Assessment of host specificity among different species of glyphosate synergistic Pythium

Mycol. Res. 100 (12):1445-1453

(1996)

1445

Printed in Great Britain

Assessment of host specificity among different species of glyphosate synergistic Pythium

R O L A N D 0 C. DESCALZO*, ZAMIR K. PUNJA, C. ANDRE LEVESQUE' A N D JAMES E. RAHE Centre for Pest Management, Department of Biological Sciences, Simon Fraser University, Burnuby, British Columbia, Canada, V5A IS6 Agriculture and Agri-Food Canada, Pacific Agriculture Research Centre, 6660 N. W. Marine Drive, Vancouver, British Columbia, Canada, V6T 1x2

A total of 39 Pythium isolates representing 14 species of Pythium was assessed for host specificity as glyphosate synergists. This was done using three groups of Pythium isolates from roots of glyphosate-treated bean (PBI), wheat (PWI), and isolates from various glyphosate-untreated hosts. PBI consisted of 15 isolates and included P. ultimum, P. sylvaficum, P. coloratum, P. irregulare and P. group 'HS'. PWI consisted of 14 isolates representing the first four of these species. Pythium from glyphosate-untreated hosts included single isolates representing P. aphanidermafum, P. spinosum, P. paroecandrum, P. hypogynum, P. splendens, P. sulcatum, P. vanterpooli, P. acanthicum, P. arrhenomanes and P. coloratum. The glyphosate synergistic potential of the Pyfhium isolates was determined by treating 2-week-old seedlings growing in soil infested with individual isolates of Pyfhium with different doses of glyphosate. LD,, values associated with each isolate were estimated by logistic regression analysis of plant mortalities recorded 4 wk after treatment with glyphosate, and compared with LD,, values for plants grown in the absence of Pythium. Host specificity was assessed by comparing the glyphosate synergistic potential of PBI on bean and PWI on wheat seedlings, with the potential of these same isolates of PBI on wheat and PWI on bean seedlings. Glyphosate synergistic potential of PBI was also estimated on sunflower and pepper, to test whether PBI were capable of glyphosate synergistic interaction (GSI) on other unrelated dicot yledonous species. Pythium isolates from glyphosate-untreated hosts were tested on bean to determine if Pyfhium species not represented in the PBI and PWI groups were capable of GSI. The glyphosate synergistic potentials of the PBI and PWI on wheat seedlings were low and inconsistent compared to those observed on dicot plants. All PWI and 12 of the 15 PBI were glyphosate synergists on beans, and all the PBI were glyphosate synergistic on sunflower and pepper seedlings. All Pythium isolates from glyphosate-untreated sources tested were also glyphosate synergists on bean seedlings. These various tests of glyphosate synergistic potential of Pythium isolates from diverse sources on various plant species revealed no evidence of host specificity among the isolates and species tested.

Soilbome species of Pythium have been shown to augment the herbicidal activity of glyphosate by colonizing the roots of glyphosate-treated plants. This enhancement of herbicidal efficacy was termed glyphosate synergistic interaction (GSI) by Johal & Rahe (1984). Soilbome Pythium spp. were found to be the first and predominant root colonizers of glyphosate treated plants grown in different soils (LCvesque et al., 1993). Several different species of Pythium were involved in GSI on bean (Descalzo et al., 1996). A possible indication of hostspecificity of some glyphosate synergistic Pythium (GSP) was suggested from the result of an experiment by Lkvesque, Rahe & Eaves (1992). in which Pythium isoIates previously collected from the roots of glyphosate-treated appIe and bean seedlings were glyphosate synergists only on the plant species from which the fungus originated. N o other tests conducted so far substantiate this initial suggestion of host specificity among glyphosate synergistic Pyfhium (GSP). This paper describes the results from research to address the host specificity of Corresponding author: R. C. Descalzo.

GSP. The specific objectives were to determine if: (i) isolates of Pythium species from the roots of 2-week-old glyphosatetreated bean and wheat seedlings were selectively synergistic on the host species from which they were isolated; (ii) isolates of Pythium species from the roots of glyphosate-treated bean seedlings were capable of GSI on various unrelated dicot yledonous species; (iii) isolates of Pythium species from glyphosate-untreated hosts were capable of GSI on bean seedlings; and (iv) the relative pathogenicity of isolates of Pythium species differed on glyphosate-untreated hosts.

