© 2012 Plant Management Network.
Documentation of an Extended Latent Infection Period by Phakopsora pachyrhizi, the Soybean Rust Pathogen
N. A. Ward, R. W. Schneider, and C. L. Robertson, Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803
Ward, N. A., Schneider, R. W. and Robertson C. L. 2012. Documentation of an extended latent infection period by Phakopsora pachyrhizi, the soybean rust pathogen. Online. Plant Health Progress doi:10.1094/PHP-2012-0321-01-RS.
Phakopsora pachyrhizi, the soybean rust pathogen, overwinters on kudzu in the southern United States. However, even with severely affected kudzu adjacent to soybean fields, disease symptoms do not occur on soybeans until plants are in mid-reproductive stages of growth during mid to late summer. These observations suggested that soybeans are exposed to airborne inoculum of the pathogen long before symptoms occur, and we hypothesized that these plants may be latently infected. This hypothesis was confirmed by using quantitative real-time PCR (qPCR) to detect the rust pathogen in soybean leaves 18 to 60 days before symptoms were observed. Additionally, DNA of P. pachyrhizi increased only slightly until one week before symptoms developed. Results from this study documented that soybeans can become infected by the rust pathogen during early stages of plant growth, but symptoms often develop during the mid-reproductive stages. This extended latent infection period may be an optimum time for fungicide applications.
Soybean rust (SBR), caused by Phakopsora pachyrhizi, was first reported in Asia in 1904 and has since spread to Africa and the Americas (12,19,30). Disease losses caused by SBR have ranged from 10 to 90%, though there are reported losses as high as 100% (1). SBR was first discovered in the United States in 2004, and yield losses between 35 and 40% were reported in Louisiana and as high as 82% in Florida on susceptible varieties that were not sprayed with fungicides (22) (D. R. Walker, personal communication).
Breeding efforts have yet to produce resistant cultivars (6); therefore, disease management studies have focused mainly on fungicide applications. These investigations demonstrated that preventative applications of fungicides must be accurately timed and applied very early in the infection process for effective control of the disease (13,14,21,24). Such practices may lead to unnecessary fungicide applications, especially throughout the southern United States because growers fear rapidly escalating epidemics such as those seen in Africa, Asia, and South America (19,20,29).
Phakopsora pachyrhizi has become endemic in the southern USA because it also infects kudzu (Pueraria spp.), which is widespread throughout the southeastern United States. The pathogen overwinters on kudzu leaves that survive winter conditions in the lower Gulf South, and these leaves are a source of urediniospores that spread to soybeans and cause SBR each year (7,8,16,17,19,23,27). Despite this source of inoculum each spring, the disease usually is not reported on soybean until the summer months. In our experience in commercial fields, research plots, and sentinel plots, SBR is not observed until mid-reproductive stages (R5) of plant growth when seeds begin to develop (2,3,5,31). Furthermore, it has been commonly observed that plants growing side by side but at different growth stages (from vegetative to late-reproductive) show symptoms only after they reach R5 (unpublished). This suggests that vegetative plants were exposed to inoculum, but symptoms developed only after plants reached a specific reproductive growth stage. However, in laboratory and greenhouse studies, SBR symptoms typically appear 7 to 14 days after inoculation (1,9,15,26). The objective of this study was to determine whether infection of soybean in the field occurred early in the growing season and whether there was an extended latent infection period. We monitored the development of the rust pathogen using quantitative real-time PCR (qPCR), a technique used to quantify DNA and hence provide a quantitative assessment of the pathogen in leaf tissue.
We evaluated three soybean fields for SBR in Louisiana and one in Florida in 2009 and 2010. Soybean plants were not inoculated with the pathogen; epidemics were initiated from naturally occurring inoculum. We were interested in whether or not plants were infected well in advance of symptom expression, and we chose the R1 growth stage as our starting point for sampling because it is easily recognizable and because signs and symptoms of soybean rust usually appear at the early-pod-fill growth stage (R5), which occurs at least 6 weeks after R1.
