Biometry of oospores and intraspecific variation of four Pythium species

Mycol. Res. 105 (10) : 1206–1215 (October 2001). Printed in the United Kingdom.

1206

Biometry of oospores and intraspecific variation of four Pythium species

Kaare MØLLER and John HOCKENHULL Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. E-mail : kaare.moller!get2net.dk or km!kvl.dk Received 14 September 1999 ; accepted 27 June 2001.

Evidence that the biometric characters of the oogonial unit of Pythium are of significant taxonomic value, allowing for a good species separation, has been presented by other workers, based on 40 species, each represented by fairly few isolates. Since broad intraspecific variation and interspecific overlapping of biometric parameters was observed in isolates of four Pythium species from Denmark, it was suspected that a such wide variability would weaken the conclusions obtained by previous biometric approaches. Species to be studied should be represented by higher numbers of isolates to reflect intraspecific variation. In the present biometric study 49 isolates were used to test the robustness of the biometric approach and to examine the taxonomic value of biometric characters from a practical perspective. Good species separation was obtained despite intraspecific variation. The biometric parameter set for the oogonium, consisting of oogonial, oospore and ooplast diameters, supplemented with the thickness of the oospore wall or the protoplast diameter and with derived indices of volume or linear ratios of the measured parameters were all of taxonomic value for the separation of species, as shown from canonical variate plots after linear discriminant analysis. More detailed information on species separation, isolate-species or isolate–isolate associations could be obtained by separation based on the Mahalanobis distance function in the linear discriminant analysis. Both the derived volume and linear indices contributed significantly to species separation and appeared to be equally satisfactory. The substrates used for oogonium production may influence the biometric parameters to an extent which may seriously affect the results of the biometric approach to taxonomy. These results stress the importance of standardizing substrates in morphological taxonomy and identification work.

INTRODUCTION A high degree of intraspecific variability was observed for a series of Pythium tracheiphilum isolates obtained from Chinese cabbage leaves attacked by leaf and head rot, and in the course of a study of the prevalence of P. tracheiphilum and other Pythium species on roots of Chinese cabbage. A similarly high variability was found in the three species most frequently isolated from roots, namely P. rostratum, followed by P. oligandrum, and, from a particularly wet field area, P. pythioides (Møller 1999). Pronounced variation in biometric parameters caused difficulties in the initial phase of identification, requiring consideration of the full range of species characters as well as comparisons with other species of similar morphology. The taxonomic value and importance of biometric parameters of the genus Pythium was regarded as minor by Middleton (1943) and Hendrix & Campbell (1974). This may be contrasted with the importance attributed to these parameters within the oomycetes by Dick (1969), Soumati & Dick (1989), and Dick (1990). The difference is due to the fact that the former authors assessed parameters only one at a time, while the latter considered the interrelationship of

several parameters. Shahzad, Coe & Dick (1992) further demonstrated that not only were the directly measured data of the individual reproductive unit of pythiaceous species of high taxonomic value, but also their corresponding volume ratios were independently of taxonomic significance. They considered, on the basis of the combined biometric data of single reproductive units, that most Pythium species would probably be separable without the use of further taxonomic data. However, their study was restricted to one to four isolates for each species. In view of the observed variation of isolates from a narrow geographical origin, larger numbers of isolates from the four Pythium species mentioned were used to evaluate intraspecific biometric variation and to investigate whether the biometric data alone would still delimit single species well, using the analytical model of Shahzad et al. (1992). Although the four species could be clearly differentiated on the basis of non-biometric characters, single biometric parameters, such as the oospore diameter seemed to constitute a continuum, isolates and species greatly overlapping. It was expected that this would result in the overlapping of species in the analytical model, resulting in a poor species delimitation.

K. Møller and J. Hockenhull

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Pythium tracheiphilum, which was of key interest in this study, was quite variable in the ability of different isolates to form oogonia on grass leaves (rye grass, Lolium pereTnne), while production was uniform and generally high on CMAw (cornmeal agar, 1n5 % Difcoj0n05 % wheat germ oil). For this reason CMAw was preferred for comparison of the four species in the present study, although Shahzad et al. (1992) used grass leaves (species not indicated) for oogonium production. However, in order to study influence of substratum on the variation of biometric parameters of P. tracheiphilum isolates, comparisons were made of isolates grown on CMAw, grass leaves (rye grass) and two other substrates (see below).

isolates of P. oligandrum and P. rostratum were obtained from 80 tap roots in isolations made in 1997, using the same methods as described by Møller & Hockenhull (1997). A total of eight root isolates of P. tracheiphilum were obtained in the latter process, but none from the former. Representative isolates (13) used in the study are deposited in CBS (Utrecht) as well as being maintained in our laboratory. Production of oogonia Oogonium production of Pythium tracheiphilum was most stable on CMAw. For species comparison all isolates were grown on this substrate at 20 m in 9-cm Petri dishes until mature oospores had formed.

