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Use of digital images to differentiate reactions of collections of yellow starthistle (Centaurea solstitialis) to infection by Puccinia jaceae
Biological Control 28 (2003) 171–179 www.elsevier.com/locate/ybcon
Use of digital images to differentiate reactions of collections of yellow starthistle (Centaurea solstitialis) to infection by Puccinia jaceae D.K. Berner* and L.K. Paxson Foreign Disease-Weed Science Research Unit, Agricultural Research Service, US Department of Agriculture, 1301 Ditto Avenue, Ft. Detrick, Frederick, MD 21702, USA Received 18 September 2002; accepted 14 April 2003
Abstract Yellow starthistle (YST) (Centaurea solstitialis) is an invasive weed in the USA and a target of classical biological control. However, the precise geographical origin(s) of YST in the USA is not known. This study was conducted to learn whether the origin(s) of YST in California, USA could be determined on the basis of digital image analysis (DIA) of YST reactions to isolates of the rust Puccinia jaceae. One trial was conducted with two isolates of P. jaceae and another trial with only one of these isolates. Six different YST collections were tested. The DIA consistently detected and measured rust pustules on YST leaves. Compared to visual assessment, DIA was a fair predictor of the number of rust pustules per leaf. There were no differences between isolates of P. jaceae in reactions of different YST collections in the first trial. Differences in plant reactions between trials were predominately due to more disease incidence and severity in the second trial, which was probably from the greater amount of inoculum. Based on analyses of variance and canonical discriminative analyses, YST from California appeared most closely related to the YST collection from Erzurum, Turkey. In examination of squared Mahalanobis distances, all collections, except the collection from Turkey, were significantly distant from the California collection in both trials. More studies are necessary to determine the precise area(s) of origin (within Turkey and neighboring countries) of YST in the USA. Digital image analysis of rust reactions can be a powerful tool to do this. Published by Elsevier Science (USA). Keywords: Carduus nutans; Carduus thoermeri; Centaurea solstitialis; Digital image analysis; Puccinia carduorum; Puccinia jaceae; Rust; Yellow starthistle
1. Introduction Yellow starthistle (YST) (Centaurea solstitialis L., Asteraceae) is an important invasive weed, predominately in rangelands and natural areas, in the western USA (Cheater, 1992). It is the focus of numerous classical biological control efforts (Smith et al., 2001). Classical biological control involves the introduction of an insect or pathogen from the area from which the weed originated. This follows the presumption that the insect or pathogen is one of the multitudes of natural enemies that keep the weed under control in its native habitat (Wapshere et al., 1989). In the case of YST, the
* Corresponding author. Fax: +1-301-619-2880. E-mail address: [email protected] (D.K. Berner).
1049-9644/$ - see front matter. Published by Elsevier Science (USA). doi:10.1016/S1049-9644(03)00096-3
native habitat of the plant is generally described as Eurasia (Maddox, 1981). However, not all insects or pathogens of YST from Eurasia have the potential to control YST in the USA. To determine what specific area, within the native habitat of the weed, might hold the most promise to find effective insects and pathogens of YST in the USA, it is desirable to determine, within a relatively small geographical area, where the introduced weed population originated. One potential biological control agent of YST is the rust fungus Puccinia jaceae Otth. that does substantial damage to the weed (Shishkoff and Bruckart, 1993) and is relatively host-specific (Bruckart, 1989). Currently, one isolate of P. jaceae from Turkey is on the verge of release in the USA for classical biological control of YST (Federal Register, 2002). Other, more damaging isolates of P. jaceae might also be found in the natural
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range of YST, once that origin is determined. These isolates could then be released in a relatively short period of time given that host-range studies and approval for release of one isolate have already been completed. Because rust pathogens can be specific to the level of single gene differences among cultivars (Flor, 1971), disease reactions of different host plants (and populations) can be used to differentiate both susceptible (and resistant) host cultivars and pathogen races (Flor, 1971; Stakman et al., 1962). Because of the high degree of specificity, reactions of geographically diverse collections of YST to infection by isolates of P. jaceae may be accurate indicators of the relatedness among YST collections and could help to determine within a relatively well-defined area: (1) the geographical origin of YST that was introduced to California and (2) where more aggressive and damaging isolates of P. jaceae, relative to the isolate planned for release in the USA, might be found. Of specific interest is the reaction of different YST collections to the isolate of P. jaceae that is planned for release in the USA. Because this isolate has been extensively tested and is virulent and aggressive on YST from California (Bruckart, 1989), and because of the aforementioned specificity, disease reactions of different YST collections to this isolate alone could accurately indicate relatedness of YST collections to YST from California. Degree of relatedness of other collections to the California collection is the only parameter of interest. Amplified fragment length polymorphism (AFLP) studies are also in progress on the YST collections used in this study, as well as on other YST collections (Luster et al., 2001), and these data could, in the future, be combined with rust-reaction data to further narrow the origin of YST in California. Objective analysis of plant reactions to rust infections has been difficult because of the various sizes and numbers of rust pustules on individual plants that otherwise might suggest a range from susceptible to resistant reactions. Consequently, different rating scales have been developed in an attempt to determine plant reactions to rust infections. In wheat, the standard rating scale for disease reaction to stem rust uses seven rating classes, in addition to Ô+Õ, Ô)Õ, Ô++Õ, and Ô ¼ Õ modifiers, to describe disease reactions of cultivar isolate based on the presence and size of uredinia (Stakman et al., 1962). Since this scale was designed to evaluate relatively genetically homogeneous cultivars, it may not be the most appropriate to evaluate disease reactions of highly heterogeneous weed populations. To evaluate rust pathogens for efficacy as biological control agents of weeds, another scale was developed to characterize disease reaction of individual plants within weed populations (Bruckart et al., 1996). This scale uses: (1) five rating classes based on the number of sori (either uredinia or telia) per square centimeter of leaf tissue and (2) the amount, as percent, of symptomatic leaf tissue. Both
scales depend, at least in part, on subjective evaluation of each sample (leaf or plant), and the consistency between samples depends heavily on the precision of the rater. Neither scale actually quantifies both number and size of the rust pustules relative to total leaf area. Digital image analysis (DIA) has been used extensively to quantify the effects of plant diseases on host plants (Nilsson, 1995). For the most part, DIA has been used to measure chlorotic and necrotic areas of diseased leaves (Lindow and Webb, 1983) and roots (Biernacki and Bruton, 2001), but it also has been used to differentiate symptoms caused by rust infections (Bacchi et al., 1992) and to differentiate isolates of rusts (Ball et al., 1992). The objectives of this study were to use DIA to evaluate disease reactions of different collections of YST to isolates of P. jaceae and then to compare the collections based on an aggregate of these disease reactions for each collection, to the collection from California, USA. It was hoped that this would enable us to determine, within a narrow geographical area, where YST that was introduced to California originated and where more aggressive isolates of P. jaceae, for eventual release in the USA, might be found.