To monitor the SBR pathogen in soybean, 10 trifoliolate leaves (30 leaflets) were sampled weekly from each field. Immediately after sampling, each apical leaflet was quantitatively assessed for symptom expression. We examined three fields of vision (5 cm²) per leaflet with a dissecting microscope at 25× magnification. Leaflets were washed for 30 s in water to remove surface inoculum and debris, allowed to air dry at room temperature for 10 m, and then stored in freezer bags at −20˚C until DNA was extracted. DNA of the SBR pathogen was quantified using qPCR as described below.
Louisiana field studies 2009. Phakopsora pachyrhizi overwintered in Baton Rouge on kudzu during the winter of 2008/2009. Symptomatic kudzu was reported on 5 June 2009 less than 10 km from the Louisiana State University Agricultural Centers Ben Hur Research Farm where test fields were located. Field 1: Soybean cultivar Asgrow 3905 (Monsanto Corp., Creve Coeur, MO) was planted on 17 April 2009. Ten trifoliolate leaves were collected weekly beginning 4 June 2009 at flowering (R1) and continued through 29 July 2009 at senescence (R7). Field 2: Soybean cultivar Asgrow 5802 (Monsanto Corp.) was planted on 20 May 2009 in a field adjacent to Field 1. Sampling began on 23 July 2009 at flowering (R1) and continued through 25 September 2009 at senescence (R7). Field 3: Soybean cultivar Asgrow 6202 (Monsanto Corp.) was planted 21 July 2009 adjacent to Fields 1 and 2. Leaf collections began on 20 August 2009 during mid-vegetative stages (V4) and continued through 22 October 2009 at senescence (R7). Nonhost controls: Corn and morning glory vine were included as nonhost controls in order to quantify urediniospores that may have landed on leaf surfaces but did not initiate infections. This provided an indication of the amount of inoculum available. Three corn leaves were sampled from the outer edge of the field on a weekly basis between 4 June and 29 July 2009. Morning glory was used as a nonhost control between 5 August and 30 September 2009. Ten leaves were collected weekly from wild vines on the outer edge of soybean plots.
Florida field study 2010. Field 4: On 14 July 2010, soybean cultivar Pioneer 95Y20 (Pioneer Hi-Bred, Johnston, IA) was planted at the University of Florida North Florida Research and Education Center in Quincy, FL. SBR was reported on kudzu approximately 180 m from soybean plots about one week after soybeans were planted. However, SBR was not detected in this field until 12 September 2010, 68 days after planting, when plants were in the R5 growth stage. Sampling began on 2 August 2010 during late vegetative stages (V6) of growth and ended on 26 September 2010 at the onset of senescence (R7). Morning glory from adjacent row was sampled as a nonhost control in order to quantify surface inoculum as described above.
All soybean plots were maintained according to common agricultural practices with regard to insect and weed control and fertilization. None of the plants in this study was sprayed with fungicides.
Primers and probe. Primers and probe were based upon previous work by Frederick et al. (4) in which primers Ppm1 and Ppa2 were bound specifically to DNA of P. pachyrhizi. FAM-probe was labeled at the 5 end with the fluorescent reporter dye 6-carboxy-fluorescein (FAM) and at the 3 end with the quencher dye 6-carboxytetramethyl-rhodamine (TAMRA) for quantification of fluorescence in qPCR assays.
DNA extractions. Freezer bags containing leaves from each plot were stored at −20˚C until they were ground in liquid nitrogen with mortar and pestle. Genomic DNA was extracted from 50 mg of ground leaf tissue using Qiagens DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. Final DNA concentrations were adjusted by dilution to 10 ng/μl for use in qPCR assays.
qPCR assay. qPCR was used to quantify the amount of DNA of the SBR pathogen. Triplicate samples were tested in a total volume of 25 μl per reaction. The qPCR instrument software package (Applied Biosystems, Carlsbad, CA) automatically calculated the critical threshold values (Ct) for each reaction (11). Ct values were converted to picograms (pg) DNA according to standard curves. Less than 1.0 pg DNA of the soybean rust pathogen was detected with this protocol.