MATERIALS AND METHODS Species and isolates examined

Substrate effects on biometric parameters

Four species of Pythium represented by a total of 49 isolates were examined (Table 1). The four geographical locations from which isolates of P. tracheiphilum were obtained were all in southern Zealand, Denmark, three of these locations were less than 3 km apart, while the fourth (Vedskølle) was located about 70 km west of the other three (Table 1). Isolates of the other three species were all obtained from the same field, also in southern Zealand (Table 1). Isolates of P. pythioides (identification in Møller 1999) were obtained with a number of other Pythium species recovered from 100 tap roots of Chinese cabbage in 1995 (Møller & Hockenhull 1997), while

For examination of substrate effects on Pythium tracheiphilum isolates were also grown in 9-cm Petri dishes at 20 m until mature oospores were formed on : (1) Rye grass leaves : 10–15 5-cm leaf segments, autoclaved in 10–15 ml distilled water in glass Petri dishes. (2) HAs : Filtered decoction of 20 g crushed hemp seedsj20 g sucrosej15 g Bacto agar, Difco, adjusted to 1 l with water. (3) V8s : 200 ml filtered Campbell’s V8 juice (dextrose content " 30 g l−")j14 g sucrosej15 g Bacto agar, Difco,j3 g CaCO adjusted to 1 l with water. $

Table 1. Isolates of Pythium species examined, their source and origin. Species P. oligandrum km13o, km14o, km21o, km29oa, km34o, km35o, km38o, km39o, km41o, km43o, km44o, km55o, km59o, km5o, km67o, km8o P. pythioides km1pRV, km2pRV, km32pRT, km33pRT P. rostratum km15r, km16r, km17r, km1r, km20r, km21r, km22r, km31r, km32r, km36r, km40r, km45r, km4r, km52r, km58r, km60r, km9r P. tracheiphilum km1at, km18t, km19t km7t km25t, km26t, km27t, km28t km30t, km31t, km32t, km33t km38t km39t km40t1, km41t1

Source

Locality (farm), Year

Roots of Chinese cabbage

Over Vindinge, 1997

Roots of Chinese cabbage

Over Vindinge, 1995

Roots of Chinese cabbage

Over Vindinge, 1997

Attacked leaves of Chinese cabbage* Vascular stem tissue of attacked Chinese cabbage* Attacked leaves of Chinese cabbage* Attacked leaves of Chinese cabbage* Attacked leaves of Chinese cabbage* Attacked leaves of Chinese cabbage* Field soil Roots of Chinese cabbage

Snertinge, 1992 Snertinge, 1992 Snertinge, 1993 Over Vindinge, 1993 Sværdborg, 1993 Vedskølle, 1993 Over Vindinge, 1996 Over Vindinge, 1997

* Disease : Leaf and head rot, causal agent P. tracheiphilum. Table 2. Oogonium production of isolates of Pythium tracheiphilum on four substrates. (j indicates successful production). Isolates Substrate

km1t

CMAw Rye grass HAs V8s

j j

km18t

j

km25t

km26t

km27t

j

j j j j

j j j j

j j

km28t j

km30t

km31t

km32t

km33t

km38t

km39t

km40t1

km41t1

j j j j

j

j j

j

j j

j

j

j

j

j

Biometry of Pythium Isolates with a successful oospore production (Table 2) were then biometrically analysed.

Biometry Cultures in agar blocks or on grass leaves in water were mounted on slides and observed at 10i100 magnification under oil immersion. All Pythium tracheiphilum isolates with the exceptions of km38t, km40t1 and km41t1 were measured using a microscope for which the eyepiece calibration corresponded to 0n640 µm per unit, while all other measurements, in a later batch, were made using a microscope with an eyepiece calibration of 0n729 µm per unit. Oogonium, oospore, protoplast and ooplast diameters were recorded, using rounding of readings to half units. Shahzad et al. (1992) examined the taxonomic value of the measured diameters of oogonia, oospores and ooplasts, and of oospore wall thickness, but also constructed indices based on the volume ratio between oospore and oogonium (aplerotic index), between ooplast and protoplast (ooplast index) and the volume ratio between wall and oospore (wall index), calculated as 1 minus the volume ratio between the protoplast and the oospore. The three ratios or indices were expressed in percent. In their study the protoplast radius required for volume calculation was derived as the difference between the measured oospore radius and wall thickness. In our study, the protoplast diameter was measured directly, and where necessary, wall thickness was derived as the difference between oospore and protoplast radius. The aplerotic, ooplast and wall indices were calculated for each oogonial unit, according to the formulas of Shahzad et al. (1992), but also linear ratios of oospore\ oogonium, ooplast\protoplast and protoplast\oospore diameters (in the following referred to as the aplerotic, ooplast and protoplast linear indices) were calculated in order to test whether these more simply calculated indices might also contribute equally well to the discrimination between isolates and species. In the following, the three index-values devised by Shahzad et al. (1992) will be referred to as the aplerotic, ooplast and wall volume indices. The rationale (Shahzad et al. 1992) for using volumes was based on the biological capacity of the spore ; much of the earlier dispute over linear measurements was due to the small micrometer differences and the direct (not normalized) comparisons between species with large or small diameters. While the volume indices are expressed as percent as defined in the referred study, the linear indices are expressed as normalized ratios.