2. Materials and methods 2.1. Plant establishment The study was conducted in the biological containment facility of USDA/ARS Foreign Disease-Weed Science Research Unit (FDWSRU) at Fort Detrick, MD. Yellow starthistle plants were grown from collections of seeds made by Dr. F. Di Cristina, Dr. M. Cristofaro, Dr. P. Pecora, Dr. S. Resnik, and Dr. C. Roche in the fall of 1999 from fields close to Limassol, Cyprus (34°460 N, 32°420 E); Bari, Italy (41°030 N, 16°150 E); Jarash, Jordan (32°150 N, 35°370 E); Krasnodar, Russia (45°030 N, 38°550 E); Palermo, Sicily (36°540 N, 13°590 E); Terragona, Spain (41°300 N, 00°500 E); and Erzurum, Turkey (40°100 N, 40°230 E). In each location, seeds of individual plants were harvested and packaged separately from seeds of other plants. Seeds of YST from a non-specific (bulk) collection in the USA were collected from Solano County, California (38°150 N, 122°020 W) in the fall of 1997 and were provided by Dr. M. Pitcairn (California Department of Food and Agriculture). Two trials of the test were conducted. In each trial, seeds of YST were pre-germinated by surface disinfesting the seeds for 5 min in 2% NaOCl and then placing them in sterile petri dishes that contained 1.5% (w/v) water agar. Seeds of individual plants from each collection were pre-germinated in separate petri dishes. After emergence of radicles, developing seedlings were transplanted into individual cells of a 32-cell (8 4 cell)
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flat filled with steam-sterilized potting soil (2:1:1, v/v, soil, sand, and peat). Each cell in the flat measured 6 6 6 cm. Seedlings from individual plants in each collection were transplanted into single rows of eight cells in each flat. One seedling was transplanted into each cell. Each of four sets of two flats contained all of the collections tested with each rust isolate in each trial. In each of the four sets of flats, eight plants (one row) of each collection were grown. Thus, 32 plants (4 rows of 8 plants in separate flats) of each collection were tested for each rust isolate in each trial. Positions of collections (rows) within each two-flat set were randomly assigned. Collections (rows) within each of the four sets of flats were the replicated experimental units and plants within rows were the sampling units. When possible, seedlings from different parent plants in the same collection were planted in successive cells within a row. In cases when transplanted seedlings died, these were replaced with seedlings from the same parent plant in the same collection, if available. In some cases there were not enough seeds or seedlings of a parent plant in a collection to replace dead plants; these were then replaced with seedlings of another parent plant in the same collection, and the position of these plants in the flats was noted.
compressed air at 34.5 kPa. All leaves of all plants were sprayed until the suspension ran off the leaves. Inoculation with each rust isolate was done separately to avoid inadvertent cross-inoculation of the plants with the unintended isolate. Immediately after inoculation, the flats were placed overnight, for 12–15 h, in dark dew chambers set at 19 °C. Flats of plants inoculated with the different isolates were placed in separate dew chambers. The following day the flats were transferred to greenhouses with ambient temperatures between 24 and 26 °C. Flats of plants inoculated with the different isolates were placed in separate greenhouses. In the evening, the flats were returned to the respective dew chambers, for 12– 15 h, and the dew chamber-greenhouse rotation was repeated for 3 days and nights, after which the plants were kept constantly in the greenhouses. This procedure was repeated for the second trial that was planted on 30 March 2001 and inoculated on 20 April 2001 with the isolate of P. jaceae from Ankara, Turkey. There were not enough urediniospores to include the isolate from Bulgaria in this trial, but the amount of inoculum for the isolate from Turkey was increased to 2.5 mg (about 7.75 104 spores) per plant.
2.2. Inoculation
2.3. Data collection
In the first trial, seedlings were transplanted on 17 November 2000, and dead plants were replaced on 20 November 2000. Approximately 2 g of a complete fertilizer (19% N, 6% P2 O5 , and 12% K2 O) were added to the soil around each plant two weeks after planting. At 28 days after planting, four, two-flat sets of plants were spray-inoculated with urediniospores of an isolate of P. jaceae that was collected from near Ankara, Turkey in 1984. This isolate will be released in the USA in the near future for classical biological control of YST. Four additional two-flat sets of plants were similarly inoculated with an isolate of P. jaceae collected from an unspecified location in Bulgaria in 1978. Spores of each isolate had been periodically increased, since collection, on YST plants in greenhouses at FDWSRU and were maintained in a viable condition in cryovials in liquid nitrogen at )196 °C. For each isolate, 6.4 mg of urediniospores (about 0.4 mg or 1.25 104 spores per plant) were weighed and suspended in 100 ml of distilled water that contained 0.125% (v/v) oxysorbic 20 polyoxyethylene sorbitan monooleate to break surface tension. The amount of inoculum applied to each plant was limited by the total amount of urediniospores available for the isolate from Bulgaria and the desire to use the same amount of inoculum of each isolate. To ensure uniformity in application, this suspension was divided into two aliquots, and each 50-ml aliquot was added to an atomizer and sprayed onto the same two, two-flat sets of plants with
In the first trial, data were collected 36 days after inoculation when abundant pustules were evident on leaves and no further pustule development was occurring. In the absence of free moisture, no secondary pustules or disease cycles occurred in the greenhouses. Each plant was uprooted, the total number of leaves was counted, and the plant number, collection, rust isolate, and flat number were recorded. Leaves with pustules were then harvested at the base of the petiole, and each diseased leaf was tagged and the position recorded. Leaf number Ô1Õ was nearest the crown at the base of the plant, and successive leaves received numbers Ô2Õ and so on. The harvested leaves were then placed pustule side down on the glass bed of a digital scanner (HewlettPackard model 6100C), scanned at 600 dpi, and saved, at the same resolution, in individual computer files named according to YST plant and collection, rust isolate, flat number, and leaf position. Data collection in the second trial also followed this procedure. 2.4. Digital analysis The number of pustules per diseased leaf, mean area of pustules per leaf, and total leaf area were collected with digital analysis software (SigmaScan Pro 5.0, SPSS Science, SPSS, Chicago, IL). Macro programming routines based on Visual Basic were written with the software to automate the digital analysis process and to provide a consistent protocol for each reading. To
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analyze pustule number and area, all of the following steps were programmed and used for the color images of each leaf: the brightness was increased by 30%, the contrast was increased by 20%, the image was converted to the 8-bit gray scale, and pixels within the light intensity range of 67–129 were highlighted with color overlays. This range corresponded to the light intensity of the pustules and was used to differentiate darker pustules from lighter shaded leaves and to determine the precise shape of each pustule. The overlays, representing pustule number and size, were counted and measured by the software. Pustule area was measured as total number of pixels within the color overlay of each pustule. Because dark areas along veinations and creases of some leaves were also overlaid, the macro was modified to restrict the areas mathematically to those areas interpreted as pustules to circular-to-slightly oval shapes within the range of pustule sizes that were visually observed. Objects that did not fit these criteria were omitted from pustule counts and measurements. Total leaf area was measured using a separate programming routine. Each leaf was first analyzed for mean pustule area and total pustule number and then reanalyzed for leaf area. A data set was created to include total leaf area, mean pustule area per leaf, total pustule number per leaf, and the appropriate leaf identifiers, e.g., YST plant and collection, rust isolate, and flat number. To compare DIA with visual assessment, 50 diseased leaves were selected at random (with a random number table), and the pustules on scanned images of these leaves were counted manually. The count was then compared, as the dependent variable in regression analysis, to the count obtained from the digital analysis software.
