APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1998, p. 3846–3853 0099-2240/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 64, No. 10
Interlaboratory Comparison of Methods To Quantify Microsclerotia of Verticillium dahliae in Soil A. J. TERMORSHUIZEN,1* J. R. DAVIS,2 G. GORT,3 D. C. HARRIS,4 O. C. HUISMAN,5 G. LAZAROVITS,6 T. LOCKE,7 J. M. MELERO VARA,8 L. MOL,9 E. J. PAPLOMATAS,10 H. W. PLATT,11 M. POWELSON,12 D. I. ROUSE,13 R. C. ROWE,14 AND L. TSROR15 Laboratory of Phytopathology, Wageningen Agricultural University, 6700 EE Wageningen, The Netherlands1; Department of Plant, Soil and Entomological Sciences, University of Idaho Research and Extension Center, Aberdeen, Idaho 831202; Subdepartment of Mathematics, Wageningen Agricultural University, 6703 HA Wageningen, The Netherlands3; Horticultural Research International East Malling, West Malling, Kent ME19 6BJ, United Kingdom4; Department of Plant Pathology, University of California, Berkeley, California 947205; Pest Management Research Centre, Agriculture and Agrifood Canada, London, Ontario N5V 4T3, Canada6; ADAS, Rosemaund, Preston Wynne, Hereford HR1 3PG, United Kingdom7; Consejo Superior de Investigaciones Cientificas, Instituto de Agricultura Sostenible, 14080 Cordoba, Spain8; Department of Agronomy, Wageningen Agricultural University, 6709 RZ Wageningen, The Netherlands9; Benaki Phytopathological Institute, 145 61 Kifissia, Athens, Greece10; Plant Pathology Research Station, Charlottetown, Prince Edward Island C1A 7M8, Canada11; Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-290212; Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 5370613; Department of Plant Pathology, Ohio Agricultural Research and Development Center, Ohio State University, Wooster, Ohio 44961-409614; and GILAT Regional Experiment Station, M. P. Negev 2, 85280 Israel15 Received 9 April 1998/Accepted 17 July 1998
In a comparison of different methods for estimating Verticillium dahliae in soil, 14 soil samples were analyzed in a blinded fashion by 13 research groups in seven countries, using their preferred methods. One group analyzed only four samples. Twelve soil samples were naturally infested, and two had known numbers of microsclerotia of V. dahliae added to them. In addition, a control was included to determine whether transport had an effect on the results. Results differed considerably among the research groups. There was a 118-fold difference between the groups with the lowest and highest mean estimates. Results of the other groups were evenly distributed between these extremes. In general, methods based on plating dry soil samples gave higher numbers of V. dahliae than did plating of an aqueous soil suspension. Recovery of V. dahliae from samples with added microsclerotia varied from 0 to 59%. Most of the variability within each analysis was at the petri dish level. The results indicate the necessity to check the performance of detection assays regularly by comparing recoveries with other laboratories, using a common set of soil samples. We conclude that wet plating assays are less accurate than dry plating assays. Verticillium dahliae Kleb. causes wilt diseases in agronomic, horticultural, and nursery plant species (18, 19). Microsclerotia, which form in the senescing tissues of the diseased plant, may persist in soil for several years in the absence of a susceptible host. Quantifying the density of microsclerotia in soil is important for the development of disease prediction systems and for assessing the performance of control tactics. Many methods have been devised for quantifying V. dahliae in soil. These include elements such as concentrating microsclerotia by flotation (2, 9), wet sieving soil samples (11), and using semiselective media, including incorporation of a selective carbon source such as ethanol (15), cellulose (23), sorbose (13), or pectate (10). Cellulose also has been added to nutrient media to improve discrimination between V. dahliae and V. tricorpus Isaac (8, 23). Wet plating (WP) (14, 15) and dry plating (DP) (3, 7) are the two methods of distributing soil onto an agar medium. Both methods involve plating a small amount of soil on a semiselective medium which usually contains pectin as
a carbon source. WP assays may include wet sieving as a step to remove particles smaller (e.g., ,20 mm) and larger (e.g., .160 mm) than microsclerotia (6). With DP assays, the Andersen air sampler (1, 4) distributes the soil particles as evenly as possible onto agar plates (3). In both assay types, soil is dried prior to analysis to reduce the number of conidia and mycelial fragments of V. dahliae (14), to reduce the inoculum of other fungi, and to facilitate grinding and mixing of the soil sample. Existing methods of analyzing soil for V. dahliae can be compared either by having a single laboratory following published methods for the same soil sample or by having different laboratories each follow their own protocols with a common set of soil samples. Although several comparative studies using the first approach have been published (3, 6, 16, 21, 25), the question of which method is best remains unresolved. A difficulty encountered with the first approach is how to reproduce exactly the published methods of other workers, since minor details may be important (6). For this reason, we took the second approach in an attempt to identify which method has the highest precision (i.e., the least variation between repeated measurements), recovery percentage (i.e., accuracy, or the nearness to the true value; bias is the reverse of accuracy), and sensitivity (i.e., the detection limit).