Statistics Sample size The measurement of the biometric data for 30 oogonia of all isolates of Pythium tracheiphilum except km38t, km40t1 and km41t1 (see below) served for a verification of the finding of Shahzad et al. (1992) that the standard error of oospore diameter measurements would be acceptably low (in their study  0n5 µm) when using a sample size of 20 oogonia per isolate. Upon this verification, applying the same standard error limit, a sample size of 20 was accepted for all remaining

1208 isolates and species. However, when all data had been collected, standard errors were calculated for oospore diameters of all isolates in order to evaluate whether the applied sample sizes were in fact sufficient for all species and isolates. The degree of continuity of different parameters over the full range of observations from all isolates on all substrates was examined by plots of the parameter ranks. Parameter evaluation An important point made by Soumati & Dick (1989) was that biometric data should be obtained as parameter sets, characterizing the individual reproductive unit, rather than as means for individual parameters, and this concept was followed in the analysis of data. The measured parameters of isolates grown on CMAw were evaluated in combination with the volume indices devised by Shahzad et al. (1992) and, for comparison, in combination with the linear indices. Linear discriminant function analysis was used for the analysis of data, deriving also their group (isolates or species) mean canonical variates. The first canonical variates of the groups represent the best discrimination between groups, the second the next best discrimination and so on. The number of possible canonical variates corresponds to the number of variables, p (parameters), or to the number of groups k1 (kk1), whichever the smaller. Solutions to the linear discriminant function are based on all included parameters, and individual parameters may have a strong or weak effect on the discrimination between groups and thus contribute strongly or weakly to the solutions. The value of a specific parameter for the group discrimination may be assessed by its correlation to the different canonical variates, and these correlations were calculated. The magnitude of a canonical variate of a specific group is determined by the parameters specific for the group, and the variate then is a collective expression for the effect of all the group parameters, dominated by those which contribute significantly to the discrimination. Since groups (species or isolates) are discriminated by their differences in canonical variates, and the variates are noncorrelated, the extent to which the parameters separate groups may be well visualized by plotting, for example, the first variates against the second variates, since the variates are collective expressions of several parameters. Such plots were made where appropriate. The correlation between parameters was also evaluated. In the linear discriminant analysis the distances (Mahalanobis distance ; e.g. Armitage & Berry 1991) separating individual observations (oogonia) from group centres in the seven-dimensional ‘ space ’ (cfr seven parameters) may be calculated, and the distance from the single observation to the different group centres may thus be used as a criterion for classification, allocating the particular observation to the least distant group. For the present set of data, group centres may be either the individual isolates or the species. When species are used as group centres in the analysis, the fractions of single observations correctly allocated to their species of origin and fractions misclassified to other species are calculated on the basis of distance evaluations, thus giving a more detailed evaluation of how well the biometric parameters separate

K. Møller and J. Hockenhull

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species compared with what could be obtained from plots of canonical variates. It is possible to evaluate how large a proportion of a species as a whole and how large a proportion of an individual isolate was misclassified. Thus, both the tendency for species to overlap in biometric criteria and the classification status of individual isolates could be evaluated. Using isolates as group centres, possible tendencies of isolate–isolate associations or of sub-grouping within a species may be evaluated in a similar way. Such analyses were carried out on the total set of data and on sub-sets, using either isolates or species as class variables, for group definitions, as appropriate. In order to test the robustness of the analysis results, cross-validation of data was performed. Crossvalidation is carried out as an iterative process, in which the parameter set of one individual observation at the time is excluded from the data set, which is then subjected to linear discriminant analysis, and subsequently the parameter set of the observation excluded is matched with the analysis result, in order to see to which group this observation is now allocated. If the analysis result is robust, there should be little discrepancy between the first results of discriminant analysis on the full data set and the results of the cross-validation process. Comparison of the two sets of results was made in order to evaluate robustness of the method and the result after cross-validation was considered the final result. The discriminant analyses were carried out using the SAS system (SAS Institute 1989). RESULTS AND DISCUSSION Isolates were markedly different in their capacity to produce oogonia as well as sporangia on the different substrates (Table 2), with the result that biometry was not possible for some isolate – substrate combinations. Variability between isolates also showed in the onset of oogonium initiation – most isolates produced oogonia in 3–5 d, and oospores in 7–8 d, while some did not produce these structures until a culture age of 30 d or more had been reached. A similar pattern of variation was seen for sporangium production. In some isolates, on some substrates (while not necessarily on others), oospore degeneration or absence of ooplasts was so extensive that full biometry was impossible. In one isolate, km39t, on CMAw, ooplasts were lacking in four observations, but the remaining 26 sets of observations were used in the analysis. The three other species were fully capable of producing oogonia and oospores on CMAw, although in some isolates a high proportion of degenerating oospores was noted (particularly in Pythium oligandrum).