unit leaf area, and transformed pustule area per unit leaf area. With these as dependent variables, differences in trials, YST collections, isolates of P. jaceae within trials, and the interactions were tested by analysis of variance (GLM procedure; SAS). Least-square means for each trial and YST collection were generated. After conducting the analyses of variance, the data were analyzed by canonical discriminative analysis (CANDISC procedure; SAS) that derived linear combinations (canonical variables) of the variables for disease reaction, i.e., number of diseased plants, transformed pustule number, and transformed pustule area per unit leaf area. The means of the first two canonical variables for each YST collection were plotted. In addition, the canonical discriminative analysis produced estimates of squared Mahalanobis distances between each YST collection and each other collection. The squared distance between any two collections was (in matrix algebra): D2 ðijjÞ ¼ ðX i X j Þ0 cov1 ðX i X j Þ, where X i and X j are the means of clusters (collections) i and j that the distance is calculated for and cov1 ðX i X j Þ is the inverted covariance matrix (Rao, 1952). The distances between the foreign YST collections and the California collection were tabulated. The Mahalanobis distance is a scale-independent estimate, based on the canonical variables, of differences between clusters (or in this case YST collections); the greater the distance, the greater the difference between collections (Asthana et al., 1998; Weatherup, 1994). Each trial was analyzed separately and then the combined data from both trials were analyzed.
2.5. Statistical analysis
3.1. Data collection and digital analysis
All statistical analyses were done using SAS software (ver. 8, Statistical Analysis System, Cary, NC). Total pustule area per leaf was calculated by multiplying the mean pustule area by total pustule number. Pustule area per unit leaf area and total pustule number per unit leaf area were calculated by dividing total pustule area per leaf and total pustule number per leaf by the area associated with the appropriate leaf. These ratios of pustule area and pustule number to total leaf area were then transformed by arc-sine square root. If pustule number was greater than zero, then the leaf was deemed diseased and a variable for number of diseased leaves was set to Ô1Õ for that leaf. Total number of diseased leaves was calculated for each plant. If the sum of diseased leaves per plant was greater than zero then the plant was deemed diseased and a variable for number of diseased plants was set to Ô1Õ for that plant. The resulting variables for each trial were: number of diseased plants, transformed number of pustules per
During data collection it was noted that the YST plants from Cyprus and Jordan were morphologically distinct from plants in other collections. The plants of these collections had large numbers of small, simple leaves without lobes. The plants were highly branched and somewhat woody. Very few plants of either collection became diseased with either isolate of P. jaceae. Plants in these collections were morphologically identical or very similar to Centaurea pallescens Delile. These collections were not analyzed further. Representative leaf images and digital overlays that corresponded to rust pustules are presented in Figs. 1 and 2. Although the images are in black and white, the leaf background in Fig. 1 was predominately green while the leaf background in Fig. 2 was predominately brown. Both leaves had similar leaf areas, and the numbers of pustules detected by DIA on both leaves were not greatly different: 51 versus 47, but the size of pustules differed greatly between the two leaves. The average
3. Results
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Fig. 1. Gray scale image (left) of scanned yellow starthistle leaf with relatively large pustules of P. jaceae and overlays of pustules (right) from digital image analysis. The original color of the leaf that was subjected to digital image analysis was green.
pustule size in Fig. 1 was 49 pixels (range 9–159), and the average pustule size in Fig. 2 was 11.2 pixels (range 9–26 pixels). From the regression analysis between visual and digital analysis assessment of pustule counts, DIA was a fair predictor of the number of pustules from visual assessment (R2 ¼ 0:80). Visual counts were about 0.83 (slope of the regression equation) of that obtained from digital images. 3.2. Statistical analysis With the analyses of variance there were no differences (P < 0:05) between isolates of P. jaceae for any of the variables in the first trial. The data for the isolates were pooled for subsequent analysis. There were, however, significant differences between trials and the trial YST collection interaction for all variables. Average values for all variables were greater in the second trial (Table 1). The proportion of diseased plants was statistically the same for the collection from Erzurum, Turkey, and California in all analyses (Table 1). In the first trial, the average
number of diseased plants for all other collections differed significantly from the number of diseased plants in the California collection. With the exception of the collections from Sicily and Spain in the second trial, all plants in the other collections were diseased. In the first trial, the collection from Turkey had almost twice as many pustules per unit leaf area as the California collection. In the second trial, the number of pustules per unit leaf area was the same for these two collections. All other collections had fewer pustules per unit leaf area than either of these two collections. There were no differences in average pustule area between any of the collections and the California collection in the first trial. Pustule areas for all collections except the one from Turkey were significantly smaller than the California collection in the second trial and in the analysis of the combined data. The average pustule area for the Turkish collection was significantly larger than the pustule area for the California collection in the analysis of the combined data but was numerically closer (0.011 versus 0.009) to the California collection than was any other collection.
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Fig. 2. Gray scale image (left) of scanned yellow starthistle leaf with relatively small pustules of P. jaceae and overlays of pustules (right) from digital image analysis. The original color of the leaf that was subjected to digital image analysis was brown.