* Corresponding author. Mailing address: Laboratory of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE Wageningen, The Netherlands. Phone: 31 317 483411. Fax: 31 317 483412. E-mail:
[email protected]. 3846
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Our objectives were as follows: (i) to inventory the methods currently used for estimating V. dahliae in soil and (ii) to compare the results of various methods over a range of soils. Preliminary results of this study have been published previously (24).
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effect of subsample k of method i and soil sample j with variance ss2, and pijkl is the random error of petri dish l of subsample k, method i, and soil sample j with variance sp2. ss2 can be interpreted as the variance among subsamples of the same soil sample, and sp2 can be interpreted as the variance among petri dishes of the same subsample; their estimates are referred to as ss2 and sp2, respectively. The analysis was performed with the SAS procedure MIXED. The effect of transportation was evaluated separately.
MATERIALS AND METHODS Exchange of soil samples. The contacts for the research groups were as follows: J. R. Davis (United States), O. C. Huisman (United States), G. Lazarovits (Canada), T. Locke (United Kingdom), J. M. Melero Vara (Spain), L. Mol (The Netherlands), M. Powelson (United States), D. I. Rouse (United States), A. J. Termorshuizen (The Netherlands), E. J. Paplomatas (Greece), H. W. Platt (Canada), R. C. Rowe (United States), and L. Tsror (Israel). Throughout this paper, the results will be attributed anonymously; groups will be referred to by uppercase letters, and the methods they used and soils they provided will be referred to by lowercase letters. Each group, with the exception of two, collected one soil sample of about 5 kg. Each sample was air dried and ground. Samples were sent by airmail to the senior author (A.J.T.), who divided and distributed the subsamples. A small portion of each sample was retained by each group to determine the effect of transportation. To treat the Dutch samples in a manner similar to the foreign samples, they were airmailed to the United Kingdom and back. Two additional soil samples were prepared by adding known numbers of microsclerotia to a sandy soil (pH 7.0; organic matter, 2.6%). Microsclerotia were obtained from diseased potato stems collected from an experimental field near Wageningen, The Netherlands. The level of germination of the collected microsclerotia on ethanol agar was 85% (15). Microsclerotia were added as aqueous suspensions to the sandy soil to densities of 5 and 60 germinable microsclerotia g of air-dried soil21 (mpg). The soils to which microsclerotia had been added were blended thoroughly in a concrete mixer and allowed to dry for 2 weeks at room temperature. Seventy-two 50-g subsamples were taken from each original bulk sample. This was done by placing the soil in a funnel, with quickly rotating flasks being placed under the outlet so that for every rotation a small amount of soil (approximately 0.1 g) was distributed to each flask (a device designed by Retsch, Ochten, The Netherlands). Subsamples were collected into plastic 100-ml containers. These were randomly assigned and sent to 12 groups; 2 groups shared one set of subsamples. Most groups analyzed a total of 90 subsamples: 6 replicate subsamples from 14 samples (12 naturally infested soil samples from the research groups and 2 soil samples with added microsclerotia) and 6 replicate subsamples from the nontransported sample. Group C analyzed only four soil samples. All groups analyzed the soils by their favored methods. Group F analyzed all the samples by its favored method (a DP assay; coded f-2) and by another method (a WP assay; coded f-1). Group B did all analyses in duplicate, referred to as b1 and b2. Details of the methods (Table 1) were provided by each group via a questionnaire. Identification of V. dahliae. Four groups reported that they could not distinguish between colonies of V. dahliae and V. tricorpus. The other groups did attempt to discriminate between the two species, but not all were totally confident in their identifications. On average, figures for V. tricorpus were 3.9% of those for V. dahliae plus V. tricorpus. Given the apparently low incidence of V. tricorpus, the uncertainty of several groups in identifying the two species, and the desirability of comparing all the data in one analysis, data for the two fungi were combined. Analysis of data. Data of two groups, L and M, were omitted from the analysis because the data were anomalous. Group L reported averages of 90 and 89 CFU g of soil21, respectively, for the samples amended with 5 and 60 mpg. The results obtained by group M were