Early Infections by the Soybean Rust Pathogen
Field 1: Infection was expressed as pg of DNA of P. pachyrhizi per 10 ng of soybean DNA. Latent infection was detected in April-planted soybeans on the first sampling (R1) in which we quantified 1.4 pg of DNA of the soybean rust pathogen (Figs. 1 and 2). However, at the R6.5 growth stage, DNA of the SBR pathogen increased significantly to 2.6 pg. One week later (R7), we detected 10.2 pg of DNA. Symptoms of SBR were not observed, but necrotic tissue and other disease lesions made leaf examinations difficult. Surface inoculum was not detected using corn as a nonhost control.
Field 2: At first flowering (R1), 2.5 pg DNA of the SBR pathogen were detected, which indicated that soybean plants were infected (Fig. 1). Amounts of DNA of the SBR pathogen increased from 7.5 pg of DNA on asymptomatic leaves at the R5 growth stage to 53.5 pg when first signs and symptoms were observed one week later (R5.5) (Fig. 2). There was a 43-day latent infection period. Surface inoculum was not detected on corn during this study.
Field 3: Latent infection (2.0 pg of the SBR pathogen) was detected at the V4 stage. Symptoms developed at R5, which was 33 days after the onset of latent infection. We detected 2.6 pg of DNA of the SBR pathogen one week before symptoms developed. After symptoms were first observed, DNA of the SBR pathogen increased to 60.5 pg. Surface inoculum (1.0 pg DNA) was detected on morning glory at the edge of Fields 2 and 3 on 4 September 2009.
Field 4: SBR was detected on kudzu, which was approximately 1.5 km from the soybean plots, at the Florida research farm one week after soybeans were planted, but only a limited amount of disease developed (less than 1% incidence) on soybeans. Latent infection on soybean was confirmed at 0.7 pg DNA 18 days before symptoms developed at R5. Epidemics of SBR were delayed throughout the Gulf South in 2010 probably because kudzu leaves were killed by extended periods of subfreezing temperatures during the previous winter (27). Surface inoculum was not detected on morning glory during the course of this experiment.
We documented that SBR symptoms first appeared during mid-reproductive stages (R5 and later) of plant growth when seeds began to develop in pods. This contrasts with previous laboratory and greenhouse studies in which symptoms typically developed 7 to 14 days after inoculation (1,10). SBR has been reported on cotyledons and unifoliolate leaves of young soybean plants under intense disease pressure in Brazil (1). However, in the southern United States there have been no reports of symptom development on field-grown plants at these early growth stages. In previous studies infected leaflets developed symptoms only after extended periods of incubation when plants were in their mid-reproductive stages of growth, which suggests that even when leaves are latently infected, symptoms do not occur until plants reach a specific reproductive growth stage. Furthermore, incubation of field-infected leaves during early reproductive stages failed to result in symptom development (unpublished). Limited research suggests that the rate of symptom development is directly related to physiological age of the plant (25,29). While we do not know the physiological changes that predispose soybean plants to the onset of symptoms when they reach R5, it is well known that soybean plants undergo substantial changes during reproductive growth (18). The most pronounced changes with regard to carbon partitioning and rates of protein and oil accumulation in the seeds occur at the mid-pod-fill stage (R5.5) (28). We speculate that these changes are associated with susceptibility in older leaves, which are the first to show symptoms (1).
In 2009, field plots in Baton Rouge were established less than 10 km from infected kudzu, and sporulating pustules were reported in early February on kudzu leaves that had been protected from frost. April-planted soybeans never developed symptoms, but latent infection was detected during early reproductive stages (R1). Collection of leaf samples did not begin in this field until R1 at which time we detected a high concentration of DNA of the SBR pathogen. Although we never observed signs or symptoms in Field 1, there was an increase in DNA of the SBR pathogen at the R7 growth stage. We attempted to find SBR pustules at the R7 growth stage, but Cercospora leaf blight and other late-season diseases hindered accurate assessment. It is possible that we overlooked signs and symptoms that may have developed late in the season. In Field 2, we detected infection in the May-planted field during early reproductive stages of plant growth. Latent infection persisted for 33 days until symptoms appeared during the R5 growth stage. We suspect that small amounts of SBR inoculum were present during early vegetative stages of growth in Field 2 because we detected DNA of the SBR pathogen in an adjacent field (Field 1) just as seedlings emerged in Field 2. A small amount of DNA of P. pachyrhizi was detected only once on morning glory. It is possible that the amount of surface inoculum was lower than the detection limits of our qPCR assay, which was about 1.0 pg DNA of the SBR pathogen. Field 3 was planted as soybean plants from Field 1 reached their highest concentration of the SBR pathogen and as latent infection was first detected in Field 2. July-planted soybeans in Field 3 became infected during vegetative stages of plant growth, but symptoms were not observed until R5. Thus, we documented latent infection periods of more than 30 days in all fields in this study, and we provide evidence that inoculum was present throughout the life cycles of the plants in Fields 2 and 3.