Shahzad et al. (1992) stated that no Pythium species is truly plerotic, which may be true, depending on the interpretation of ‘ plerotic ’ (oospore filling the entire oogonial cavity). However, in only very rare cases could an oogonial sac or oogonial wall be discerned as separate from the oospore wall in P. tracheiphilum. Since the oogonium wall thickness was not measurable in light microscopy, equal diameters were recorded for oogonium and oospore diameters for all but a very few observations. This situation may be contrasted with taxa such as Pachymetra, in which the oospore wall is laid down against the entire inner surface of the oogonial cavity, truly filling that cavity, though not necessarily confluent with the oogonial wall layer (Dick et al. 1989). In the few cases where different oospore and oogonium diameters could be recorded, the oospore was clearly free of the oogonial sac. In another few cases the oogonial sac was just visible as a slight ‘ neck ’ at the oogonium base, free of the oospore wall in less than 1\4 of its circumference, and identical diameters were recorded for the two units. The apparently plerotic condition of isolates did not depend on the substrates used. Matta (1965) reported a diameter difference between oogonium (indicated as 13– 16–18 µm) and oospore (indicated as 12–15–17 µm) of 1 µm for Italian isolates of P. tracheiphilum grown on oat meal agar, and Tortolero & Sequira (1978) reported a mean difference of 2 µm for Wisconsin-isolates, grown on V8 agarjsitosterol (45 ppm). Other literature reports the diameter of either the oospore or the oogonium, reporting P. tracheiphilum as plerotic (e.g. Zinkernagel & Kro$ ber 1978, Kro$ ber 1985). Thus, the Danish isolates of P. tracheiphilum apparently differ in this respect from those described by Matta (1965) and by Tortolero & Sequira (1978) by proximating to a plerotic condition, at least on the substrates involved in this study. The wide, intraspecific variation of the biometric parameters, with observations for oogonial and oospore diameters overlapping from species to species, is seen from Table 3, and may also be visualized by rank plotting of data (cumulation of observations). From such plots in Figs 1–5 it is seen that discontinuities between isolate means are few and extremely narrow. The linear index rank plots presented in Figs 1–5 exhibit the same pattern as the ranks of measured data and are also comparable to the volume index rank plots presented by Shahzad et al. (1992). When disregarding some extreme values, the range of oospore diameter variation of the four Pythium species represented in this study closely matches the range covered by the 40 species represented in their study. While the standard error level was acceptably low for sample size n l 20 (as well as for n l 30) for the isolates of P. tracheiphilum initially examined, the final evaluation of

Table 3. Range and means of oogonial and oospore diameters for isolates of four Pythium species. Oogonium diam (µm)

Oospore diam (µm)

Species

Number of isolates

min. obs.

min. mean

max. mean

max. obs.

min. obs.

min. mean

max. mean

max. obs.

P. P. P. P.

16 4 17 12

13n9 13n9 8n8 12n2

21n1 18 18n6 15n4

24n9 19n8 22n5 17n8

31n4 26n2 29n9 22n1

10n6 12n4 8n8 12n7

16n5 16n1 17n3 15n4

22 17n2 21n2 17n8

27n7 20n4 27 22n1

oligandrum pythioides rostratum tracheiphilum

Biometry of Pythium

1210 Rank for protoplast diameter

Rank for oospore diameter

1 1 0.8 0.6 0.4 0.2 0

14

16

18 20 Oospore diam (lm)

22

0.8 0.6 0.4 0.2

10

12

14 16 Protoplast diam (lm)

18

20

1 Rank for ooplast linear index

Rank for ooplast diameter

2

0

24

1 3 0.8 0.6 0.4 0.2 0

1

5

6

7

8 9 10 Ooplast diam (lm)

11

12

13

4 0.8 0.6 0.4 0.2 0 0.4

0.45

0.5

0.55 0.6 0.65 Ooplast linear index

0.7

0.75

Rank for protoplast index

1 5 0.8 0.6 0.4 0.2 0 0.74 0.76 0.78

0.8 0.82 0.84 0.86 0.88 Protoplast linear index

0.9

0.92

Figs 1–5. Continuity in distributions shown as ranked mean values (68 means) of different features. Fig. 1. Oospore diameters. Fig. 2. Protoplast diameters. Fig. 3. Ooplast diameters. Fig. 4. Ooplast linear indices. Fig. 5. Protoplast indices for all isolates and all substrates. Table 4. Standard error levels of oospore measurements in µm for four Pythium species on CMAw for sample size l 20. All values are based on isolate means. Species

Minimum

Mean

Maximum

P. P. P. P.

0n40 0n21 0n46 0n18

0n70 0n30 0n87 0n29

1n00 0n37 1n13 0n51

oligandrum pythioides rostratum tracheiphilum

standard errors showed a wider variation in P. oligandrum, and notably in P. rostratum. From Table 4 it is seen that the standard errors of oospore diameters for single isolates ranged from 0n40 to 1n13 µm for these two species. Although the standard error was acceptable for a number of individual

isolates of the latter two species, the species standard error mean for P. rostratum exceeded both the eyepiece unit of 0n729 µm and, of course, the half-unit to which readings were made, while for P. oligandrum standard errors were generally less, yet still not quite acceptable. Thus for these species both the intraspecific variation and the within-isolate variation are larger than for P. tracheiphilum and P. pythioides (Table 3), and it can be concluded that larger sample sizes than 20 should be used for P. oligandrum and P. rostratum. Of the four species treated here, only P. rostratum was included in the work of Shahzad et al. (1992), represented by two isolates. The standard deviations of different parameters found for these two isolates were less than standard deviations found for all but one of the Danish isolates of P. rostratum in the present study (data not presented).