extremely variable and therefore regarded as unreliable. For example, their data showed the largest mean value (490 CFU g of soil21) of all methods for densities of V. dahliae plus V. tricorpus, but their median was 0. Although group B indicated that its usual analysis involved a complete repetition of the measurement of each sample, the two analyses of this group are treated here separately so that standard deviations for all groups are based on six subsamples per soil sample. Due to a nonuniformity of variance, data were transformed to log10(x 1 1), where x is the number of microsclerotia of V. dahliae plus V. tricorpus g of soil21 per petri dish. This transformation was better at achieving a homogeneity of variance than several others tried. Effects of transporting samples, analysis method, soil sample source, and interactions on the means of transformed units per subsample (yijk) were investigated by using the fixed-effects analysis of variance (ANOVA) model yijk 5 m 1 ai 1 bj 1 abij 1 eijk, where m is the mean, ai is the effect of method i, bj is the effect of soil sample j, abij is the interaction, and eijk is the error term, with k indicating the subsample per method per soil. Analysis was performed with the SAS procedure GLM (SAS System version 6.12; SAS Institute Inc., Cary, N.C.). Variability between petri dishes within subsamples and between subsamples was quantified by using the variance component model yijkl 5 m 1 ai 1 bj 1 abij 1 sijk 1 pijkl, where yijkl is the transformed value per petri dish, sijk is the random
RESULTS Differences between transported and nontransported samples were significant (P , 0.05) for three groups by t tests. After correcting for the number of comparisons, using the Bonferroni approach (testing at a significance level of 0.05), two comparisons showed significant effects. Group H reported that numbers of V. dahliae plus V. tricorpus in the transported samples were 3.3 times lower than those in nontransported soil (see Fig. 4). Results of group E (see Fig. 4) and of method b1 (see Fig. 3) of group B showed the opposite effect: the numbers of V. dahliae plus V. tricorpus were 1.8 times (not significant) and 7.8 times (significant) higher in the transported samples than in the nontransported samples. The high degree of recovery in the transported sample measured by method b1 is due to a single extreme value reported for one subsample (Fig. 3). The naturally infested soil samples differed widely in their content of V. dahliae, which ranged on average from 1.1 to 120 CFU g of soil21 (Table 2). The results obtained by the different groups for the same soils differed markedly (Fig. 1 to 4). The means of the nontransformed data ranged from 0.57 CFU for method a to 67 CFU for method k (Table 3). The duplicate analyses b1 and b2 by group B yielded generally similar data. Group F reported twice as many colonies with its DP assay (method f-2) as with its WP assay (method f-1) (Table 3). Numbers obtained by DP assays were generally higher than those obtained by methods based on WP, although group G (method g) reported obtaining relatively low values by the DP-based method (Table 3). Median and backtransformed values per method were considerably lower than the arithmetic means (Table 3). This was due to the high frequency of zero values in the subsamples (DP assays, 14%; WP assays, 7.7%) and the petri dish counts (DP assays, 39%; WP assays, 37%). An ANOVA of log-transformed subsample means with method, sample, and method 3 soil interaction as sources of variation explained 88% of the total variation (r2), and all means were highly significant (Table 4). The interaction indicates that certain methods work better for certain soils, although this effect is relatively small compared to the main effects (Table 4). Residual plots, used to check the goodness of fit of the model, showed a few studentized residuals with absolute values of larger than 4. This appeared to be due to some outliers of groups B (method b1) and H (Fig. 1 to 4). For the samples containing 5 or 60 mpg, maximum mean recoveries were 59% (group H, by the DP assay) and 41% (groups H, I, and K, by the DP assay), respectively (Table 5). The maximum mean recoveries determined by WP assays were 39 and 25% (both group E), respectively. There was an insignificant trend toward a lower percent recovery at the higher population level. Large fluctuations among groups occurred for the estimates of variance among subsamples (ss2) and among petri dishes (sp2) (Table 6). For all methods sp2 was larger than ss2. In all cases except method g, methods based on DP resulted in larger sp2 values than did those based on WP. The large residual for method h seems to be due to variation among subsamples rather than variation among petri dishes.
