management strategy for orange rust of sugarcane (Puccinia kuehnii) is the use of resistant cultivars. Nevertheless, canes with intermediate resistance are still grown over large areas in the Brazilian sugarcane industry, with varying degrees of disease severity. Factors governing the severity of orange rust in these crops are largely unknown. Therefore, the present work aimed to examine the influence of pathogen concentration, period of leaf wetness, and urediniopore survival on sugarcane residue on disease severity. Thirty-day-old plants of cultivars RB72454, RB855156, RB867515, RB92579, RB935744, RB975201, SP81–3250, and SP89–1115 were inoculated with 50 ml of spore suspension with varying urediniospore concentrations (from 1.101 to 107 spores.ml−1) or period of leaf wetness (12, 24, and 48 h, pathogen concentration 1.106 spores.ml−1). Reaction of cultivars was evaluated by assessing leaf area affected. Survival of urediniospores was examined on sugarcane residues at 0, 10, 15, 20 days after separation from the cane plant. Only RB72454, RB92579, and RB855156, classified as susceptible or intermediate, exhibited higher disease levels as pathogen concentration increased. Extended periods of leaf wetness only influenced susceptible/intermediate cultivars, where higher severity occurred with longer period of leaf wetness. Viability of urediniospores collected in winter always had a greater loss of viability compared to spring but both season harbored viable urediniospores on 20-day-old sugarcane residues, suggesting they potentially could play a role in initiation of disease epidemics.

A Multiplexed Immunofluorescence Method Identifies Phakopsora pachyrhizi Urediniospores and Determines Their Viability

Soybean rust, caused by Phakopsora pachyrhizi, occurs concomitantly wherever soybean is grown in the tropical and subtropical regions of the world. After reports of its first occurrence in Brazil in 2001 and the continental United States in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capturing urediniospores, primarily by rain collection or wind traps, and detecting them either by microscopic observations or by immunological or molecular techniques. This system of detection has been touted for use as a potential warning system to recommend early applications of fungicides. One shortcoming of the method has been an inability to determine whether the spores are viable. Our study developed a method to detect viable P. pachyrhizi urediniospores using an immunofluorescence assay combined with propidium iodide (PI) staining. Antibodies reacted to P. pachyrhizi and other Phakopsora spp. but did not react with other common soybean pathogens or most other rust fungi tested, based on an indirect immunofluorescence assay using fluorescein isothiocyanate-labeled secondary antibodies. Two vital staining techniques were used to assess viability of P. pachyrhizi urediniospores: one combined carboxy fluorescein diacetate (CFDA) and PI, and the other utilized (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide] (FUN 1). Using the CFDA-PI method, viable spores stained green with CFDA and nonviable spores counterstained red with PI. Using the FUN 1 method, cylindrical intravacuolar structures were induced to form within metabolically active urediniospores, causing them to fluoresce bright red to reddish-orange, whereas dead spores, with no metabolic activity, had an extremely diffused, faint fluorescence. An immunofluorescence technique in combination with PI counterstaining was developed to specifically detect viable P. pachyrhizi urediniospores. The method is rapid and reliable, with a potential for application in forecasting soybean rust based on the detection of viable urediniospores.

Métodos de preservação in vitro de urediniósporos de Puccinia kuehnii

Com o objetivo de avaliar métodos de preservação de urediniósporos de Puccinia kuehnii, conduziram-se dois bioensaios sendo o primeiro (B1) com diferentes métodos de desidratação e o segundo (B2), com diferentes métodos de reidratação. Em B1 foi adicionado um grânulo de sílica gel para preservação dos urediniósporos nos tubos de microcentrífuga. Foram coletadas folhas com sintomas de ferrugem alaranjada, P. kuehnii, da cultivar de cana-de-açúcar SP89 1115. Os urediniósporos do agente causal de ferugem foram extraídos das folhas com o auxílio de bomba a vácuo. Posteriormente, estes foram acondicionados em tubos de microcentrífuga. Os tratamentos para B1 foram: l- desidratação em sílica gel, liofilização e sem desidratação; ll- temperatura ambiente (20ºC), geladeira (5ºC), congelador (-20ºC) e deep-freezer (-80ºC). Para B2 os tratamentos foram: l- desidratação em sílica gel e sem desidratação; ll- temperatura ambiente (20ºC), geladeira (5ºC), congelador (-20ºC) e deep-freezer (-80ºC); lll- com reidratação e sem reidratação nas avaliações. Para ambos os bioensaios foi realizada a germinação inicial, outras aos 15 e 30 dias de armazenamento e posteriormente a cada 30 dias, até 180 dias. Prepararam-se suspensões de urediniósporos em água e uma alíquota de 0,1 mL foi transferida para placas de Petri contendo meio ágar-água (15g L-1). Essas permaneceram a 20ºC, no escuro. Para a avaliação da viabilidade, procedeu-se a contagem de 200 urediniósporos por placa. Os dados foram submetidos à análise de variância não paramétrica de Kruskal-Wallis e complementadas com o teste de Dunn. Os resultados demonstraram que a viabilidade decresceu em função do tempo, sendo que os melhores tratamentos atingiram 27,6% e 6,6% aos 30 dias, e 12,0% e 1,9% aos 60 dias, para B1 e B2, respectivamente. O método da desidratação em sílica gel seguido do armazenamento a -80ºC foi o único que apresentou urediniósporos viáveis (1,2%) aos 180 dias, para B1. No B2, o melhor método foi preservação com desidratação, armazenados a 5ºC, sem reidratação. Esse método apresentou melhor porcentagem de germinação aos 120 dias (0,4%). Não foi observada influência do fator choque térmico na recuperação de urediniósporos viáveis em B2. A inclusão de grânulo de sílica gel no tubo de microcentrífuga permitiu a recuperação da viabilidade dos urediniósporos aos 180 dias.