Table 1 Least-square means of variables from digital image analysis of disease reactions of different yellow starthistle collections Yellow starthistle collection
Average proportion of diseased plantsa
Average number of pustules per unit leaf area (pustules/pixel)b
Average pustule area per unit leaf area (pixels/pixel)c
Trial 1
Trial 2
Both trials
Trial 1
Trial 2
Both trials
Trial 1
Bari, Italy Krasnodar, Russia Palermo, Sicily Terragona, Spain Erzurum, Turkey California, USA
0.375d 0.321 0.118 0.227 0.824 ns 0.813
1.000 ns 1.000 ns 0.875 ns 0.333 1.000 ns 1.000
0.565 0.472 0.360 0.240 0.880 ns 0.875
0.00030 ns 0.00040 ns 0.00004 ns 0.00050 ns 0.00170 0.00090
0.005 0.004 0.001 0.002 0.007 ns 0.007
0.0020 0.0020 0.0008 0.0006 0.0050 0.0040
0.0006 0.0009 0.0001 0.0001 0.0004 0.0002
Mean
0.446
0.868
0.557
0.00060
0.004
0.0030
0.001
a
ns ns ns ns ns
Trial 2
Both trials
0.008 0.008 0.002 0.003 0.018 ns 0.017
0.004 0.004 0.001 0.001 0.011 0.009
0.009
0.006
A total of 32 plants were tested in each collection in each trial. Proportions are tabulated for ease of interpretation, but results of meanseparation tests are based on numbers of diseased plants. b Number of pustules per pixel of leaf area; transformed (arc-sine square-root) data. c Pustule area in pixels per pixel of leaf area; transformed (arc-sine square-root) data. d Significantly different than California population based on single-degree-of-freedom contrasts; P < 0:05; ns, not significant.
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Plots of the means of the first two canonical variables from the canonical discriminative analyses are presented in Figs. 3–5. The California collection grouped closest to the collection from Turkey in the second trial and in the analysis of the combined data. In the first trial, the collection from Italy appeared to be slightly closer than the Turkish collection to the California collection. However, the squared Mahalanobis distances between the California collection and the collection from Turkey (Table 2) were not different in either trial or in the combined analysis. Distances between the other collec-
Fig. 5. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of diseased plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from the combined data of two trials in which yellow starthistle collections were inoculated with isolates of P. jaceae from Bulgaria and Turkey.
tions and the California collection were significant in all analyses.
4. Discussion Fig. 3. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of infected plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from a trial in which yellow starthistle collections were inoculated with P. jaceae isolates from Bulgaria and Turkey.
Fig. 4. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of diseased plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from a trial in which yellow starthistle collections were inoculated with an isolate of P. jaceae from Turkey.
In general, visual acuity was more accurate than DIA to detect pustules. However, once the software detected the pustules and overlaid each with a proportionately sized color overlay, the calculation of the pustule size was a simple computer procedure that required only the summation of pixels in the color overlays of each pustule. The same was true for leaf area, which was a summation of all pixels in the color overlay of the leaf. Because these were solely mathematical processes, pustule-size measurement was consistent within and among leaves. The regression of pustule numbers from image analysis had a fair fit to pustule numbers from visual assessment. It was hoped that by using two different isolates of P. jaceae, different plant reactions would result and enable the collections to be differentiated on the basis of reaction to both isolates. This was not the case according to analyses of variance, for there were no significant isolate effects or isolate by YST collection interactions for any of the variables. There were significant effects of trial and trial by YST collection interactions for all of the variables. For the most part, differences in trials were from the magnitude of plant reactions, which was greater in the second trial. These differences in plant reactions were likely from the different amounts of inoculum applied in the two trials; the amount applied in the first trial was reduced because
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Table 2 Squared Mahalanobisa distances, from canonical discriminative analyses of data of plant reactions to P. jaceae,b between five yellow starthistle collections and a yellow starthistle collection from California, USA Source of yellow starthistle collection
Squared distances from California collection (Trial 1)
Squared distances from California collection (Trial 2)
Squared distances from California collection (both trials combined)
Bari, Italy Krasnodar, Russia Palermo, Sicily Terragona, Spain Erzurum, Turkey
1.05c 1.38 2.61 1.90 0.29 ns
10.20 3.67 8.15 15.34 0.02 ns
3.00 1.54 2.65 2.82 0.03 ns
Squared distance in matrix algebra: D2 ðijjÞ ¼ ðX i X j Þ0 cov1 ðX i X j Þ. Data on plant reactions to P. jaceae were number of diseased plants, number of pustules per unit leaf area, and area of pustules per unit leaf area. c Probability of a greater Mahalanobis distance for squared distance to the California, USA collection of yellow starthistle: P ¼ 0:05, P ¼ 0:01, ns, not significant. a
b
of the limited amount of urediniospores of the isolate of P. jaceae from Bulgaria. The Mahalanobis distances generated by canonical discriminative analyses indicated no significant differences in distances between YST from Turkey and California in either trial or from the analysis of the combined data. Distances between YST from other countries and YST from California were significant in both trials and analysis of the combined data. Because trials were conducted on individual offspring of separate plants in each non-US collection, the data on these collections should accurately reflect the overall disease reaction of each collection. Since no attempt was made, during collection, to separate (or distinguish) morphologically distinct plants from California, it was not clear that the overall disease reaction of this bulk collection accurately describes the general disease reaction of YST in California (or the USA). It was possible that the seeds from the California collection may have been from offspring of plants introduced from different geographical areas, i.e., multiple introductions, and the observed disease reactions of this bulk collection may have been a mix of characteristics of each of these originally introduced parents. Any such theoretical mix could be either that of originally introduced genotypes or natural hybrids among introduced genotypes. To test these possibilities, this study should be repeated with seeds from plants of morphologically distinct YST collections in the USA. Another possibility is that the California collection may be more closely related to an YST collection from an area close to, but other than, Erzurum, Turkey. Because of the relatively close relationship observed between these collections in this study, areas near Erzurum would be good sources for more closely related YST collections and insects and pathogens of YST in the USA. Furthermore, it would be desirable to include YST collections from these areas and YST collections in the USA in another similar study. If digital image analysis of rust reactions was combined with data on
DNA fragment patterns from different YST collections (Luster et al., 2001), this could help narrow the origin (within Turkey and neighboring countries) of YST in the USA. Leaf-to-leaf consistency in pustule measurements makes digital image analysis powerful to measure disease severity and to distinguish between plant collections based on disease reactions. Since these data will be used to narrow the geographical area for potential biological control agents of YST and to reduce exploration time spent to collect effective biological control agents, the amount of time spent to collect consistent data to accomplish this seems well spent.