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Description of: Method
a b c d e f-1 f-2 g h i j k l m
Assay type
WP WP WP WP WP WP DP DP DP DP DP DP WP WP
Handling of soil sample prior to platinga
Plating method
Medium
Sievingb
Waterc
Amt of soil used (g)d
Additione
Other concn method appliedf
Amt of soil suspension used (ml)g
Amt of soil plated (g)h
Petri dish typei
No. of petri dishes usedj
38–130 NDt 20–120 20–130 ND ND ND ND 0–8,000 0–250 0–1,000 ND 0–1,000 44–150
Dis Dem Dis Tap Dis Dis NAw NA NA NA NA NA Dis Dis
15 20 25 25 10 27v NA NA NA NA NA NA 20 10
None None None Agar None None NA NA NA NA NA NA Agar Agar
Sucrose None None None None None None None None None None None None None
1.0 1.0 1.0 0.8 1.0 1.0 NA NA NA NA NA NA 0.5 1.0
1.5 0.20 0.25 0.20 0.10 0.27 0.09x 0.10 0.09 0.05 0.05 0.025 0.10 0.10
p g p p1c g p p p1c p p p p p1c p
10 10 10 10 6 5 5 4 9y 5 3 10 5 6
Carbon source(s)k
P P P P P P P P E, S P E, S P E E, S
Incubation conditions
pHl
Antibioticsm
Detection leveln
Drying time (min)o
Temp (°C)p
Lightq
Storage positionr
Storages
Duration (days)
5.0 5.6 6.4u 5.8 6.0 4.5 4.5 4.5 ND 5.8 6.8 7.0 7.9 ND
S, T S, T C, O, S, T O, T C, O, S, T C, O, S, T C, O, S, T C, O, S, T C, E, PC, S C, O, S, T C, E, O, P, S, T C, E, O, S, T E, O C, E, PC, S
0.067 0.5 0.4 0.5 1.7 0.74 2.2 2.5 1 4 6.7 4 2 1.7
20 30 30 0 15 0 NA NA NA NA NA NA 0 0
21* 25* 22 22* 22 23* 24 22* 24* 18 22* 24 23 27
DL D D D D DL D D DL D D D D D
Up Up Up Up Up Up Down Up Down Up Up Down Up Down
Co 2 2 2 P2 2 P 2 2 P P P P2 Ce
42 14 28 14 28 10 14 14 21 21 14 14 21 18
TERMORSHUIZEN ET AL.
TABLE 1. Description of methods used to determine the density of microsclerotia of V. dahliae in soil
a
Prior to being shipped, soil samples were air dried (14 days), ground, and sieved (2-mm mesh size). Shown are minimum and maximum sieve sizes (in micrometers); soil was wet sieved for WP and dry sieved for DP. Kind of water used for preparation of the soil suspension. Dem, demineralized water; Dis, distilled water; Tap, tap water. d Weight of soil per 100 ml of water. e Additions to the soil suspension. Agar was added at 0.08 to 0.1% agar. f Other methods used to concentrate microsclerotia prior to plating. Suc, sucrose flotation method (described in reference 2). g Volume of soil suspension plated on each petri dish. h Weight of air-dried soil plated on each petri dish. With WP, the soil weight is that per petri dish if no loss occurred due to wet sieving. With DP, the soil weight indicated is the actual amount present per petri dish. i p, standard plastic petri dishes, without cams; p1c, plastic petri dishes with cams; g, glass petri dishes. j Number of petri dishes employed per soil sample. k P, pectate; E, ethanol; S, sucrose. l Unless otherwise noted, values are mean pHs of the nutrient medium after autoclaving. m Antibiotics and other growth inhibitors used in the nutrient medium. C, chloramphenicol; E, ethanol; O, oxy/chlortetracycline; PC, pentachloronitrobenzene; S, streptomycin; T, tergitol. n Number of microsclerotia g of air-dried soil21 when only one microsclerotium was found on a petri dish. o After addition of the soil suspension, the agar surface was often allowed to dry before the lid was placed on a dish. p Mean incubation temperature. The presence of an asterisk indicates ambient incubation conditions; the absence of an asterisk indicates temperature-controlled incubation conditions. q Light conditions during incubation. D, constant darkness; L, constant light; DL, alternating darkness and daylight. r Placement of the petri dishes during incubation. Up, upright; Down, upside down. s Petri dish storage conditions. Ce, in cellophane; Co, in transparent containers; P, in plastic bags; P2, first 3 days in plastic bags, then without any kind of packaging; 2, without any kind of packaging. t ND, not done. u Mean pH before autoclaving. v Mean value; range, 16 to 37 g. A 20-cm3 sample of soil was taken, weighed, and added to 80 cm3 of water. w NA, not applicable. x Mean value; range, 0.05 to 0.12 g. Samples were taken on a volume basis. y During plating of the 9 petri dishes, a 10th dish was placed at the bottom of the Andersen sampler to retain soil that escaped the other dishes. b c