Detection of Variants of Wheat Stem Rust Race Ug99 (Puccinia graminis f. sp. tritici) in Zimbabwe and Mozambique.

The migration of Ug99 variants of Puccinia graminis f. sp. tritici is of concern to global wheat production (1). Seven races have been characterized in the Ug99 lineage (3), three of which occur in South Africa (4). During surveys of wheat fields for Ug99 in Zimbabwe and Mozambique in August and September 2010, high stem rust severities were found at Chiredzi, Chisumbanje, and Birchenough in Zimbabwe and at Rotanda in Mozambique. Stem rust was widespread in the lowlands (<800 m above sea level) of Zimbabwe and trace amounts were present in the mid-altitude areas. In Mozambique, stem rust was only observed at Rotanda (sample Moz1001). Collections from Chiredzi (samples Zim1004 and Zim1005), Chisumbanje (Zim1006), and Birchenough (Zim1009 and Zim1010) yielded viable urediniospores for infection studies. According to race analysis conducted on seedlings of the North American stem rust differential set (2) in a greenhouse at 18 to 25°C, Zim1005 and Zim1006 were typed as PTKST and Zim1004 and Zim1009 as TTKSF. Both TTKSF and PTKST were detected in the Zim1010 sample. Race analysis experiments were conducted three times. Urediniospores of isolate Moz1001 were not viable in infection studies, but yielded fungal DNA for simple sequence repeat (SSR) analysis. Using eight selected SSR primer combinations (4), all six isolates clustered within the Ug99 lineage. Isolates Zim1005, Zim1006, Zim1009, Zim1010, and Moz1001 and the stem rust control races TTKSF, TTKSK, and PTKST grouped into two main clusters, with Zim1009 and Zim1010 clustering together and sharing 88% similarity with the rest of the isolates. Zim1005 and Zim1006 were identical to TTKSF and TTKSK, respectively. Zim1004 shared 96% genetic similarity with the TTKSP control, with these two sharing 74% genetic similarity with the remaining isolates. The SSR data correlated with the infection data, except for Zim1004, which was typed as TTKSF but clustered close to TTKSP. Wheat cvs. SC Nduna, SC Shine, SC Stallion, SC Smart, Kana, Insiza, and Dande are predominant in Zimbabwe. Cv. SC Stallion and other unidentified cultivars were susceptible to P. graminis f. sp. tritici in the field in Zimbabwe. In Mozambique, the tall, local cv. Sitsonko was susceptible to P. graminis f. sp. tritici but no infections were observed on SC Nduna or SC Shine. The similarity in P. graminis f. sp. tritici races in Zimbabwe, South Africa, and Mozambique suggests that inoculum is exchanged within the region and explains the detection of race PTKST in South Africa in 2009. Trajectory models showed winds originating at Birchenough in October 2009, where stem rust was observed, passing directly over KwaZulu-Natal, South Africa within 48 to 72 h. Race PTKST was confirmed from collections in KwaZulu-Natal in November 2009 (4). The confirmation of Sr31 virulence in race PTKST in Zimbabwe is important because it provides new geographical records for an Ug99-related race and puts Southern African cultivars with 1B.1R resistance at risk.