MATERIALS A N D METHODS Sources of Pythium isolates Isolates of five Pythium species previously collected from the roots of glyphosate-treated bean and wheat seedlings grown separately in various soils were used (LCvesque et al., 1993; Descalzo et al., 1996). Pythium isolates obtained from glyphosate-treated beans or wheat are referred to hereafter as

Glyphosate synergistic Pythium

1446

Table I. Pythium species and the numbers of genotypes represented among isolates obtained from the roots of glyphosate-treated bean and wheat seedlings Number of genotypes from Bean"

P. ultimum Trow P. sylvaticum W. A. Campb. & F. F. Hendrix P. coloratum Vaartaja P. irreguhre Buisman P. group 'HS' Total

Wheatb

3

4

6 2 2 2

7 2 I 0

15

14

" Bean seedlings were grown in soils collected from Summerland and Fraser Valleys, B.C., Canada. Wheat seedlings were grown in soils collected from Fraser Valley, B.C., Canada.

Pythium bean isolates (PBI) and Pyfhium wheat isolates (PWI). The Pythium genotypes were defmed on the basis of restriction fragment length polymorphisms (RFLP) of the total DNA (LCvesque et al., 1993; Descalzo et al., 1996). The number of genotypes represented per Pythium species tested is shown in Table 1. To indicate the original source of the isolates of the same Pythium species obtained from glyphosate-treated bean or wheat seedlings in the succeeding Tables and Figures, letters B or W was placed after the RFLP type designation. Except for P. acanthicum which was isolated from soil, all other Pythium species in these collections were originally isolated from the roots of various host plants that were untreated with glyphosate (Table 2).

Table 2. Isolates of different Pythium species obtained from hosts and soil that were not treated with glyphosate

P. aphanidermatum (Edson) Fitzp. P. sulcatum R. G. Pratt & J. E. Mitch. P. hypogynum Middleton P. coloratum Vaartaja P. splendens Braun P. acanthicum Drechsler P. arrhenomanes Drechsler P. spinosum Sawada P. paroecandrum Drechsler P. vanterpoolii V. Kouyeas & H. Kouyeas

Source

Place of origin

English cucumber

B.C., Canada

Carrot

B.C., Canada

Lamb's-quarters

Quebec, Canada

Alfalfa

Ontario, Canada

Blueberry

Nova Scotia, Canada

Soil

Ontario, Canada

Oat

Manitoba, Canada

Turfgrass

North Carolina, U.S.A.

Turfgrass

North Carolina, U.S.A.

Turfgrass

North Carolina, U.S.A.

Pythium isolates from Canada were provided by Dr Donald J. S. Barr of Biosystematic Research Institute, Ottawa, Canada, and isolates from U.S.A. were from Dr Gloria Abad, North Carolina, State University, U.S.A.

25:19OC temperature regimes. The light intensity at plant height was 230 m o l m-' s-l. The soil was kept moist by addition of distilled water as needed. The plants were watered with a dilute solution made up of 4 g 20-20-20 fertilizer 1-1 d.lst~lled . water 1 wk after planting. Inoculum was made by growing individual Pyfhiurn isolates in stationary culture in Vb-cholesterol broth (Ayers & Lumsden, 1975) for 8 d. The cultures were incubated in the Determination of glyphosate LD,, values for plants dark at 20-23'. The mycelium was decanted into a Biichner grown in soils infested with various Pythium isolates funnel lined with filter paper and rinsed with sterile distilled The relative efficacy of the different Pythium isolates to act as water. The mycelial mat was aseptically cut into nominal glyphosate synergists on various plant species was assessed 1 cm2 square pieces with a scalpel. The mycelium pieces were by estimating the glyphosate LD,, values associated with suspended in 0.08% sterile water agar (WA) at I g 100 ml-', each Pythium isolate on each of the host species. LD,, values and macerated in a Somall blender for 20 s. Inoculum was were estimated using PBI on bean seedlings, Phaseolus vulgaris applied I d before glyphosate treatment by drenching 25 ml L. cv. Topcrop (Descalzo e f al., 1996) and PWI on winter of the mycelial suspension between each row of seedlings wheat seedlings, Triticum aestivum L. cv. Northstar. Reciprocal when the seedlings had two fully expanded primary leaves tests were then conducted using PBI on wheat and PWI on (2 wk after seeding). bean seedlings to determine whether the Pythium isolates Glyphosate treatments were made 1 d later as two I pl were specific glyphosate synergists for the host plant species droplets of aqueous dilutions of Roundup@ herbicide from which they were initially isolated. GSI tests were also (360 g a.i. 1-l) applied to the stem at the cotyledonary node for done using PBI on two dicot hosts: sunflower, Helianfhus dicot yledonous plants and in the whorl of the wheat plants. annuus L. cv. Sunwheat 101 and pepper, Capsicumfrutescens cv. Different glyphosate doses were assigned at random to each CaliforniaWonder. The final GSI tests were done to determine row of plants, but all plants in a row received the same dose. whether other Pythium species from various glyphosate- Doses of 0, 2, 6, 20, 50 and 150 vg glyphosate a.i. per plant untreated hosts and from soil were also capable of GSI on were used for the Pythium inoculated bean, sunflower and bean seedlings. pepper plants. A lower range of doses (0, 0.5, 1.5, 5, 15 and The method used for all GSI tests consisted of planting 40 pg glyphosate a.i. per plant) was used for wheat seedlings. surface-sterilized seeds in plastic trays (54 x 28 x 7 cm) filled To obtain 100% mortality at the highest glyphosate dose, with 3.5 kg autoclaved clay loam soil (OM = 22 %, pH = 5.0). Pythium uninoculated plants (controls) had to be treated with Twenty seeds were planted in each of six equally spaced rows glyphosate ranging from 0 to 1200 vg glyphosate a.i. per oriented across the width of each tray. The plants were grown plant for dicot yledonous seedlings and 0-60 pg glyphosate in a growth room under a 16 :8 h, day :night photoperiod and a.i. per plant for wheat seedlings. The proportions of plants