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TABLE 3. Mean and median inoculum densities of V. dahliae plus V. tricorpus by method Method
Assay type
a b1 b2 cc d e f-1 f-2 g h i j k
WP WP WP WP WP WP WP DP DP DP DP DP DP
Inoculum density (CFU g of soil21) Mean
a
0.57 19 21 28 17 26 18 36 16 36 41 54 67
Backtransformed meanb
Mediana
0.33 7.2 7.3 6.7 6.9 8.0 4.0 9.9 3.1 8.7 9.4 18 12
0.27 10 8.6 4.6 14 13 4.9 15 2.5 18 20 40 36
a
Calculated from average densities per subsample. Log(x 1 1) subsample means were calculated and backtransformed 2 1). (10 c Group C analyzed only part of the soil samples, and their data therefore cannot be compared to the data of the other groups. b
log(x11)
FIG. 1. Densities of V. dahliae plus V. tricorpus in six subsamples per soil sample (squares) and their averages (asterisks) per method for soil samples that were artificially infested with 5 and 60 mpg. The methods are described in Table 1.
DISCUSSION Our results show that laboratories differ widely in their ability to quantify V. dahliae in soil. One problem which had not been anticipated was the difficulty that some groups experiTABLE 2. Mean and median inoculum densities of V. dahliae plus V. tricorpus for each soil sample Soil sample
a b c d e f g h i k l m 5d 60e a
Inoculum density (CFU g of soil21) Mean c
a
24 17 1.1 30 57 8.0 120 58 17 42 23 3.3 1.4 12
Backtransformed meanb
Median
7.9 4.7 0.24 16 21 2.1 63 31 7.6 13 6.9 0.92 0.37 4.3
13 6.9 0.29 25 47 4.1 94 48 14 28 14 1.9 0.62 10
Calculated from average densities per subsample. Log (x 1 1) subsample means were calculated and backtransformed log(x11) 2 1). (10 c Calculated without the data of group C, who analyzed only part of the soil samples. d Soil sample with 5 mpg. e Soil sample with 60 mpg. b
enced in distinguishing colonies of V. dahliae from those of V. tricorpus. V. tricorpus is a commonly occurring saprophyte or weak pathogen (12) that can be distinguished from V. dahliae by its dark hyphae and chlamydospores (12, 20). Those confident in making this distinction reported low numbers of V. tricorpus compared to V. dahliae on plates; therefore, for the purpose of analyzing the data, we assumed that including this species would have no substantive effect on the conclusions drawn. Methods were based on either DP or WP of soil, but there were many other variations (Table 1), probably including several which are insignificant—for example, the precise manner in which the Andersen air sampler was handled. Nevertheless, it is clear that with the exception of group G, the DP assays (methods f2 to k) yielded higher numbers of CFU per gram of soil than did the WP assays (methods a to f1). The parallel comparison by group F of WP and DP assays (methods f-1 and f-2) strongly supports this conclusion. The difference in the recovery rates of DP and WP assays may be explained in part by the loss of microsclerotia from wet sieving (16, 21). However, the results of group E, which did not sieve, suggest that other factors also may be involved. Thus, the conditions for germination of microsclerotia may be better if dry, small, discrete aggregates of soil, rather than an aqueous suspension of aggregates, are dispersed over the agar surface. In their comparison of methods, Nicot and Rouse (16) also found that recoveries from naturally infested soils by a DP-type assay were at least twofold higher than those of dilution plating (a WP-type assay). However, the DP-type assay was found to be biased in contrast to the dilution plating method. This remark-
TABLE 4. ANOVA for transformed data Source
df
Sum of squares
Mean squares
Method Soil Method 3 soil Error
11 13 143 840
78.753 242.810 101.623 55.543
7.159a 17.678a 0.711a 0.066
a
P , 0.001.