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D.K. Berner, L.K. Paxson / Biological Control 28 (2003) 171–179 populations using AFLP fragment pattern analysis. In: Smith, L. (Ed.), Proceedings of the First International Knapweed Symposium of the Twenty-First Century, March 15–16, 2001, Coeur dÕAlene, Idaho, USDA-ARS, Albany, CA, p. 88. Maddox, D.M., 1981. Introduction, phenology, and density of yellow starthistle in coastal, intercoastal, and central valley situations in California. US Department of Agriculture, Agricultural Research Service, Agricultural Research Results, ARR-W-20. Nilsson, H.-E., 1995. Remote sensing and image analysis in plant pathology. Annu. Rev. Phytopathol. 15, 489–527. Rao, C.R., 1952. Statistical Methods in Biometric Research. Wiley, New York. Shishkoff, N., Bruckart, W., 1993. Evaluation of infection of target and nontarget hosts by isolates of the potential biocontrol agent
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Puccinia jaceae that infect Centaurea spp. Phytopathology 83, 894– 898. Smith, L., Story, J.M., DiTomaso, J.M., 2001. Bibliography of spotted knapweed, yellow starthistle and other weedy knapweeds. In: Smith, L. (Ed.), Proceedings of the First International Knapweed Symposium of the Twenty-First Century, March 15–16, 2001, Coeur dÕAlene, Idaho, USDA-ARS, Albany, CA, pp. 1–62. Stakman, E.C., Stewart, D.M., Loegering, W.Q., 1962. Identification of physiological races of Puccinia graminis var. tritici. U.S. Dept. Agric./Agric. Res. Serv. E, 617. Wapshere, A.J., Delfosse, E.S., Cullen, J.M., 1989. Recent developments in biological control of weeds. Crop Prot. 8, 227–250. Weatherup, S.T.C., 1994. Use of Mahalanobis distance to measure varietal distinctness. Plant Var. Seeds 7, 107–119.
Use of digital images to differentiate reactions of collections of yellow starthistle (Centaurea solstitialis) to infection by Puccinia jaceae D.K. Berner* and L.K. Paxson Foreign Disease-Weed Science Research Unit, Agricultural Research Service, US Department of Agriculture, 1301 Ditto Avenue, Ft. Detrick, Frederick, MD 21702, USA Received 18 September 2002; accepted 14 April 2003
Abstract Yellow starthistle (YST) (Centaurea solstitialis) is an invasive weed in the USA and a target of classical biological control. However, the precise geographical origin(s) of YST in the USA is not known. This study was conducted to learn whether the origin(s) of YST in California, USA could be determined on the basis of digital image analysis (DIA) of YST reactions to isolates of the rust Puccinia jaceae. One trial was conducted with two isolates of P. jaceae and another trial with only one of these isolates. Six different YST collections were tested. The DIA consistently detected and measured rust pustules on YST leaves. Compared to visual assessment, DIA was a fair predictor of the number of rust pustules per leaf. There were no differences between isolates of P. jaceae in reactions of different YST collections in the first trial. Differences in plant reactions between trials were predominately due to more disease incidence and severity in the second trial, which was probably from the greater amount of inoculum. Based on analyses of variance and canonical discriminative analyses, YST from California appeared most closely related to the YST collection from Erzurum, Turkey. In examination of squared Mahalanobis distances, all collections, except the collection from Turkey, were significantly distant from the California collection in both trials. More studies are necessary to determine the precise area(s) of origin (within Turkey and neighboring countries) of YST in the USA. Digital image analysis of rust reactions can be a powerful tool to do this. Published by Elsevier Science (USA). Keywords: Carduus nutans; Carduus thoermeri; Centaurea solstitialis; Digital image analysis; Puccinia carduorum; Puccinia jaceae; Rust; Yellow starthistle
1. Introduction Yellow starthistle (YST) (Centaurea solstitialis L., Asteraceae) is an important invasive weed, predominately in rangelands and natural areas, in the western USA (Cheater, 1992). It is the focus of numerous classical biological control efforts (Smith et al., 2001). Classical biological control involves the introduction of an insect or pathogen from the area from which the weed originated. This follows the presumption that the insect or pathogen is one of the multitudes of natural enemies that keep the weed under control in its native habitat (Wapshere et al., 1989). In the case of YST, the
* Corresponding author. Fax: +1-301-619-2880. E-mail address: [email protected] (D.K. Berner).
1049-9644/$ - see front matter. Published by Elsevier Science (USA). doi:10.1016/S1049-9644(03)00096-3
native habitat of the plant is generally described as Eurasia (Maddox, 1981). However, not all insects or pathogens of YST from Eurasia have the potential to control YST in the USA. To determine what specific area, within the native habitat of the weed, might hold the most promise to find effective insects and pathogens of YST in the USA, it is desirable to determine, within a relatively small geographical area, where the introduced weed population originated. One potential biological control agent of YST is the rust fungus Puccinia jaceae Otth. that does substantial damage to the weed (Shishkoff and Bruckart, 1993) and is relatively host-specific (Bruckart, 1989). Currently, one isolate of P. jaceae from Turkey is on the verge of release in the USA for classical biological control of YST (Federal Register, 2002). Other, more damaging isolates of P. jaceae might also be found in the natural
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range of YST, once that origin is determined. These isolates could then be released in a relatively short period of time given that host-range studies and approval for release of one isolate have already been completed. Because rust pathogens can be specific to the level of single gene differences among cultivars (Flor, 1971), disease reactions of different host plants (and populations) can be used to differentiate both susceptible (and resistant) host cultivars and pathogen races (Flor, 1971; Stakman et al., 1962). Because of the high degree of specificity, reactions of geographically diverse collections of YST to infection by isolates of P. jaceae may be accurate indicators of the relatedness among YST collections and could help to determine within a relatively well-defined area: (1) the geographical origin of YST that was introduced to California and (2) where more aggressive and damaging isolates of P. jaceae, relative to the isolate planned for release in the USA, might be found. Of specific interest is the reaction of different YST collections to the isolate of P. jaceae that is planned for release in the USA. Because this isolate has been extensively tested and is virulent and aggressive on YST from California (Bruckart, 1989), and because of the aforementioned specificity, disease reactions of different YST collections to this isolate alone could accurately indicate relatedness of YST collections to YST from California. Degree of relatedness of other collections to the California collection is the only parameter of interest. Amplified fragment length polymorphism (AFLP) studies are also in progress on the YST collections used in this study, as well as on other YST collections (Luster et al., 2001), and these data could, in the future, be combined with rust-reaction data to further narrow the origin of YST in California. Objective analysis of plant reactions to rust infections has been difficult because of the various sizes and numbers of rust pustules on individual plants that otherwise might suggest a range from susceptible to resistant reactions. Consequently, different rating scales have been developed in an attempt to determine plant reactions to rust infections. In wheat, the standard rating scale for disease reaction to stem rust uses seven rating classes, in addition to Ô+Õ, Ô)Õ, Ô++Õ, and Ô ¼ Õ modifiers, to describe disease reactions of cultivar isolate based on the presence and size of uredinia (Stakman et al., 1962). Since this scale was designed to evaluate relatively genetically homogeneous cultivars, it may not be the most appropriate to evaluate disease reactions of highly heterogeneous weed populations. To evaluate rust pathogens for efficacy as biological control agents of weeds, another scale was developed to characterize disease reaction of individual plants within weed populations (Bruckart et al., 1996). This scale uses: (1) five rating classes based on the number of sori (either uredinia or telia) per square centimeter of leaf tissue and (2) the amount, as percent, of symptomatic leaf tissue. Both
scales depend, at least in part, on subjective evaluation of each sample (leaf or plant), and the consistency between samples depends heavily on the precision of the rater. Neither scale actually quantifies both number and size of the rust pustules relative to total leaf area. Digital image analysis (DIA) has been used extensively to quantify the effects of plant diseases on host plants (Nilsson, 1995). For the most part, DIA has been used to measure chlorotic and necrotic areas of diseased leaves (Lindow and Webb, 1983) and roots (Biernacki and Bruton, 2001), but it also has been used to differentiate symptoms caused by rust infections (Bacchi et al., 1992) and to differentiate isolates of rusts (Ball et al., 1992). The objectives of this study were to use DIA to evaluate disease reactions of different collections of YST to isolates of P. jaceae and then to compare the collections based on an aggregate of these disease reactions for each collection, to the collection from California, USA. It was hoped that this would enable us to determine, within a narrow geographical area, where YST that was introduced to California originated and where more aggressive isolates of P. jaceae, for eventual release in the USA, might be found.