R. C. Descalzo and others killed by each dose were recorded 4 wk after treatment with glyphosate. One tray was used for each Pythium isolate per experiment and each experiment was repeated a minimum of two times. Plant mortality data for each experiment were modelled by standard backward regression method with logistic regression program (Baker & Nelder, 1987) using the computation method for LD,, and the associated variance described previously (Rahe, LCvesque & Johal, 1990; LCvesque, Rahe & Eaves, 1992). Glyphosate LD,, values for plants inoculated with different Pythium isolates were compared with the control using Bonferroni's test (Neter, Wasserman & Kutner, 1990).

1447

coloratum RFLP B1; P. 'HS' RFLP type BI; and P. sylvaticum RFLP types B3 and B6; as well as those inoculated with P. ultimum RFLP type B l b ; P. coloratum RFLP type B2 and P. sylvaticum RFLP type B4 required similar amounts of glyphosate to kill 50% of the plant population as was needed for the control in one out of three, and two out of three tests respectively. It was also noted that in some instances plants inoculated with P. ultimum RFLP type Bla and B l b ; P. ultimum RFLP type B2; P. irregulare RFLP type B1; P. sylvaticum RFLP types BI, B2, B4 and B5 had higher LD,, values than the control.

The effects of PWZ on wheat and bean seedlings Pathogenicity of Pythium isolates

The ability of different Pythium isolates to cause infection of germinating seeds was assessed by growing individual isolates for 5 d on 16 ml solidified potato dextrose agar (PDA) contained in 100 x 15 mm Petri dishes. The culture surface was then covered with sterilized peat-based potting mix (Metro-mixa, grade 290) to a depth of 5 - 6 mm. Twenty surface sterilized bean, wheat, sunflower, or pepper seeds were put on the surface of the Metro-mix and additional Metro-mix was added to barely cover them. Approximately 20 ml of sterilized distilled water was added and the plates were placed in individual plastic bags and kept for 7 d at the same conditions used to grow plants, after which the numbers of ungerminated seeds, and dead and living seedlings in each Petri dish were recorded. Three to five plates were used for each Pythium isolate in each replicate experiment. The dead plants were counted 4 wk after inoculation and ANOVA was performed on arcsine transformed data using SAS Statistical Package (1985).