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Table 5. Correlation coefficients for parameters including volume indices. Pooled within-isolate correlations (upper right triangles of table contents), between-isolate correlations (lower left triangles of table contents). Brackets indicate non-significance.

Parameters

Oogonium

Oospore

Ooplast

Wall

Aplerotic index

Ooplast index

Wall index

Oogonium Oospore Ooplast Wall Aplerotic index Ooplast index Wall index

1n00 0n83 0n78 k0n51 k0n84 0n48 k0n77

0n88 1n00 0n89 k0n32 k0n42 0n53 k0n69

0n78 0n86 1n00 k0n41 k0n39 0n84 k0n72

0n45 0n48 0n38 1n00 0n55 (k0n23) 0n90

k0n34 0n12 (0n03) (k0n01) 1n00 (k0n21) 0n62

k0n16 k0n26 0n22 0n10 k0n16 1n00 k0n42

k0n17 k0n24 k0n22 0n73 k0n09 0n33 1n00

Table 6. Correlation coefficients for parameters including linear indices. Pooled within-isolate correlations (upper right triangles of table contents), between-isolate correlations (lower left triangles of table contents). Brackets indicate non-significance.

Parameters

Oogonium

Oospore

Ooplast

Protoplast

Aplerotic index

Ooplast index

Protoplast index

Oogonium Oospore Ooplast Protoplast Aplerotic index Ooplast index Protoplast index

1n00 0n83 0n78 0n87 k0n81 0n44 0n76

0n88 1n00 0n89 0n98 k0n35 0n51 0n69

0n78 0n86 1n00 0n90 k0n35 0n82 0n72

0n86 0n99 0n85 1n00 k0n43 0n51 0n81

k0n28 0n20 0n12 0n21 1n00 (k0n18) k0n57

k0n15 k0n24 0n25 k0n28 k0n17 1n00 0n40

0n17 0n23 0n22 0n39 0n11 k0n32 1n00

Table 7. Correlation coefficients of measured parameters including volume indices to canonical variates derived from discriminant analysis of isolate means. Non-significant values in brackets.

Variates

Oogonium

Oospore

Ooplast

Wall

Aplerotic index

Ooplast index

Wall index

CAN1 CAN2 CAN3 CAN4 CAN5 CAN6 CAN7

k0n71 k0n04 0n11 0n21 0n16 0n58 k0n28

k0n41 0n23 k0n25 0n08 0n24 0n76 k0n28

k0n59 0n58 k0n18 0n17 k0n03 0n49 k0n11

0n48 (k0n05) k0n14 0n76 0n14 0n38 k0n10

0n80 0n38 k0n34 k0n26 0n14 0n06 k0n06

k0n36 0n69 0n08 0n44 k0n12 k0n23 0n37

0n73 k0n19 0n06 0n63 k0n07 (k0n03) 0n17

Table 8. Correlation coefficients of measured parameters including volume indices to canonical variates derived from discriminant analysis of isolate means. Non-significant values in brackets.

Variates

Oogonium

Oospore

Ooplast

Protoplast

Aplerotic index

Ooplast index

Protoplast index

CAN1 CAN2 CAN3 CAN4 CAN5 CAN6 CAN7

0n7 k0n15 (k0n00) (k0n036) k0n23 0n56 0n35

0n43 0n14 k0n12 0n26 k0n13 0n76 0n34

0n67 0n44 k0n17 0n13 k0n28 0n45 0n15

0n53 0n13 k0n21 0n26 (k0n02) 0n69 0n33

k0n68 0n56 k0n17 0n34 0n24 0n16 (k0n04)

0n49 0n65 0n12 k0n2 k0n39 k0n21 k0n29

0n73 0 k0n43 0n16 0n46 (k0n03) 0n19

The taxonomic value of the biometric parameters was evaluated by discriminant analysis of those isolates and species, which were grown on the same substrate, i.e. CMAw. Two analytical procedures were carried out using either the measured parameter set including the volume indices or the measured parameter set including the linear indices. As part of the analyses, correlations between parameters were calculated. From Tables 5–6 it is seen that the parameter correlations both within and between isolates are generally high between

the four measured parameters. Correlations between measured parameters and their derived indices are low within isolates and generally low between isolates, although higher than those found by Shahzad et al. (1992). This difference may possibly be due to the inclusion of a larger number of isolates, defining only four species in our study, versus the much wider span of species represented by only few isolates in the study of Shahzad et al. (1992). However, the generally low correlations between measured and derived parameters

Biometry of Pythium

1212 5

5

8

6 4

4

3

3

2

2 1 CAN 3

CAN 2

1 0

0 –1

–1

–2 –2 –3 –3

–4

–4

–5 –5

–5 –5

–4

–3

–2

–1

0

1

2

3

4

–4

–3

–2

–1

–3

–2

–1

5

0 1 CAN 1

2

3

4

5

2

3

4

5

CAN 1

5

5 7

9

4

4

3

3

2

2 1 CAN 3

CAN 2

1 0

0 –1

–1 –2 –2

–3

–3

–4

–4

–5

–5 –5

–4

–3

–2

–1

0

1

2

3

4

5

CAN 1

Figs 6–7. Plots of first (CAN1) and second (CAN2) canonical variates derived from measuredjvolume index parameters (Fig. 6) and from measuredjlinear index parameters (Fig. 7) for isolates of Pythium oligandrum (o), P. pythioides (p), P. rostratum (r) and P. tracheiphilum (t), respectively.