Culturing Phakopsora pachyrhizi on Detached Leaves and Urediniospore Survival at Different Temperatures and Relative Humidities.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important foliar diseases of soybean worldwide. In a series of experiments, multiple objectives were addressed to determine the (i) longevity of detached soybean leaves, (ii) reproductive capacity of uredinia on leaves inoculated and/or incubated on the abaxial versus adaxial side of the leaf, (iii) reproductive capacity of uredinia and urediniospore germination when spores were harvested at regular intervals or all at once, and (iv) effect of temperature and relative humidity (RH) on urediniospore germination. A detached-leaf assay using agar medium amended with 6-benzylaminopurine performed better in retarding leaf chlorosis than filter paper alone among five soybean genotypes. Among the three susceptible genotypes tested, detached leaves of cv. Williams 82 had the lowest level of leaf chlorosis and often allowed for the greatest urediniospore production and germination rate. Temperature and RH played significant roles in survival of urediniospore as measured by germination rates. Viable urediniospores were harvested from infected soybean leaves maintained at room temperature (23 to 24°C at 55 to 60% RH) for up to 18 days, whereas freshly harvested urediniospores that were desiccated for 12 h before being placed in vials and maintained at room temperature remained viable for up to 30 days. Urediniospore hydration was the major factor for the dormancy reversion; thermal shock with hydration and no thermal shock with hydration treatments had consistently similar urediniospore germination rates. In the RH experiment, urediniospores harvested from inoculated leaf pieces maintained at 85% RH had the highest germination rates compared with higher and lower RH. Improvement in P. pachyrhizi cultural techniques and understanding of urediniospore survival will enhance our knowledge of the pathogen biology, host-plant relationship, and conditions that favor the infection, reproduction, and survival of the pathogen.

Sources of primary inoculum ofTranzschelia discolorin Australian almond orchards

Sources of primary inoculum of almond rust (Tranzschelia discolor) were investigated. Retained leaves, fruit not removed during harvest, leaf litter on soil and green or lignified shoots were monitored for viable urediniospores between harvest and the emergence of new leaves. Retained leaves were confirmed as the primary source of overwintering inoculum in almonds with up to 27% of the urediniospores viable at the time of new leaf emergence. Viable urediniospores were also found on symptomless green shoots, retained fruit and lignified shoots. The viability of urediniospores on leaf litter rapidly declined to nil within two months. Inoculation of almond stems and fruit showed that both were capable of producing uredinia, but were less frequently infected compared with leaves. Urediniospores from stem or fruit lesions, symptomless green or lignified shoots, or leaf litter are not considered to play a major role in the epidemiology of almond rust. Inoculation of cherry, peach, nectarine and plum plants with urediniospores collected from almond leaves did not produce disease symptoms indicating that other stone fruit are unlikely to provide an alternative overwintering host for almond rust. Removal of retained leaves in combination with strategic fungicide use may help growers to optimise rust control in almonds.

Winter Survival of the Soybean Rust Pathogen, Phakopsora pachyrhizi, in Florida.

Soybean rust (SBR) survival and host availability (kudzu, Pueraria spp.) were assessed from November 2006 through April 2007 at six sites from the panhandle to southwest Florida. Micro loggers recorded both temperature and relative humidity hourly at each location. Periods of drought and cumulative hours below 0°C correlated with kudzu defoliation. Inoculum potential from detached kudzu leaves was evaluated in vitro under various temperature and relative humidity levels. Kudzu leaves with SBR kept at 4°C produced viable urediniospores with the highest germination at all moisture levels over time. Freezing temperatures (-4 and -20°C) drastically reduced spore germination. However, when leaves were incubated at low (<35%) relative humidity, inoculum potential was prolonged. Results from this study demonstrate that both temperature and relative humidity impact P. pachyrhizi in the field and in vitro, and that detached kudzu leaves have the potential to serve as an inoculum source in kudzu stands.

Disease cycles of Puccinia menthae on commercial Mentha species growing in north-east Victoria, Australia

Mint oil production from peppermint (Mentha × piperita) is a new industry in the river valleys of north-east Victoria, Australia. In this study, the disease cycles on M, × piperita, M. × gracilis (Scotch spearmint) and M. spicata (spearmint) growing in the Ovens Valley, north-east Victoria, were monitored for four years. The complete life cycle was observed annually on M. × gracilis and M. spicata, but only urediniospores and teliospores were observed on M. × piperita. Viable urediniospores were always present on M. × piperita, confirming that urediniospores carry the disease over winter on peppermint grown in north-east Victoria. On the two spearmint species, viable urediniospores were observed only on M. × gracilis during the 1997 winter. Reports on the disease cycle of P menthae in the mint-growing regions of the United States of America, the United Kingdom and New Zealand have shown that P. menthae is macrocyclic, with teliospores as the mechanism for overwintering, but the present study concludes that it is microcyclic on peppermint growing under local conditions.