The effect of different PWI on glyphosate LD,, values on wheat seedlings is shown in Fig. 1. Plants inoculated with P. ultimum RFLP types W l , W2, W3 and W4; P. colorafum RFLP types W 1 and W2; P. sylvaticum RFLP type W5 significantly (P Q 0.05) enhanced the herbicidal activity of glyphosate on 2 wk old wheat seedlings in each of two tests done at different times. In one out of two tests, plants inoculated with P. irregulare RFLP type W1; P. sylvaticum RFLP types W I , W2, W3, W4, W6 and W7 significantly enhanced the herbicidal efficacy of glyphosate on wheat seedlings. In one of two tests, plants inoculated with P. sylvaticum RFLP types W2, W3 and W7 needed similar amounts of glyphosate to kill 50% of the plant population as was required in the control treatment, and in the remaining instances, plants inoculated with P. sylvaticum RFLP types W I , W4 and W6; and P. iwegulare RFLP type W l had higher LD,, than the control treatment. The effect of PWI on glyphosate LD,, values on beans is shown in Fig. 2. All PWI consistently and significantly enhanced the herbicidal efficacy of glyphosate on 2-week-old bean seedlings in two experiments. Mortality of 50% was observed at doses 1/10 to 1/30 of those required to cause 50% mortality in the absence of Pythium (control).

RESULTS There were significant interactions between the replicate experiments and various Pythium isolates in all GSI tests. It was, therefore, necessary to analyse each experiment separately.

Effect of PBZ on bean and wheat seedlings

The effect of different Pythium isolates on glyphosate LD,, values on beans was described by Descalzo et al. (1996). The PBI GSI tests on wheat seedlings gave an inconsistent GSI response in three replicated experiments (Fig. I). In two out of three experiments, plants inoculated with P. irregulare RFLP type BI; P. sylvaticum RFLP types Bl and B2; and P. coloratum RFLP type B2 had significantly ( P < 0.05) lower LD,, values than the control. In one out of three experiments, plants inoculated with Pythium ultimum RFLP types Bl a and Bl b; P. irregulare RFLP type B2; P. 'HS' RFLP types BI and B2; P. sylvaticum RFLP types 83, B4, B5 and B6; and P. coloratum RFLP type BI had significantly lower LD,, values than the control. However, it was also observed that plants inoculated with P. ultimum RFLP type B2; P. iwegulare RFLP type B2; P.

The effects of PBI on sunflower and pepper

All PBI enhanced the herbicidal activity of glyphosate on 2week-old sunflower seedlings by 20 to 50 fold (Fig. 3). All PBI except P. 'HS' group RFLP type BI and P. coloratum RFLP type B2 enhanced herbicidal activity on pepper seedlings by six to 30-fold. P. 'HS' group RFLP type B1 and P. coloratum RFLP type B2 caused smaller but significant reductions of glyphosate LD,, on pepper seedlings (Fig. 3).

GSZ of Pythium species obtained from soil and glyphosate-untreated hosts on beans

The effect of different Pythium species obtained from soil and from glyphosate-untreated plants on glyphosate LD,, values on beans are shown in Fig. 4. All Pyfhium species tested significantly enhanced ( P < 0.05) the herbicidal effect of glyphosate on 2 wk old bean seedlings, and 50% mortality was observed at doses seven to 80-fold less than of those required to cause 50% mortality in the absence of Pyfhium. Pythium coloratum, P. hypogynum, P. aphanidermatum and

Glyphosate synergistic Pythium I? ultimum B 1a I? ultirnum B 1 b

I? ultimum B2 I? irregulare B 1

P irregulare B2 I? group 'HS' B1 I? group 'HS'B2 I? coloratum B 1 I? colomtum B2 I? sylvaticum B 1 I? syfvaticum B2

I? sylvaticum B3 I? sylvaticum B4

I! sylvaticum B5 I? sylvaticum B6

Control

t? ultimum W1 I? ultimum W2 I? ultimum W3 I? ultirnum W4 I? irregulare W 1 I? coloratum W 1 I? coloratum W2 I? sylvaticum W 1 I? sylvaticum W2 I? sylvaticum W3

I? sylvaticum W4 I? sylvaticum W5 I? sylvaticum W6

t? sylvaticum W7 Control 0.2

Glyphosate LDSo(pg a.i. per plant) Fig. I. Effect of isolates of Pyfhium obtained from the roots of glyphosate-treated bean (top panel) and wheat (bottom panel) seedlings on glyphosate LD,, on wheat seedlings. Means (vertical bars) and standard errors (horizontal bars) from two or three experiments shown on each line.