(volume as well as linear) obtained in this study support the conclusion of Shahzad et al. (1992) that the derived indices function as independent parameters : they are not just linear projections of the measured parameters. With a group number of 49 (isolates) and 7 parameters the analysis allowed for the calculation of 7 canonical variates. Likelihood tests showed that for both types of parameters all seven variates were significant, and correlation tests showed that all parameters significantly contributed to one or more of the variates (Tables 7–8). Both volume indices (Table 7) and linear indices (Table 8) give important contributions to the first two canonical variates and these indices are thus valuable for the group separation. Plots of isolate mean values of the second or third canonical variates against the first variates are shown in Figs 6–7 and

–5

–4

0

1

CAN 1

Figs 8–9. Plots of first (CAN1) and third (CAN3) canonical variates derived from measuredjvolume index parameters (Fig. 8) and from measuredjlinear index parameters (Fig. 9) for isolates of Pythium oligandrum (o), P. pythioides (p), P. rostratum (r) and P. tracheiphilum (t), respectively.

Figs 8–9, respectively, from which clustering of isolates within species and thus separation of species is apparent. Again it is seen that clustering patterns for variates of the two types of parameter sets (volume vs linear indices) are almost identical, but form mirror images of one another, since wall thickness and protoplast diameter, as well as wall and protoplast indices, are complementary parameters. From Figs 6–9 it is seen that although mean values of P. tracheiphilum and P. pythioides oogonium\oospore diameters are close, clusters of each of the two species are well separated from the other species. Pythium rostratum and P. oligandrum also form clusters, which, however, tend to merge (Figs 6–9), both when volume and linear indices are used, and when plotting either variate 2 or 3 against variate 1. These plots only permit a two-dimensional evaluation of separation of species, while in fact the availability of seven variates allows for separation in seven dimensions.

K. Møller and J. Hockenhull

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Table 9. Classification and misclassification of single reproductive units of four Pythium species, after cross-validation, based on individual unit parameter sets including volume index parameter sets. % observations classified into Observations from

P. oligandrum

P. pythioides

P. rostratum

P. tracheiphilum

P. P. P. P.

67n2 2n5 26n2 0n0

12n8 96n3 2n7 0n3

19n4 1n3 61n2 4n3

0n6 0n0 10n0 95n4

oligandrum pythioides rostratum tracheiphilum

Table 10. Classification and misclassification of single reproductive units of four Pythium species, after cross-validation, based on individual unit parameter sets including volume index parameter sets. % observations classified into Observations from

P. oligandrum

P. pythioides

P. rostratum

P. tracheiphilum

P. P. P. P.

71n3 2n5 24n7 0n0

5n9 95n0 2n1 0n6

21n9 1n3 63n2 6n4

0n9 1n3 10n0 92n9

oligandrum pythioides rostratum tracheiphilum

Table 11. Classification and misclassification of single reproductive units and of single isolates (in brackets) after cross-validation, using linear indices. Substrate effect on classification of Pythium tracheiphilum. % observations and number of isolates (in brackets) classified into P. tracheiphilum on Observations from P. oligandrum P. pythioides P. rostratum P. tracheiphilum on CMAw Rye grass HAs V8s

P. oligandrum on CMAw

P. pythioides on CMAw

P. rostratum on CMAw

CMAw

67n8 (15) 2n5 24n4 (3)

8n8 95n0 (4) 2n4

21n3 (1) 1n3 57n1 (14*)

1n6 0n0 12n4

0n0 0n0 0n0 0n0

0n0 0n0 0n0 0n0

3n4 0n0 1n7 0n4

66n9 (10) 11n9 17n5 (1) 13n3

Rye grass 0n3 1n3 2n7 8n3 77n6 (7) 0n0 12n9

HAs

V8s

0n0 0n0 1n2

0n3 0n0 0n0

16n0 (2) 0n0 70n8 (3) 24n6 (1)

5n5 10n5 10n0 48n8 (5)

* One isolate equally split between P. oligandrum and P. rostratum.