2. Materials and methods 2.1. Plant establishment The study was conducted in the biological containment facility of USDA/ARS Foreign Disease-Weed Science Research Unit (FDWSRU) at Fort Detrick, MD. Yellow starthistle plants were grown from collections of seeds made by Dr. F. Di Cristina, Dr. M. Cristofaro, Dr. P. Pecora, Dr. S. Resnik, and Dr. C. Roche in the fall of 1999 from fields close to Limassol, Cyprus (34°460 N, 32°420 E); Bari, Italy (41°030 N, 16°150 E); Jarash, Jordan (32°150 N, 35°370 E); Krasnodar, Russia (45°030 N, 38°550 E); Palermo, Sicily (36°540 N, 13°590 E); Terragona, Spain (41°300 N, 00°500 E); and Erzurum, Turkey (40°100 N, 40°230 E). In each location, seeds of individual plants were harvested and packaged separately from seeds of other plants. Seeds of YST from a non-specific (bulk) collection in the USA were collected from Solano County, California (38°150 N, 122°020 W) in the fall of 1997 and were provided by Dr. M. Pitcairn (California Department of Food and Agriculture). Two trials of the test were conducted. In each trial, seeds of YST were pre-germinated by surface disinfesting the seeds for 5 min in 2% NaOCl and then placing them in sterile petri dishes that contained 1.5% (w/v) water agar. Seeds of individual plants from each collection were pre-germinated in separate petri dishes. After emergence of radicles, developing seedlings were transplanted into individual cells of a 32-cell (8 4 cell)
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flat filled with steam-sterilized potting soil (2:1:1, v/v, soil, sand, and peat). Each cell in the flat measured 6 6 6 cm. Seedlings from individual plants in each collection were transplanted into single rows of eight cells in each flat. One seedling was transplanted into each cell. Each of four sets of two flats contained all of the collections tested with each rust isolate in each trial. In each of the four sets of flats, eight plants (one row) of each collection were grown. Thus, 32 plants (4 rows of 8 plants in separate flats) of each collection were tested for each rust isolate in each trial. Positions of collections (rows) within each two-flat set were randomly assigned. Collections (rows) within each of the four sets of flats were the replicated experimental units and plants within rows were the sampling units. When possible, seedlings from different parent plants in the same collection were planted in successive cells within a row. In cases when transplanted seedlings died, these were replaced with seedlings from the same parent plant in the same collection, if available. In some cases there were not enough seeds or seedlings of a parent plant in a collection to replace dead plants; these were then replaced with seedlings of another parent plant in the same collection, and the position of these plants in the flats was noted.
compressed air at 34.5 kPa. All leaves of all plants were sprayed until the suspension ran off the leaves. Inoculation with each rust isolate was done separately to avoid inadvertent cross-inoculation of the plants with the unintended isolate. Immediately after inoculation, the flats were placed overnight, for 12–15 h, in dark dew chambers set at 19 °C. Flats of plants inoculated with the different isolates were placed in separate dew chambers. The following day the flats were transferred to greenhouses with ambient temperatures between 24 and 26 °C. Flats of plants inoculated with the different isolates were placed in separate greenhouses. In the evening, the flats were returned to the respective dew chambers, for 12– 15 h, and the dew chamber-greenhouse rotation was repeated for 3 days and nights, after which the plants were kept constantly in the greenhouses. This procedure was repeated for the second trial that was planted on 30 March 2001 and inoculated on 20 April 2001 with the isolate of P. jaceae from Ankara, Turkey. There were not enough urediniospores to include the isolate from Bulgaria in this trial, but the amount of inoculum for the isolate from Turkey was increased to 2.5 mg (about 7.75 104 spores) per plant.
2.2. Inoculation
2.3. Data collection
In the first trial, seedlings were transplanted on 17 November 2000, and dead plants were replaced on 20 November 2000. Approximately 2 g of a complete fertilizer (19% N, 6% P2 O5 , and 12% K2 O) were added to the soil around each plant two weeks after planting. At 28 days after planting, four, two-flat sets of plants were spray-inoculated with urediniospores of an isolate of P. jaceae that was collected from near Ankara, Turkey in 1984. This isolate will be released in the USA in the near future for classical biological control of YST. Four additional two-flat sets of plants were similarly inoculated with an isolate of P. jaceae collected from an unspecified location in Bulgaria in 1978. Spores of each isolate had been periodically increased, since collection, on YST plants in greenhouses at FDWSRU and were maintained in a viable condition in cryovials in liquid nitrogen at )196 °C. For each isolate, 6.4 mg of urediniospores (about 0.4 mg or 1.25 104 spores per plant) were weighed and suspended in 100 ml of distilled water that contained 0.125% (v/v) oxysorbic 20 polyoxyethylene sorbitan monooleate to break surface tension. The amount of inoculum applied to each plant was limited by the total amount of urediniospores available for the isolate from Bulgaria and the desire to use the same amount of inoculum of each isolate. To ensure uniformity in application, this suspension was divided into two aliquots, and each 50-ml aliquot was added to an atomizer and sprayed onto the same two, two-flat sets of plants with
In the first trial, data were collected 36 days after inoculation when abundant pustules were evident on leaves and no further pustule development was occurring. In the absence of free moisture, no secondary pustules or disease cycles occurred in the greenhouses. Each plant was uprooted, the total number of leaves was counted, and the plant number, collection, rust isolate, and flat number were recorded. Leaves with pustules were then harvested at the base of the petiole, and each diseased leaf was tagged and the position recorded. Leaf number Ô1Õ was nearest the crown at the base of the plant, and successive leaves received numbers Ô2Õ and so on. The harvested leaves were then placed pustule side down on the glass bed of a digital scanner (HewlettPackard model 6100C), scanned at 600 dpi, and saved, at the same resolution, in individual computer files named according to YST plant and collection, rust isolate, flat number, and leaf position. Data collection in the second trial also followed this procedure. 2.4. Digital analysis The number of pustules per diseased leaf, mean area of pustules per leaf, and total leaf area were collected with digital analysis software (SigmaScan Pro 5.0, SPSS Science, SPSS, Chicago, IL). Macro programming routines based on Visual Basic were written with the software to automate the digital analysis process and to provide a consistent protocol for each reading. To
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analyze pustule number and area, all of the following steps were programmed and used for the color images of each leaf: the brightness was increased by 30%, the contrast was increased by 20%, the image was converted to the 8-bit gray scale, and pixels within the light intensity range of 67–129 were highlighted with color overlays. This range corresponded to the light intensity of the pustules and was used to differentiate darker pustules from lighter shaded leaves and to determine the precise shape of each pustule. The overlays, representing pustule number and size, were counted and measured by the software. Pustule area was measured as total number of pixels within the color overlay of each pustule. Because dark areas along veinations and creases of some leaves were also overlaid, the macro was modified to restrict the areas mathematically to those areas interpreted as pustules to circular-to-slightly oval shapes within the range of pustule sizes that were visually observed. Objects that did not fit these criteria were omitted from pustule counts and measurements. Total leaf area was measured using a separate programming routine. Each leaf was first analyzed for mean pustule area and total pustule number and then reanalyzed for leaf area. A data set was created to include total leaf area, mean pustule area per leaf, total pustule number per leaf, and the appropriate leaf identifiers, e.g., YST plant and collection, rust isolate, and flat number. To compare DIA with visual assessment, 50 diseased leaves were selected at random (with a random number table), and the pustules on scanned images of these leaves were counted manually. The count was then compared, as the dependent variable in regression analysis, to the count obtained from the digital analysis software.