P. spinosum were significantly more effective as synergists than were other Pythium species in this group. Pathogenicity of PBI on germinating seeds Symptoms of pre-emergence damping-off caused by various species of Pythium were similar in all types of plant tested. Pythium entered the germinating seeds in the soil by infecting the emerging radicle. After the initial infection, the mycelia colonized the growing embryo and cotyledons. Susceptible seeds were eventually killed and were covered with white mycelia 1wk after infection. Infected seeds seldom emerge from the soil, and those that did emerge from the soil were

weak and had obvious stem necrosis immediately above the soil surface. Infected seedlings usually topple down 3-4 d after emergence and showed symptoms of stem girdling. The 15 PBI varied in their virulence on germinating bean seedlings (Table 3). All isolates of P. ulfimum and P. iwegulare were highly virulent, causing 100% and 88-98% preemergence damping-off on beans, respectively. P. sylvaficum RFLP types B2 and B3, and Pythium 'HS' group RFLP types B 1 and B2 were moderately virulent, causing 35-52% preemergence damping-off. Other isolates of P. sylvaticum and all isolates of P. coloratum were moderately to weakly virulent, causing a range of pre-emergence damping-off from as high as 27% to as low as 9%.

R. C. Descalzo and others P ultimum W 1 F! ultimum W2

P ultimum W3 F! ultimum W4

P irregulare W 1 F! coloratum W 1

P coloratum W2 l? sylvaticum W 1 P sylvaticum W2

P sylvaticum W3 P sylvaticum W4 F! sylvaticum W5

P sylvaticum W6 P sylvaticum W7

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Fig. 2. Effect of isolates of Pythium obtained from glyphosate-treated wheat on glyphosate LD,, tests on bean seedlings. Means

(vertical bars) and standard errors (horizontal bars) from two experiments shown on each line. The same isolates were also pathogenic to varying degrees on germinating wheat seeds. P. ulfimum RFLP type Bla was the most virulent isolate, causing 95 % pre-emergence damping-off. P. ultimum RFLP types Blb and B2; P. irregulare RFLP types B1 and B2; and Pytkium 'HS' group RFLP type B2 caused pre-emergence damping off ranging from 63 % to 93 %. P. sylvaticum RFLP types BI, B2 and B3 caused 76, 63 and 50% respectively. The least virulent isolates, P. coloratum RFLP types BI and B2; Pytkium 'HS' group RFLP type B l ; and P. sylvaficum RFLP types B4, B5 and B6 caused a range of pre-emergence damping-off ranging from 9 to 26%. The different PBI were pathogenic to varying degrees on germinating pepper seeds (Table 3). All isolates of P. ultimum, P. irregulare and P. colorafum as well as Pyfhium 'HS' RFLP type B2 and P. sylvaficum RFLP type BI caused 100% preemergence damping-off. P. sylvaticum types B2, B3 and B5 caused mortalities ranging from 72 to 90%. Pytkium 'HS' group RFLP type B1 and P. sylvaficum RFLP types B4 and B6 were the least pathogenic, causing 15 to 30% mortality on pepper seedlings. PBI were generally less pathogenic to germinating sunflower seeds than to bean, wheat and pepper. The most pathogenic isolates were P. ultimum RFLP type B2 and P. irregulare RFLP type B2, causing 48% and 47% mortalities respectively. P. ulfimum RFLP types Bla and Bl b; P. iwegulare RFLP type Bl; P. sylvaficum RFLP type Bl, B2 and B3; and P. coloratum types B1 and B2 caused mortalities ranging from 10 to 35%. Mortalities caused by the least pathogenic isolates, Pytkium 'HS' RFLP types Bl and B2, and P. sylvaticum RFLP types B4, B5 and B6, ranged from 0 to 5%. Pathogenicity of PWI on germinating bean and wheat seeds

All isolates of PWI were pathogenic to varying degrees on germinating wheat seeds (Table 4). P. ulfimum RFLP types

WI, W2 and W3 were the most pathogenic, causing 95 % mortality. P. coloratum RFLP type W l and P. irregulare RFLP type W l were the least pathogenic causing 20% mortality. All the other PWI were intermediate in pathogenicity, causing mortality ranging from 25 to 63%. When the same Pytkium isolates were tested on beans, all except P. coloratum type W2 were pathogenic to varying degrees. P. ulfimum RFLP types W l , W2, W3 and W4 were the most pathogenic, causing 85 % pre-emergence damping-off while P. colorafum RFLP type W1, P. irregulare RFLP type W l and P. sylvaticum types W2 and W6 were the least pathogenic, causing 8-23 % mortality. P. sylvaticum types W l , W3, W4, W5 and W7 were intermediate in pathogenicity, causing 25-45 % mortality on germinating bean seeds.

