Occurrence and pathogenicity of Pythium spp. in seedling roots of winter rye

. Seedlings of winter rye collected from yellowing patches during October to November 1985—1987 showed oospores of Pythium species in apparently healthy as well as in discolored roots. Examination of 1550 root pieces of rye on CMA yielded fungi belonging to 35 genera. The most commonly isolated ones were Fusarium spp, Penicittium spp, Mucor spp, Mortierella spp. and Cladosporium spp. Pythium spp. were isolated from 35 root pieces on CMA. Identified species were P. splendens, P. irregulare, P. dissimile, a species resembling P. ahstosporum and a speciesresembling P. ultimum. In in vitro and in vivo, tests on the cereals winter rye, spring wheat, oats and barley the pathogenicity of some Pythium isolates varied from high (P. splendens, P. irregulare) to moderate (P. irregulare, P. dissimile) and low (a species resembling P. ultimum).


Introduction
Pythium species are common in agricultural soils and they parasitize a wide range of hosts (Domsch et al. 1980). All spring and winter small grains and forage grasses are susceptible to root rot caused by one or several Pythium species acting singly or in combination (Wiese 1977).
Root rot on rye caused by species of Pythium has been reported only in a few cases. The species Pythium aphanidermatum (Edson) Fitz (Sechler & Luke 1967) and P. myriotylum Drechs. (Littrell & McCarter Subr., P. irregulare Buisman, P. splendens Braun, P. tardicrescens Vanterpool and P. volutum Vanterpool & Truscott are the most important species reported to be pathogenic on barley. Browning root rot of wheat also has a world-wide distribution, and it has caused damage to wheat seedlings in the U.S.A. (Kilpatrick 1968, Chamswarng andCook 1985), England (Waller 1979), Austria (Glaeser 1979) and Australia (Dewan and Sivasithamparam 1988). More than ten species of Pythium have been reported to cause root rot, seed rot and damping-off in wheat seedlings. The most extensively documented wheat pathogens are P. arrhenomanes, P. aphanidermatum, P. graminicola, P. myriotylum Drechs. and P. volutum (Wiese 1977).
In autumn 1984, patches of yellowed and stunted rye plants were observed in rye fields in southern Finland (Bremer and Vestberg 1986). Electron microscopy revealed two types of virus particles in the leaves and roots of the yellowed rye seedlings. No virus vector, e.g.
Polymyxa graminis Ledingham, was observed, only indications of fairly abundant occurrence of Pythium spp in roots of yellowed rye seedlings.
The aim of this study was to isolate and to identify species of Pythium from roots of rye seedlings. Introductory pathogenicity tests were conducted with some isolates on rye, barley, oats and winter wheat.

Materials and methods
Sampling and sample treatment Seedling samples were collected from yellowing patches of winter rye fields during October to November 1985-87 andin May 1987 in southern and central Finland. The localities numbered 12, 23, 9 and 19 in autumn 1985, autumn 1986, autumn 1987 and spring 1987, respectively. The roots were rinsed thoroughly in tap water. One half of the root sample was examined with a compound microscope. From the other half of the sample, discoloured pieces (approx. 0.5 cm long) of fine roots, including healthy and discolored ones, were transferred to water agar (WA) without any surface sterilization or antibiotic treatment. The fungi were transferred from the WA to corn meal agar (CMA) for identification.

Studies on Pythium strains
Morphological characteristics of the strains of Pythium spp. were determined on 4-week cultures on CMA and on 9-day cultures in water. The water cultures were established by transferring a small piece of culture (1 cm in diameter) on CMA beneath an autoclaved piece of young rye leaf, cv 'Dan Kowskie Zlote' into a Petri dish containing autoclaved water. The water consisting of "pond water" and destilled water (1:1) (Van der Plaatsniterink 1981) was changed twice daily. On these occasions sexual and asexual structures of Pythium isolates were distinguished on the piece of rye.