Clusters overlapping in a two-dimensional view may thus actually be separated when more dimensions are considered. Tables 9–10 show the frequencies of allocation of individual observations to either their own group of origin (species) or to other groups, when all seven parameters are included in the analysis (volume indices in Table 9, linear indices in Table 10), after cross-validation of data. The allocation of observations to the different species following analysis of the complete datasets was virtually unchanged by the subsequent crossvalidation of data, indicating stability of the results. The majority of observations are correctly classified to species (Tables 9–10), but the tendency of P. rostratum and P. oligandrum to merge is still evident, as also seen in the plots of canonical variates (Figs 6–9), since for these two species from 28 to 36 % of the individual observations are misclassified and about 20 to 26 % of observations are exchanged between the two species. However, when looking to the proportion of correctly classified to misclassified observations within the individual isolate, the absolute majority of observations is generally correctly classified to species. If the individual isolate is allocated to the species to which its absolute majority of single observations are allocated, by this rule 46

isolates of 49 are correctly allocated to their proper species, when the analysis is based on parameters including linear indices, while when including volume indices 43 isolates are correctly classified. The details of allocation of individual observations are not presented. Of the misclassified isolates, one was P. oligandrum (km13o) while the rest were P. rostratum (linear indices : km21r and km45r ; volume indices : km1r, km20r, km21r, km36r and km45r). The wide intraspecific variation of especially P. rostratum and its thus less welldetermined mean values (cfr sample size) probably both contribute to the higher number of misclassifications in this species. Although this analysis is entirely based on biometry, P. rostratum has smooth oogonia while P. oligandrum has spiny oogonia, so the chance of misidentification would, in practice, be greatly reduced. The effect of substrate on biometric parameters of P. tracheiphilum was examined, using the complete biometric data on all isolates of the four species as a frame of reference. The merging of biometric data from P. tracheiphilum on rye grass leaves, HAs and V8s with the data of the four species on CMAw caused only minor changes in the pattern of classification of P. oligandrum, P. pythioides and P. rostratum.

Biometry of Pythium

1214 5

10

4 3 2

CAN 2

1 0

–1 –2 –3 –4 –5 –5

–4

–3

–2

–1

0 1 CAN 1

2

3

4

5

Fig. 10. Plot of canonical variates 2 (CAN2) against variates 1 (CAN1) derived from measuredjvolume index parameters of isolates of Pythium tracheiphilum on different substrates (G l grass leaves, H l HAs, V l V8s, T l CMAw) and of P. oligandrum (l o), P. pythioides (l p) and P. rostratum (l r) on CMAw.

Thus, after analysis and cross-validation of the data, using the linear index parameter set, misclassification increased by one P. rostratum isolate, when compared to the analysis of linear index data of species only on CMAw, while the classification of another isolate of this species became insecure (Table 11). Exchange by misclassification between P. tracheiphilum and the three other species remained minor after introduction of data from other substrates (Table 11), and P. tracheiphilum isolates had a clear tendency to cluster according to substrate background in a plot of canonical variates 1 and 2 (Fig. 10). The four substrate groups of P. tracheiphilum behaved as wellseparated groups, with only little tendency to overlapping of groups, although 6 ‘ isolates ’ out of 31 crossed over to other substrate groups (Table 11). The tendency of substrate groups to merge was largest between isolates on HAs and V8s, while isolates on rye grass leaves were remarkably well-defined as one independent group (Table 11 and Fig. 10). From an analysis, using isolates as group definition, it was seen that there was no tendency in particular for single observations of any isolate on one substrate to be classified with the same isolate on any other substrate (details not presented). It was thus demonstrated that if, in the perspective, an unknown isolate were to be identified on biometric parameters alone, through comparisons to reference parameters of already mapped species, it would be likely that a mis-match of parameters and thus wrong or missing identification would be the consquences of a difference in substrates used for the unidentified isolate and for the species to which comparisons were made. CONCLUSIONS The present study adds to the findings of Shahzad et al. (1992) by showing that even when species are represented by a wider range of isolates and display a broad intraspecific

variation, as do Pythium rostratum and P. oligandrum, the biometric parameters and their derived indices are valuable taxonomic criteria. The two types of derived indices were both useful taxonomic parameters. While the volume indices may be more comprehensible than the linear indices, the latter are more simple to calculate, and in our study they gave a slightly better species differentiation. However, so far, the most sound conclusion seems to be that the two types of indices are equally good. Species separation by biometric parameters may be visualized by pairwise plots of canonical variates or parameters, but such plots do not allow for a detailed evaluation of the extent to which species overlap, when these tend to merge. A more detailed evaluation may be obtained from the allocation\classification of single observations and individual isolates to groups (species), which is based on an evaluation of the distances separating these observations\isolates from group centres. The approach followed in this study was to allocate an isolate to the species the least distant from the absolute majority of the individual isolate observations, and this showed that overlapping was less than suggested by variate plots. A prerequisite to taxonomy based on biometry is the standardization of conditions for production of the structures to be measured. The importance of this common knowledge was demonstrated by the substrate-directed separation into rather well-defined groups of P. tracheiphilum isolates. It was found that the minimum acceptable sample size of 20 units as found by Shahzad et al. (1992) was not necessarily sufficient, since with this sample size standard errors were too high due to intraspecific variation for several isolates of P. rostratum and P. oligandrum. For the practical application of biometric criteria in taxonomy, Shahzad et al. (1992) pointed to the neccessity of procuring the relevant data for the different Pythium species of