Pathogenicity of Pythium species from soil and various glyphosate-untreated hosts on germinating bean seeds

The pathogenicities of Pyfhium species obtained from glyphosate-untreated hosts and soil were tested on germinating bean seeds (Table 5). P. aphanidematum caused the highest pre-emergence damping-off (94%), followed by P. spinosum (66%) and P. splendens (27%), P. paroecandrum (19%) and P. arrhenornanes (5 %). P. coloratum, P. vanterpoolii, P. hypogynum, P. sulcatum and P. acanthicum isolates did not cause mortality on germinating bean seeds.

DISCUSSION The significant differences sometimes observed between the results of repeated experiments probably reflects the complexity of interactions between numerous components of the soil ecosystem during GSI. Since it is impossible to completely control all of the variables in the soil ecosystem, conclusions

Glyphosate synergistic Pythium l? ultimum Bla I? ultimum B l b

l? ultimum B2 l? irregulare B 1 I? irregulare B2 I? group 'HS' B1 I? group 'HS' B2 I? coloratum B 1 I? colomtum B2 I? sylvaticum B 1 I? sylvaticum B2 I? sylvaticum B3 I? sylvaticum B4

P sylvaticum B5 I? sylvaticum B6

Control

... ... ... ... ... .... .. .. .. ....

I? ultimum Bla I? ultimurn B 1b I? ultimum B2

I? irregulare B 1 I? irregulare B2 I? group 'HS' B1 I? group 'HS' B2

I? colomtum B1 I? coloratum B2 I? sylvaticurn B 1

I? sylvaticum B2

P sylvaticum B3 I? sytvaticum B4 I? sylvaticum B5

l? sylvaticum B6 Control 0.1

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regarding Pyfhium-plant species specificities during GSI must be based on the overall trend of several replicated GSI tests. In general, the significance of GSI, as evidenced by the magnitude of differences in glyphosate LD,, values obtained in the absence and presence of different Pythium isolates, was greater on dicot yledonous than on monocot yledonous test plants. This may be due to the substantial difference in glyphosate sensitivity observed between the two plant groups, as seen in the lower glyphosate dosages needed to cause equivalent plant mortalities on monocot yledonous than on dicot yledonous plant species. The GSI tests of PWI on wheat and reciprocal GSI tests of

PBI on wheat were generally not conclusive with regard to the question of host specificity of these isolates as glyphosate synergists. However, it was interesting to observe that seven PWI Pythium isolates consistently caused significantly lower glyphosate LD,, values on wheat compared with the control. In contrast, no PBI isolates gave any evidence of consistent GSI on wheat. The lack of specificity among Pythium isolates during GSI was clearly evident in the results of the GSI tests done on various herbaceous dicot yledons. All PWI were glyphosate synergistic on bean seedlings, and PBI that were glyphosate synergistic on beans were also synergists on sunflower and

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Table 3. Mortalities observed in pathogenicity tests on beans, wheat, sunflower, and pepper seedlings using PBI Pythium isolates

Table 4. Mortalities observed in pathogenicity tests on germinating wheat and bean seeds using PWI Pythium

Pre-emergence damping-off mortality (%)'

Pre-emergence damping-off mortality (%)'

P. ultimum B l a P. ultimum Bl b P. ultimum 8 2 P. irregulare BI P. inegulare BZ P. 'HS' group B1 P. 'HS' group B2 P. sylvaticum B1 P. sylvaticum B2 P. sylvaticum 83 P. syluaticum 84 P. sylvaticum B5 P. syloaticum 8 6 P. coloratum B1 P. coloraturn 8 2

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Mean mortalities from three trials, with 3-5 replicate plates for each isolate per trial. Twenty seeds per plate and mortality recorded 2 wk after inoculation. Values within column followed by the same letter($ are not significantly different from each other (P < 0.05) according to Bonferroni's test.

Mean mortalities from three trials, with 3-5 replicate plates for each isolate per trial. Twenty seeds per plate and mortality recorded 2 wk after inoculation. Values within column followed by the same letter($ are not significantly different from each other (P < 0.05) according to Bonferroni's test.

pepper seedlings. Results from these tests showed no strong evidence for host specificity during GSI involving Pythium species on herbaceous dicot seedlings. A differential GSI response was observed on apple seedlings for two isolates of Pythium (LCvesque et al., 1992). In their experiment the isolate obtained from bean was P. ultimum, but the isolate obtained from apple was not identified. It was possible that the contrasting GSI effect that they observed on apple seedlings was due to different Pythium species used. It was also likely that the difference between a woody and an

herbaceous dicot yledonous species might confer specificities not observed among herbaceous species. This possibility notwithstanding, our results clearly support the conclusion that many different isolates and species of Pythium are capable of GSI with a general lack of specificity on diverse species of herbaceous dicot seedlings. The type of Pythium species that were isolated from the roots of glyphosate-treated bean and wheat seedlings were generally similar except for the presence of Pyfhiurn 'HS' group among PBI but not PWI. Thus, the GSI tests that