unit leaf area, and transformed pustule area per unit leaf area. With these as dependent variables, differences in trials, YST collections, isolates of P. jaceae within trials, and the interactions were tested by analysis of variance (GLM procedure; SAS). Least-square means for each trial and YST collection were generated. After conducting the analyses of variance, the data were analyzed by canonical discriminative analysis (CANDISC procedure; SAS) that derived linear combinations (canonical variables) of the variables for disease reaction, i.e., number of diseased plants, transformed pustule number, and transformed pustule area per unit leaf area. The means of the first two canonical variables for each YST collection were plotted. In addition, the canonical discriminative analysis produced estimates of squared Mahalanobis distances between each YST collection and each other collection. The squared distance between any two collections was (in matrix algebra): D2 ðijjÞ ¼ ðX i X j Þ0 cov1 ðX i X j Þ, where X i and X j are the means of clusters (collections) i and j that the distance is calculated for and cov1 ðX i X j Þ is the inverted covariance matrix (Rao, 1952). The distances between the foreign YST collections and the California collection were tabulated. The Mahalanobis distance is a scale-independent estimate, based on the canonical variables, of differences between clusters (or in this case YST collections); the greater the distance, the greater the difference between collections (Asthana et al., 1998; Weatherup, 1994). Each trial was analyzed separately and then the combined data from both trials were analyzed.
2.5. Statistical analysis
3.1. Data collection and digital analysis
All statistical analyses were done using SAS software (ver. 8, Statistical Analysis System, Cary, NC). Total pustule area per leaf was calculated by multiplying the mean pustule area by total pustule number. Pustule area per unit leaf area and total pustule number per unit leaf area were calculated by dividing total pustule area per leaf and total pustule number per leaf by the area associated with the appropriate leaf. These ratios of pustule area and pustule number to total leaf area were then transformed by arc-sine square root. If pustule number was greater than zero, then the leaf was deemed diseased and a variable for number of diseased leaves was set to Ô1Õ for that leaf. Total number of diseased leaves was calculated for each plant. If the sum of diseased leaves per plant was greater than zero then the plant was deemed diseased and a variable for number of diseased plants was set to Ô1Õ for that plant. The resulting variables for each trial were: number of diseased plants, transformed number of pustules per
During data collection it was noted that the YST plants from Cyprus and Jordan were morphologically distinct from plants in other collections. The plants of these collections had large numbers of small, simple leaves without lobes. The plants were highly branched and somewhat woody. Very few plants of either collection became diseased with either isolate of P. jaceae. Plants in these collections were morphologically identical or very similar to Centaurea pallescens Delile. These collections were not analyzed further. Representative leaf images and digital overlays that corresponded to rust pustules are presented in Figs. 1 and 2. Although the images are in black and white, the leaf background in Fig. 1 was predominately green while the leaf background in Fig. 2 was predominately brown. Both leaves had similar leaf areas, and the numbers of pustules detected by DIA on both leaves were not greatly different: 51 versus 47, but the size of pustules differed greatly between the two leaves. The average
3. Results
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Fig. 1. Gray scale image (left) of scanned yellow starthistle leaf with relatively large pustules of P. jaceae and overlays of pustules (right) from digital image analysis. The original color of the leaf that was subjected to digital image analysis was green.
pustule size in Fig. 1 was 49 pixels (range 9–159), and the average pustule size in Fig. 2 was 11.2 pixels (range 9–26 pixels). From the regression analysis between visual and digital analysis assessment of pustule counts, DIA was a fair predictor of the number of pustules from visual assessment (R2 ¼ 0:80). Visual counts were about 0.83 (slope of the regression equation) of that obtained from digital images. 3.2. Statistical analysis With the analyses of variance there were no differences (P < 0:05) between isolates of P. jaceae for any of the variables in the first trial. The data for the isolates were pooled for subsequent analysis. There were, however, significant differences between trials and the trial YST collection interaction for all variables. Average values for all variables were greater in the second trial (Table 1). The proportion of diseased plants was statistically the same for the collection from Erzurum, Turkey, and California in all analyses (Table 1). In the first trial, the average
number of diseased plants for all other collections differed significantly from the number of diseased plants in the California collection. With the exception of the collections from Sicily and Spain in the second trial, all plants in the other collections were diseased. In the first trial, the collection from Turkey had almost twice as many pustules per unit leaf area as the California collection. In the second trial, the number of pustules per unit leaf area was the same for these two collections. All other collections had fewer pustules per unit leaf area than either of these two collections. There were no differences in average pustule area between any of the collections and the California collection in the first trial. Pustule areas for all collections except the one from Turkey were significantly smaller than the California collection in the second trial and in the analysis of the combined data. The average pustule area for the Turkish collection was significantly larger than the pustule area for the California collection in the analysis of the combined data but was numerically closer (0.011 versus 0.009) to the California collection than was any other collection.
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Fig. 2. Gray scale image (left) of scanned yellow starthistle leaf with relatively small pustules of P. jaceae and overlays of pustules (right) from digital image analysis. The original color of the leaf that was subjected to digital image analysis was brown.