K. Møller and J. Hockenhull more geographical origins in order to adequately cover intraspecific variation. Obviously provision of data on ooplast diameters and wall thickness or protoplast diameters is also required, since normally these data do not appear in species diagnoses. As an approach to the practical application of biometry in Pythium taxonomy, Dick (1990) introduced the use of cut-points separating, for example, small oospores from large oospores by a 1 µm discontinuity zone ; no doubt this is a useful approach, which may be further improved through a more profound biometric mapping of the genus. However, the use of a dichotomous key is based on the stepwise matching of single observed parameters to the limits set by the key, and since true discontinuity intervals appear not to exist for any parameter, parameters matching the grey zone of discontinuities will commonly occur, no matter how cut-points are selected. In consequence repeated erroneous selections will also be common, although in the end the correct identification may be reached. The advantage of simultaneous analysis of multiple parameters was stressed by Shahzad et al. (1992), and by such analysis fundamental documentation of the taxonomic value of biometric parameters was provided, and the perspective of constructing an improved dichotomous key based on biometry was outlined. In the present study it was possible to correctly identify 46 of 49 isolates by simultaneous analysis of multiple parameters in one and the same process of analysis. This opens the perspective of constructing a computerized key, combining a suitable statistical programme component and a data base, containing the biometric data of the genus, adequately covering intraspecific variation. Data of non-identified isolates could then easily be merged and analysed with the database data, and suggestions of their species classification retrieved. Such a programme could also be constructed to handle and match morphological data etc., which would possibly be required since, as it seems from the present study, some overlapping of species does occur, even when applying all biometric parameters. Supplementary biometric data of zoosporangia and zoospores may possibly also be of value, and Dick (1990) introduced such biometric data in his Keys to Pythium. However, both the perspectives of a dichotomous and of a computerized key demand for their fulfilment detailed and broad mapping of the genus, but may then offer good tools, comparable to those sought through serological and molecular methods.

1215 A C K N O W L E D G E M E N TS We are thankful to Lars Erichsen, Department for Biostatistics, The Panum Institute, København N., who has kindly provided useful advice on the statistical treatment of data.

REFERENCES Armitage, P. & Berry, G. (1991) Statistical Methods in Medical Research. 2nd. edn. Blackwell Scientific Publications, London. Dick, M. W. (1969) Morphology and taxonomy of the Oomycetes, with special reference to Saprolegniaceae, Leptomitaceae and Pythiaceae. I. Sexual reproduction. New Phytologist 68 : 751–775. Dick, M. W. (1990) Keys to Pythium. Published by the author, Reading. Dick, M. W., Croft, B. J., Magarey, R. C., De Cock, A. W. A. M. & Clark, G. (1989) A new genus of Verrucalvaceae (Oomycetes). Botanical Journal of the Linnean Society 99 : 97–113. Hendrix, F. F. & Campell, W. A. (1974) Taxonomic value of reproductive cell size in the genus Pythium. Mycologia 66 : 681–684. Kro$ ber, H. (1985) Erfahrungen mit Phytophthora de Bary und Pythium Pringsheim. Mitteilungen aus der Biologischen Bundesanstal fuW r Land- und Forstwirtschaft (Berlin Dahlem) 225 : 1–175. Matta, A. (1965) Una malattia della lattuga prodotta da una nuova specie di Pythium. Phytopathologia Mediterranea 4 : 48–53. Middleton, J. T. (1943) The taxonomy, host range and geographical distribution of the genus Pythium. Memoirs of the Torrey Botanical Club 20 : 1–171. Møller, K. & Hockenhull, J. (1997) Leaf and head rot of Chinese cabbage, – a new field disease caused by Pythium tracheiphilum Matta. European Journal of Plant Pathology 3 : 245–249. Møller, K. (1999) Studies of the infection biology and biocontrol of leaf and head rot of Chinese cabbage (causal agent : Pythium tracheiphilum) and of some taxonomical aspects of the genus Pythium. PhD thesis, Royal Veterinary and Agricultural University of Copenhagen. SAS Institute (1989) SAS\STAT User’s Guide. Version 6. 4th edn. SAS Institute, Cary, NC. Shahzad, S., Coe, R. & Dick, M. W. (1992) Biometry of oospores and oogonia of Pythium (Oomycetes) : the independent taxonomic value of calculated ratios. Botanical Journal of the Linnean Society 108 : 143–165. Soumati, B. A. & Dick, M. W. (1989) Biometry of oogonia and oospores of Saprolegniaceae. Botanical Journal of the Linnean Society 100 : 219–235. Tortolero, O. & Sequeira, L. (1978) A vascular wilt and leaf blight disease of lettuce in Wisconsin caused by a new strain of Pythium tracheiphilum. Plant Disease Reporter 62 : 616–620. Zinkernagel, V. & Kro$ ber, H. (1978) Pythium tracheiphilum als Erreger einer Wurzelfa$ ule und Tracheomykose an Kopfsalat. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes (Braunschweig) 30 : 33–36. Corresponding Editor : M. W. Dick