Glyphosate synergistic Pythium Table 5. Mortalities observed in pathogenicity tests on germinating bean seeds using isolates of Pythium species obtained from glyphosate-untreated hods and soil

1452

The antifungal metabolites sesquiterpene lactones (Spring, Albert & Hager, 1982) present in sunflower probably have contributed to its natural resistance against Pythium. The relative pathogenicity of a Pythium isolate to Pre-emergence damping-off glyphosate-untreated plants could not be used to predict its mortality (%)' glyphosate synergistic potential on the same host. This was apparent from the results of GSI tests conducted using P. aphanidermatum 94 a Pythiurn isolates from soil and various glyphosate-untreated P. spinosum 66 b P. splendens 27 c plant hosts. P. coloratum and P. hypogynum were non-virulent P. paroecandrum 19 c on germinating bean seedlings; however, their efficiencies as P. anhenomanes 5d glyphosate synergists were similar to those of P. aphaniP. coloratum 0d dermatum, the most virulent isolate. The ability of the nonP. vanterpoolii 0d virulent Pythium species to act as glyphosate synergists on P. hypogynum 0d P. sulcatum 0d bean seedlings clearly suggests that pathogenicity is not the P. acanthicum 0d only factor involved in GSI. Mean mortalities from three trials, with 3-5 replicate plates for each The evidence that diverse Pyfhium species are capable of isolate per trial. Twenty seeds per plate and mortality recorded 2 wk after GSI and are generally non-host specific, at least on herbaceous inoculation. Values within column followed by the same letter($ are not dicot seedlings, and that these same isolates are pathogenic on significantly different from each other (P< 0.05) according to Bonfemoni's germinating seeds highlights possible risks that might be test. associated with recurrent use of glyphosate as a herbicide and crop desiccant. Such risks might also be associated with the utilized Pythium species not represented in the PBI and PWI use of glyphosate on genetically engineered glyphosategroups were needed to confirm whether other Pythium species tolerant crops. There is both direct and indirect evidence that were also capable of GSI. So far, we have tested 14 different the population dynamics of soiI microbes in the soil can be Pythiurn species for GSI (five from glyphosate-treated plants, affected for short periods by glyphosate treatment (Blowes, eight from glyphosate-untreated hosts, and one from soil). 1987; LPvesque, Rahe & Eaves, 1987; Smiley, Ogg & Cook, The finding that at least some isolates of all 14 species are 1992). However, the small number of published reports in capable of GSI suggests that other species of Pythium in nature relation to the extensive use of glyphosate-containing may also behave as glyphosate synergists on herbicide- herbicide over the past two decades suggests that noticeable treated plants in the field. enhancement of root disease potential by glyphosate is not The different species of Pythium varied in their patho- common. Pythium infections on mature plants are usually not genicity. This is an expected result since Pyfhium in general discernible due to the insidious nature of pathogen decauses diseases on diverse plant species, both monocotyledons velopment; as a result, significant effects that are reflected in and dicotyledons ranging from herbaceous to woody (Hendrix overall yield may also go unnoticed. Research to assess the & Campbell, 1973). Pyfhium causes different diseases impact of glyphosate on long-term root rot disease potential depending on the g o w t h stage of the host. Mature plants are of field soils and of sublethal doses on the activity of affected by Pyfhium at root tips and root hairs, causing a subtle deleterious root microflora in perennial crops is clearly overall decline of plant health over time (Mircetich, 1971). needed. Succulent and juvenile stem and root tissues of seedlings and germinating seeds are commonly attacked below the soil line, We acknowledge the assistance of Dr D. J. S. Barr in the which results in girdling of the seedling stem and a watery confirmation of Pythium identification. This work was decay of germinating seeds (Singh & Singh, 1984). The most supported by the Natural Sciences and Engineering Research prominent indication of severe Pythium attack on seedlings is Council of Canada. manifested by damping-off, characterized by toppling-over of affected seedlings on the soil surface. Pythium can also be part of a disease complex on mature trees by causing synergistic REFERENCES interaction with other types of soil-bone microorganisms, as Ayers, W. A. & Lumsden, R. D. (1975).Factors affecting ~roductionand in apple replant disease (Braun, 1991), crown rot of apple trees germination of oospores of three Pythium species. Phytopathology 65. 1094-1 100. (Jeffers ef al., 1982) and root rot of young apple trees (Utkhede Baker, R. J. & Nelder, J. A. (1987).The GLZM System, Release 3.77, Generalized & Smith, 1991). Linear Interactive Modelling. Numerical Algorithms Group: Oxford. The pathogenicity of the isolates of Pyfhium species Blowes, W. M. (1987).Effect of rye grass residues, knockdown herbicides, and from PWI and PBI groups on their respective reciprocal hosts fungicides on the emergence of barley in sandy soils. Australian Journal of showed indications of host preferences when the most Experimental Agriculture 27, 785-790. vimlent Pythium isolates were considered. The most virulent Braun, P. G. (1991).The combination of Cyiindrocarpon luridum and Pythium inegulare as a possible cause of apple replant disease in Nova Scotia. isolate of P. ulfimurn in the PBI group appeared to be more Canadian Journal of Plant Pathology 13, 291-297. virulent on germinating bean seeds than on wheat. In contrast, Descalzo, R. C., Punja, Z. K., LCvesque, A. C. & Rahe, R. E. (1996). the most virulent isolate of P. ultimum in the PWI group was Identification and role of Pythium species as glyphosate synergists on bean less virulent on beans than on wheat. PBI were generally less (Phaseolw vulgaris) grown in different soils. 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R. C.Descalzo and others Hendrix, F. F. Jr & Campbell, W. A. (1973). Pythium as plant pathogens. Annual Review of Phytopathology 11, 77-98. Jeffers, S. N., Aldwinckle, H. S., Burr, T. J. & Aneson, P. A. (1982).Phytophthora and Pythium species associated with crown rot in New York apple orchards. Phytopathology 72, 533-538. Johal, G.S. & Rahe, J. E. (1984). Effect of soilborne plant pathogenic fungi on the herbicidal action of glyphosate on bean seedlings. Phyfopathology 74, 950-955. LCvesque, C. A., Beckenbach, K., Baillie, D. A. & Rahe, J. E. (1993). Pathogenicity and DNA restriction fragment length polymorphisms of isolates of Pythium spp. from glyphosate-treated seedlings. Mycological Research 97, 307-312. LCvesque, C. A,, Rahe, J. E. & Eaves, D. M. (1987). Effects of glyphosate on Fwarium spp.: its influence on root colonization of weeds, propagule density in the soil, and crop emergence. Canadian Journal of Microbiology 3 3 , 354-360. LCvesque, C. A,, Rahe, J. E. & Eaves, D. M. (1992). The effect of soil heat treatment and microflora on the efficacy of glyphosate in seedlings. Weed Research 3 2 , 363-373. Mircetich, S. M. (1971). The role of Pythium in feeder roots of diseased trees and orchard soils in peach tree decline. Phytopathology 61,357-360. (Accepted 13 March 1996)