Pathogenicity of Pythium strains in vitro
The pathogenicity of four Pythium strains (PI, P 2, P 3, P 9) isolated from winter rye was tested in vitro in four replicates on barley cv 'Kymppi', oats cv 'Hankkijan Vouti', spring wheat cv 'Tähti' and winter rye cv 'Dan Kowskie Zlote'. A system of hanging file folders made of filter paper was arranged in a water bath with 5 cm of water at the bottom. Due to water suction the filter paper remained wet when the water level was maintained at 5 cm. In each folder five surface sterilized seeds (1 min in 1°Io NaOCl) were applied between the two layers, 2 cm from the top. Inocula of Pythium (1 cm in diameter), 1-month culture on CMA, were placed 2 cm 3 beneath the cereal seed. Paper clips were used to bring the two layers of the hanging folder close to each other. The whole experiment consisted of six water baths with 16 hanging file folders in each. After three days of incubation in darkness the baths were placed into a growth chamber with a 16-h daylength and a light intensity of approx 5000 lux. The shoot lengths were measured seven days after onset of the experiment. Final observations of root and shoot lengths, disease symptoms and infection of Pythium were made four days later. Disease symptoms in shoots and roots of seedlings were estimated visually on a scale from 0 to 2: O = Healthy 1 = Moderately discolored 2 = Highly discolored The disease index (DI) of a treatment was calculated as the mean of altogether 20 seedlings belonging to four replicates.

Pathogenicity of Pythium strains in vivo
The pathogenicity of three Pythium isolates (P2, P 9, PI7) was tested in vivo in six replicates on the same species and cultivars of cereals as in the in vitro experiment. Surface sterilized seeds (1 min in 1 % NaOCl) were pregerminated for five days at room temperature (about 22°C) until the seedling was approx. 4 cm long; equally long seedlings were thereafter planted into white plastic pots (3 dl, 5.5 cm deep) at a depth of 2 cm, five in each.
The pots were filled with a sand-vermiculiteleca gravel mixture (1:1:1). The inoculum, a piece of Pythium culture on CMA (1 cm in diameter, 14-day old culture) was placed 1 cm beneath the seed. The pots were fertilized with the slow-release fertilizer osmocote, 2 kg/m 3 , and were kept in a glass house at a temperature of about 20 0 C throughout the experiment. Supplementary light was given to get a daylength of 16 h.
The experiment started on the 19th of October, 1987, and ended eight days later, when the disease symptoms in roots and on leaves, as well as seedling lengths and dry weights of above ground plant parts and roots were determined.
The disease symptoms in roots were estimated visually in the same way as in the in vitro experiment. The DI of a treatment was calculated as the sum of disease symptoms in all seedlings in that treatment, altogether 30 seedlings.

Statistical analysis
Statistical analysis of the results of the pathogenicity experiments was done using the analysis of variance. Means were compared with Duncan's multiple range test.

Direct microscopy of seedling roots of rye
Although all seedling samples of rye collected in autumn 1985-1987 were taken from yellow patches in the field, the majority (70.5 %) of samples contained apparently healthy roots (Table 1). Highly discolored roots were found in five samples out of 44 samples. Oospores of Pythium were detected in healthy as well as in moderately or highly discolored roots. However, severe infection of Pythium was found only in samples with highly discolored roots. On the other hand, no oospores of Pythium were detected in two samples with highly discolored roots (Table 1).
Including the seedling samples collected in spring 1987, which makes a total of 63 samples studied by direct microscopy, the samples containing Pythium spp. in roots numbered as follows:  (Table 2).

Studies on Pythium spp
Twenty-seven fungal isolates on CMA were identified as Pythium spp. Eight of these representing several species of Pythium were obtained in pure culture (Table 3).