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TERMORSHUIZEN ET AL.
FIG. 2. Densities of V. dahliae plus V. tricorpus in six subsamples per soil sample (squares) and their averages (asterisks) per method for soil samples with low infestation levels. Soil and method codes are explained in the text. The methods are described in Table 1. Method labels marked with an asterisk indicate that the results of those of nontransported soil samples.
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FIG. 3. Densities of V. dahliae plus V. tricorpus in six subsamples per soil sample (squares) and their averages (asterisks) per method for soil samples with medium infestation levels. Soil and method codes are explained in the text. The methods are described in Table 1. Method labels marked with an asterisk indicate that the results are those of nontransported soil samples.
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FIG. 4. Densities of V. dahliae plus V. tricorpus in six subsamples per soil sample (squares) and their averages (asterisks) per method for soil samples with high infestation levels. Soil and method codes are explained in the text. The methods are described in Table 1. Method labels marked with an asterisk indicate that the results are those of nontransported soil samples.
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able result may be due to an artifact caused by the use of steamed silica sand and artificially produced inoculum to quantify bias. Certain aspects of the methodology appear to be less important than had been thought, while others may be more important than was realized. Thus, groups I and K, using a pectin carbon source in the plating medium, obtained results similar to those of groups H and J, which used sucrose. The results of group E indicate that wet sieving of soil samples in the WP assays is not as advantageous as previously reported (5, 21). The pH of the plating medium was highly correlated with the percent recovery from the soil to which 60 microsclerotia were added per gram (Pearson correlation coefficient 5 0.76; P , 0.01). A medium of pH 5.6 or less was associated with low counts, and media of pH ca. 7.0 were associated with the highest recoveries. The analysis method 3 soil sample interaction was significant (Table 4). Thus, for soil sample l, group B obtained counts higher than all other methods, whereas with all of the other soils, the counts were generally low (Fig. 1 to 3). Similarly, for soils i and k, group I obtained lower mean counts than did groups J and K, whereas groups I, J, and K had similar counts for the other soils. The method 3 soil interaction may be caused by different microbial communities that act according to, for instance, the plating medium used. Recovery percentages could be used to obtain less-biased estimates of the densities in soil samples containing unknown inoculum densities, but this approach is precarious if the analysis 3 method interaction is significant. For example, the best estimate for the overall mean for group G would be 552 CFU g of soil21 (the mean [Table 3] divided by R60/100 [Table 5] 5 16/0.029), whereas for group K it would be 163 CFU g of soil21 (67/0.41). Thus, the noncritical use of percent recoveries from samples with known densities is not necessarily the correct approach for improvement of detection assays. In general, however, the interaction effect was small compared to the effect of the analysis method, indicating that the performance of most methods is soil type independent. The comparison of transported with nontransported samples gave inconsistent results. However, transport of samples was not a major factor influencing the results of this study. The degree to which V. dahliae was affected during transportation would depend on the conditions to which it was subjected, and these conditions would have been unique to each consignment for each destination. In addition, the mode of separating the sample into the transported and nontransported subsamples was not prescribed and may have been biased. Variation among plate counts was 1.3 to 36 times greater than that among subsamples (Table 6). The generally higher degree of variation among petri dishes for the DP assay than for the WP assay may be attributed to the small subsample (25 to 100 mg) leading to more zero values for the former. Therefore, at levels below 1 CFU g of soil21, which may still be epidemiologically significant (5, 