1453 Neter, J., Wasserman, W. & Kutner, M. H. (1990). Applied Linear Statistical Models, regression, analysis of variance, and experimental designs 3rd ed. Irwin: Homewood, Illinois. Rahe, J. E., LCvesque, C. A. & Johal, G. S. (1990). Synergistic role of soil fungi in the herbicidal efficacy of glyphosate. In Microbes and Microbial Products as Herbicides (ed. R. E. Hoagland), pp. 26C-275. ACS Symposium Series 439.

SAS Institute INC. (1985). SASR User's Guide, Version 5 Mition. Cary: North Carolina. Singh, R. S. & Singh, U. S. (1984). Pythium in host and soil. In Progress in Microbial Ecology (ed. K. G . Mukerji, V. P. Agnihotri & R. P. Singh), pp. 22F271. Printhouse: India. Smiley, R. W., Ogg, A. G., Jr & Cook, R. J. (1992). Influence of glyphosate on Rhiwctonia Root Rot, growth, and yield of barley. Plant Disease 76, 93 7-942. Spring, O., Albert, K. & Hager, A. (1982). Three biologically active Heliangolides from Helianthus annuus. Phytochemistry 21,2551-2553. Utkhede, R. S. & Smith, E. M. (1991). Phytophthora and Pythium species associated with root rot of young apple trees and their control. Soil Biology and Biochemistry 2 3 , 1059-1063.