Pathogenicity of Pythium strains in vitro
The "hanging file folder" method was successful. Infection of Pythium was noticed in all cereals tested. Of the isolates, PI (P. splendens) caused very extensive infection in all cereals, 87.5°7o of seedlings in this treatment being affected. On the other hand, P 3 (isolate resembling P. ultimum) caused very low or no infection at all. The isolates P 2 (P. irregulare) and P 9 (P. dissimile) gave rise to infection in about 50 % of the seedlings (Table   4).
Oats was the healthiest cereal, infection occurring in 37.5°7o of seedlings, while spring wheat was the most extensively infected, (60.3 %).
Host specificity was observed especially for isolates P 2 and P3. P 2 infected winter rye by 67 %, while it caused no infection in barley.
P 3 caused no infection in oats but a slight or moderate infection in barley (Table 4).
Out of four isolates tested, only PI caused a significant reduction in shoot and root length (Table 5). It also caused more leaf symptoms than the control treatment. Inverse- ly, isolate P 9 gave rise to the highest root symptom index, causing brown roots especially in barley. Both P 9 and PI increased the severity of root damage.

Pathogenicity of Pythium strains in vivo
Isolate P 9 significantly decreased shoot dry weight, while the isolates P 2 and Pl 7 (P. ir-regulare) caused an increase as compared to the control. In root dry weight there were no decreases due to Pythium isolates, but Pl 7 significantly increased root dry weight (Table 6).
All isolates slightly decreased seedling length, isolates Pl 7 and P 9 significantly so, as compared to the control.
The leaves and seedling bases showed no symptoms. with a thick wall. Antheridia seldom visible, with a long bent and irregu-Oospore resembling lar stalk, 1/oogonium. Club-shaped appressoria at bottom of Petri dish.
P. ultimum Good growth on rye leaf in water. No asexual structures. Seedlings showed few shoot and root symptoms in the in vivo experiment. The disease severity index of roots (maximum 60) was calculated as the sum of DI of 30 seedlings. Isolate Pl 7 had a somewhat higher root DI than the control and the other Pythium isolates, 3.8 as compared to 2.6, 2.8 and 2.5 for the control, P 2 and P 9, respectively. Barley had on average the most diseased roots, while spring wheat had the healthiest ones (Table 6).

Discussion
The aim of this investigation was to study the occurrence and the importance of Pythium spp. in roots of rye seedlings. Because Pythium spp. are often relatively infrequently isolated from surface sterilized roots (Waller 1968), an isolation method using no chemical compounds or antibiotics was chosen. The method yielded 35 genera of soil borne fungi. Most of them were classified as common saprophytic soil fungi, while other genera such as Fusarium spp., which was found quite frequently, and Pythium spp. often act as pathogens or minor pathogens in cereal roots (Salt 1979).
With the exception of P. dissimile, all the Pythium species identified, i.e. P. splendens. P. irregulare, P. dissimile, a species resembling P. aristosporum and a species resembling P. ultimum occur in roots of cereals in various parts of the world (Chamswarng & Cook 1985Dewan & Sivasithamparam 1988, Kilpatrick 1968Singleton & Ziv 1981.   Direct microscopy revealed easily oospores of Pythium in fine roots of rye seedlings, but a lower percentage of Pythium species was obtained on CMA and only a few isolates could be studied in pure culture. This indicates that on CMA the growth of the Pythium was disturbed or even overgrown by the saprophytic fungi, and some kind of surface sterilization of root pieces could have increased the number of isolates obtained in pure culture.
Oospores of Pythium were detected in brown as well as in apparently healthy roots, a fact which supports the findings of Waller (unpublished, ref. Salt 1979). This also suggests that in the roots of rye seedlings Pythium may rather be a minor pathogen than a Table 6. The effect of inoculation with three strains of Pythium isolated from winter rye on shoot and root dry weight, seedling length and root disease index (DI) in four cereal crops in vivo. real damage causing pathogen. This role of Pythium was supported also by the findings in the in vivo and in vitro pathogenicity experiments, in which the pathogenicity of some Pythium isolates varied from pathogenic to beneficial. This kind of variation in pathogenicity is commonly documented (Dewan and Sivasithamparam 1988, Kilpatrick 1968, Singleton and Ziv 1981.