17), the higher degree of accuracy of the DP assay than the WP assay may be outweighed by its poorer precision and sensitivity limits. Variability was slightly higher among subsamples tested by WP methods than with DP assays, probably because of the sub-subsampling needed to prepare the soil dilutions for the WP assays. The variance of an estimator for the infestation of a sample can be expressed as the sum of the estimated variances among subsamples, ss2, divided by the number of subsamples, I, and the estimated variance among petri dishes, sp2, divided by I and divided by the number of petri dishes per subsample J. If the number of petri dishes is held constant, the variance decreases only if I is increased. It is possible to calculate the amount of
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TABLE 5. Percentages of recovery of V. dahliae from soil specimens artificially infested with known densities of microsclerotia % Recovery from soil infested at: Method
a b1 b2 d e f-1 Meand f-2 g h i j k Meand
Assay type
5 mpg
60 mpg
Meana
Rangeb
Mediana
Backtransformed meanc
WP WP WP WP WP WP
0.9 14.6 15.6 33 39 11
0–4 0–25 6.3–25 11–44 0–100 0–26
0 16 13 39 33 13
0.70 7.8 7.7 14 10 5.6
WP
20
0.9–39e
DP DP DP DP DP DP
42 0 59 40 44 40
0–150
DP
38
0–59e
0–110 0–160 0–130 0–80
18 0 56 0 0 40
12 0 14 7.1 8.1 4.1
Meana
Rangeb
Mediana
Backtransformed meanc
1.3 8.0 6.8 16 25 3.2
0–2.6 5.7–13 3.6–9.4 4.6–21 17–36 0–8.0
1.8 6.8 6.5 17 22 3.1
0.79 5.3 4.2 9.8 14 1.6
11
1.3–25e
18 2.9 41 41 30 41
15–20 0–5.8 24–72 27–60 11–44 20–73
18 3.8 38 40 28 33
9.3 1.5 23 23 15 9.8
29
2.9–41e
a
Mean calculated from average densities per subsample. Minimum and maximum subsample values. c Log(x 1 1) subsample means were calculated and backtransformed (10log(x11) 2 1). d Prior to calculation of mean values per assay type, method b1 and b2 data were averaged. e Range of the means of subsamples. b
subsamples (I) needed to reach a certain variability at a given probability. Snedecor and Cochran (22) showed that, assuming a normal distribution and known variance s2, half the width of a 0.95 confidence interval L 5 1.96 3 s 3 I20.5. For a given L, this results in I 5 s2 3 1.962 3 L22 5 (s/m)2 3 1.962 3 (L/m)22 5 SMR2 3 1.962 3 (L/m)22, where SMR is the standard deviation-to-mean ratio, m is the (unknown) expectation, and L/m is half the width of the confidence interval expressed as a fraction of the mean. The SMR per sample (n 5 6) varied from 0.3 to 0.7 per group. At these extremes, the numbers of samples required to realize an L/m of 0.3 would be 4 and 21, respectively. It is apparent from Fig. 5 that it is highly advantageous to select for methods that produce low SMR values. Our results lead to the conclusion that the data available in
literature presenting densities of V. dahliae in soil are difficult to interpret. For this reason, methods should be compared with those of other laboratories and control soil samples containing known densities of microsclerotia should be included. Ideally, research workers should settle on a single well-defined protocol to increase the comparability of results obtained at different laboratories. In conclusion, it is apparent that WP assays are less accurate than DP assays. Additional experimental analysis of the conditions required for microsclerotia to germinate and to develop new microsclerotia in the plating medium is needed to explain the substantial variation noted within the two assay types. Method components that probably deserve experimental attention are plating medium composition and petri dish incubation conditions. The low recovery percentages for both assay types indicate that there is room for improvement of the methodologies.
TABLE 6. Estimates of variances for inoculum densities of V. dahliae plus V. tricorpus among soil subsamples (ss2) and petri dishes with the same subsample (sp2) Assay
ss2
sp2
sp2/ss2
WP a b1 b2 c d e f-1
0.0090 0.0604 0.0338 0.0042 0.0393 0.0357 0.0360
0.0293 0.0809 0.0786 0.0258 0.1086 0.2029 0.1238
3.3 1.3 2.3 6.2 2.8 5.7 3.4
DP f-2 g h i j k
0.01595 0.01436 0.15756 0.00906 0.00000 0.01811
0.23837 0.06165 0.22756 0.32756 0.30272 0.42394
15 4.3 1.4 36 23
FIG. 5. Sample size as a function of standard deviation-to-mean ratio at four different values of L/m (i.e., half the width of the confidence interval [L] expressed as a fraction of the mean [m]).
QUANTIFICATION OF V. DAHLIAE MICROSCLEROTIA
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