Survival ofPuccinia reconditaandP. graminisUrediniospores Exposed to Temperatures from Subfreezing to 35 C

Viable urediniospores and dormant mycelial infections are the principal inoculum sources that contribute to the establishment and development of destructive wheat rust epidemics in the central Great Plains. Viability of urediniospores of Puccinia recondita and P. graminis was measured by exposure in environmental chambers set at constant temperatures ranging from −6 to 35 C. Exposure of P. recondita and P. graminis urediniospores for up to 120 h to constant temperatures of 5-35 C did not significantly (P ≤ 0.05) affect spore viability among isolates. However, P. recondita and P. graminis urediniospore viability as measured by germination observed after exposure to constant temperatures of -6, −4, −2, 0, 5, 10, 20, 25, 30, or 35 C for 24, 48, 72, 96, or 120 h indicated significant differences in viability among temperature treatment means (P ≤ 0.05). Urediniospores remained viable up to 864 h at constant temperatures between 10 and 30 C and up to 504 h at constant temperatures of 5 and 35 C. Viability of urediniospores exposed at temperatures ranging from −2 to 0 C decreased rapidly after the first 4 h of exposure. At below-freezing temperatures, viability of spores declined rapidly after 1-2 h of exposure. Viability of urediniospores exposed to freezing or below-freezing temperatures was not restored by heat shocking at temperatures typically encountered in the field during an epidemic. Infectivity of P. recondita and P. graminis urediniospores exposed at 5-35 C for 120 h was not significantly different from infectivity of unexposed urediniospores; an average of 58% of the viable urediniospores produced appressoria

Survival ofPuccinia reconditaandP. graminisUrediniospores as Affected by Exposure to Weather Conditions at One Meter

Viable urediniospores and dormant mycelia are the principal inoculum sources that contribute to the establishment and development of destructive wheat rust epidemics in the central Great Plains of the United States. Inoculum survival was measured by exposing urediniospores of Puccinia recondita and P. graminis to field conditions occurring at 1 m above ground level throughout two crop years. Four phases (summer, fall, winter, and spring) important in survival of urediniospores as potential inoculum in epidemic development were used in data analysis. Survival of P. recondita and P. graminis urediniospores during wheat dormancy (winter) was reduced to 10-11 and 17-21%, respectively, within 24 h [...]

Survival and Germination of Urediniospores of Puccinia recondita and P. graminis

Reproducible, uniform inoculum distribution was obtained using a settling tower and transfer paper technique to measure effect of temperature, moisture, light intensity, and host : parasite combination on viability and survival of urediniospores of Puccinia recondita and P. graminis. Significant differences were observed on water agar plates at the P = 0.05 level in percentage of germination among cultures of both species. Significant differences were observed on host leaves at the P = 0.10 level in percentage germination among host : parasite combinations. Germination percent decreased with age of the urediniospore in both species. Increasing light intensity from levels found in the canopy on cloudy days to the intensity found in full sunlight at the top of the canopy significantly increased germination of spores. Survival of urediniospores suspended in the atmosphere was significantly longer during summer conditions than during winter conditions. Wheat leaf and stem rust, incited by Puccinia recondita Rob. ex Desm. f. sp. tritici and P. graminis Pers. f. sp. tritici, respectively, have reduced wheat (Triticum aestivum L.) yields by as much as 50% in the Great Plains region of the USA (Samborski and Peterson, 1960; Eversmeyer and Bender, 1974). Viable urediniospores and dormant mycelia are the principal inoculum sources (Burleigh et al., 1969) that contribute to the early spring establishment of the disease and the eventual development of an epidemic in the Central Great Plains. Given the early occurrence of the disease (Eversmeyer et al., 1988), favorable environments and compatible host: parasite inter

Studies on the mechanism of cross-protection in cereal rusts

Using a spore settling tower to inoculate oat leaves uniformly with known densities of urediniospores of oat and wheat rusts, a quantitative study was made to investigate the mechanism of cross-protection in cereal rusts. The number of pustules produced per unit area of leaf surface by oat rusts was markedly reduced by prior inoculation with viable urediniospores of either wheat or oat rusts. Non-viable spores failed to induce cross-protection. The degree of cross-protection varied directly with the inoculum density of the preinoculated rust. Maximal cross-protection occurred when the interval between successive inoculations was 4 days or more. Microscopic examination of the infection process showed that if an oat rust produced appressoria over stomata which were already occupied by appressoria of the preinoculated rust, penetration failed to occur and the infection process was stopped. If, however, appressoria were produced over stomata not already occupied by appressoria of the previously inoculated rust, the frequency of penetration was the same as that on uninoculated control leaves. It would seem that the halting of the infection process at the penetration phase was responsible for cross-protection in cereal rusts.