Besides East Baton Rouge Parish, SBR typically overwintered in south-central Louisiana in Acadia, Iberia, and St. Martin Parishes. The first reported instances of SBR in 2009 in Louisiana were from soybean sentinel plots in these parishes. These soybeans did not develop symptoms until the R4 to R6 stages of growth despite nearby sources of SBR inoculum on kudzu (27). These observations support our conclusion that SBR symptoms usually do not develop until plants enter pod filling stages of reproductive growth even though they may be infected several weeks before signs or symptoms are observed.
In 2010, we evaluated soybean plants in northern Florida, where the pathogen is known to overwinter on kudzu, and where the first symptoms of SBR are observed on soybean in the United States each year. Temperatures reached record lows throughout the Gulf South during the winter of 2009/2010, and SBR was reported on kudzu and soybean later than normal. In the Florida field (Field 4), the SBR pathogen was detected on soybeans by qPCR during early reproductive stages of growth, but symptoms did not develop until the R6 growth stage at which time very few pustules were observed (disease incidence was less than 1%). Nevertheless, there was an extended latent infection period.
Sporulation was not as prolific on kudzu as observed in soybean. In fact, we did not detect surface inoculum on our nonhost control plants until 2 weeks after symptoms appeared on soybean. Because our qPCR assay was able to detect as little as 1.0 pg of DNA of the soybean rust pathogen, we conclude that inoculum load was extremely low when kudzu served as the primary source of inoculum in the area. In one instance, surface inoculum was detected on morning glory during the peak of the 2009 SBR epidemic. It is possible that urediniospores do not adhere well to leaves of corn and morning glory.
In these studies, we determined that even after P. pachyrhizi infected soybean, DNA of the pathogen increased only minimally in leaves. Approximately one week before symptom development, there were significant increases in amounts of DNA of the SBR pathogen. Additionally, as disease severity increased, we documented a concomitant increase in DNA of the pathogen. Given the extended time span that plants were exposed to inoculum and the protracted latent infection period, it seems likely that physiological changes in soybean leaves are required for rampant colonization by the pathogen followed by the production of uredinia within about one week.
In field studies, fungicides applied during the latent infection period, as compared to applications at first symptom development, were most effective in disease suppression (Schneider, unpublished). Current practice is to spray at 5% incidence, but our results show that by the time symptoms occurred, the pathogen had entered a logarithmic increase in biomass within leaf tissue, and fungicide efficacy may be compromised during this phase of the infection process. Lamour et. al. (10), working with inoculated greenhouse-grown plants that were incubated in a moist chamber, demonstrated that DNA of P. pachyrhizi could be detected 6 days after leaves were inoculated with a urediniospore suspension but before symptoms were apparent. Their objective was to demonstrate that the pathogen could be detected for diagnostic purposes using qPCR protocols before the development of symptoms. They concluded that qPCR assays may be useful for deciding when to apply fungicides. Results from our field studies extend their conclusion with the provision that it may be possible to delay the onset of disease by applying fungicides shortly after flowering but several weeks in advance of symptom development.
During the past few years, sentinel plots have been planted in advance of the commercial soybean crop. These plots were then monitored on a weekly basis, and fungicide spray advisories were issued when pustules were first observed in these plots. Our findings suggest that widespread sampling of leaves in commercial fields for qPCR analyses beginning during late vegetative stages of plant growth may provide a more reliable and quantitative indicator of imminent disease development.
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