Table 1 Least-square means of variables from digital image analysis of disease reactions of different yellow starthistle collections Yellow starthistle collection
Average proportion of diseased plantsa
Average number of pustules per unit leaf area (pustules/pixel)b
Average pustule area per unit leaf area (pixels/pixel)c
Trial 1
Trial 2
Both trials
Trial 1
Trial 2
Both trials
Trial 1
Bari, Italy Krasnodar, Russia Palermo, Sicily Terragona, Spain Erzurum, Turkey California, USA
0.375d 0.321 0.118 0.227 0.824 ns 0.813
1.000 ns 1.000 ns 0.875 ns 0.333 1.000 ns 1.000
0.565 0.472 0.360 0.240 0.880 ns 0.875
0.00030 ns 0.00040 ns 0.00004 ns 0.00050 ns 0.00170 0.00090
0.005 0.004 0.001 0.002 0.007 ns 0.007
0.0020 0.0020 0.0008 0.0006 0.0050 0.0040
0.0006 0.0009 0.0001 0.0001 0.0004 0.0002
Mean
0.446
0.868
0.557
0.00060
0.004
0.0030
0.001
a
ns ns ns ns ns
Trial 2
Both trials
0.008 0.008 0.002 0.003 0.018 ns 0.017
0.004 0.004 0.001 0.001 0.011 0.009
0.009
0.006
A total of 32 plants were tested in each collection in each trial. Proportions are tabulated for ease of interpretation, but results of meanseparation tests are based on numbers of diseased plants. b Number of pustules per pixel of leaf area; transformed (arc-sine square-root) data. c Pustule area in pixels per pixel of leaf area; transformed (arc-sine square-root) data. d Significantly different than California population based on single-degree-of-freedom contrasts; P < 0:05; ns, not significant.
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Plots of the means of the first two canonical variables from the canonical discriminative analyses are presented in Figs. 3–5. The California collection grouped closest to the collection from Turkey in the second trial and in the analysis of the combined data. In the first trial, the collection from Italy appeared to be slightly closer than the Turkish collection to the California collection. However, the squared Mahalanobis distances between the California collection and the collection from Turkey (Table 2) were not different in either trial or in the combined analysis. Distances between the other collec-
Fig. 5. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of diseased plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from the combined data of two trials in which yellow starthistle collections were inoculated with isolates of P. jaceae from Bulgaria and Turkey.
tions and the California collection were significant in all analyses.
4. Discussion Fig. 3. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of infected plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from a trial in which yellow starthistle collections were inoculated with P. jaceae isolates from Bulgaria and Turkey.
Fig. 4. Plot of means of first and second canonical variables for six yellow starthistle collections. Canonical variables for each collection were derived from number of diseased plants, average number of pustules per unit leaf area, and average pustule area per unit leaf area. Results are from a trial in which yellow starthistle collections were inoculated with an isolate of P. jaceae from Turkey.
In general, visual acuity was more accurate than DIA to detect pustules. However, once the software detected the pustules and overlaid each with a proportionately sized color overlay, the calculation of the pustule size was a simple computer procedure that required only the summation of pixels in the color overlays of each pustule. The same was true for leaf area, which was a summation of all pixels in the color overlay of the leaf. Because these were solely mathematical processes, pustule-size measurement was consistent within and among leaves. The regression of pustule numbers from image analysis had a fair fit to pustule numbers from visual assessment. It was hoped that by using two different isolates of P. jaceae, different plant reactions would result and enable the collections to be differentiated on the basis of reaction to both isolates. This was not the case according to analyses of variance, for there were no significant isolate effects or isolate by YST collection interactions for any of the variables. There were significant effects of trial and trial by YST collection interactions for all of the variables. For the most part, differences in trials were from the magnitude of plant reactions, which was greater in the second trial. These differences in plant reactions were likely from the different amounts of inoculum applied in the two trials; the amount applied in the first trial was reduced because
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Table 2 Squared Mahalanobisa distances, from canonical discriminative analyses of data of plant reactions to P. jaceae,b between five yellow starthistle collections and a yellow starthistle collection from California, USA Source of yellow starthistle collection
Squared distances from California collection (Trial 1)
Squared distances from California collection (Trial 2)
Squared distances from California collection (both trials combined)
Bari, Italy Krasnodar, Russia Palermo, Sicily Terragona, Spain Erzurum, Turkey
1.05c 1.38 2.61 1.90 0.29 ns
10.20 3.67 8.15 15.34 0.02 ns
3.00 1.54 2.65 2.82 0.03 ns
Squared distance in matrix algebra: D2 ðijjÞ ¼ ðX i X j Þ0 cov1 ðX i X j Þ. Data on plant reactions to P. jaceae were number of diseased plants, number of pustules per unit leaf area, and area of pustules per unit leaf area. c Probability of a greater Mahalanobis distance for squared distance to the California, USA collection of yellow starthistle: P ¼ 0:05, P ¼ 0:01, ns, not significant. a
b
of the limited amount of urediniospores of the isolate of P. jaceae from Bulgaria. The Mahalanobis distances generated by canonical discriminative analyses indicated no significant differences in distances between YST from Turkey and California in either trial or from the analysis of the combined data. Distances between YST from other countries and YST from California were significant in both trials and analysis of the combined data. Because trials were conducted on individual offspring of separate plants in each non-US collection, the data on these collections should accurately reflect the overall disease reaction of each collection. Since no attempt was made, during collection, to separate (or distinguish) morphologically distinct plants from California, it was not clear that the overall disease reaction of this bulk collection accurately describes the general disease reaction of YST in California (or the USA). It was possible that the seeds from the California collection may have been from offspring of plants introduced from different geographical areas, i.e., multiple introductions, and the observed disease reactions of this bulk collection may have been a mix of characteristics of each of these originally introduced parents. Any such theoretical mix could be either that of originally introduced genotypes or natural hybrids among introduced genotypes. To test these possibilities, this study should be repeated with seeds from plants of morphologically distinct YST collections in the USA. Another possibility is that the California collection may be more closely related to an YST collection from an area close to, but other than, Erzurum, Turkey. Because of the relatively close relationship observed between these collections in this study, areas near Erzurum would be good sources for more closely related YST collections and insects and pathogens of YST in the USA. Furthermore, it would be desirable to include YST collections from these areas and YST collections in the USA in another similar study. If digital image analysis of rust reactions was combined with data on
DNA fragment patterns from different YST collections (Luster et al., 2001), this could help narrow the origin (within Turkey and neighboring countries) of YST in the USA. Leaf-to-leaf consistency in pustule measurements makes digital image analysis powerful to measure disease severity and to distinguish between plant collections based on disease reactions. Since these data will be used to narrow the geographical area for potential biological control agents of YST and to reduce exploration time spent to collect effective biological control agents, the amount of time spent to collect consistent data to accomplish this seems well spent.
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