Growth of Gaeumannomyces graminis var. tritici in soil: Effects of temperature and water potential

SoilBiol. Eiochem. Vol. 16, No. 3, pp. 211-216, 1984 Printed in Great Britain. All rights reserved

GROWTH TRITICI

OF GAEUMANNOMYCES GRAMINIS VAR. IN SOIL: EFFECTS OF TEMPERATURE AND WATER POTENTIAL

MARGARET Department

0038-0717/84$3.00 + 0.00 Copyright c 1984Pergamon Press Ltd

J. GROSE,

of Soil Science

C. A.

PARKER

and K.

and Plant Nutrition, University Western Australia 6009

SIVASITHAMPARAM

of Western

Australia,

Nedlands,

20 July 1983)

(Accepted

Summary-The effects of temperature and water on the growth of the take-all fungus, Gueumannotnyces graminis var. tritici (Ggt), were examined in two factorial experiments. The first examined the effects of temperature and water potential on the growth of two isolates of Ggr on agar media, using osmoticallyadjusted water potentials. The second experiment was concerned with the growth of the Ggt isolates in one sterile and two natural soils at two water regimes in the absence of a living host. Three temperatures (10, 18 and 26°C) were used in these experiments. A third experiment determined growth through soil. Growth was greatest at high temperatures and low water potential in axenic culture, but in unsterile soil growth at different temperatures and water potentials was strongly influenced by competition from the soil biota. The best temperature for growth in unsterile soil was 18°C. Growth at 26°C in unsterile soil was greatly reduced, this being attributed to more intense microbial competition. In sterile soil Ggr grew equally well at 18 and 26°C. At 10°C both isolates of Ggt grew better in unsterile soil than in sterile soil. Under suitable conditions Ggr grew out readily from infected straw into unsterile soil (up to 5 cm in 10 days) in the absence of a host plant, forming melanized, hyaline and branched hyphae. These hyphae were infectious after dry storage for 5 months in the laboratory. Ggr thus appears to be a more successful soil inhabitant than is widely believed. Our experiments could explain many of the host-based concepts related to field expression of disease. The technique presented here could be of value for testing the suppressiveness or conduciveness of soils by measuring fungal growth in soil.

INTRODUCTION

In most studies of Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker (henceforth referred to as Ggt) the natural behaviour of the fungus in the soil has been judged from information obtained from either growth on artificial media or from disease expression in host plants. Little work has been done to measure the capacity of Ggt to grow in the soil. It has been generally assumed that this fungus grows in the soil only along the roots of cereal and grass host plants, the fungus having been described by Garrett (1963) as a specialized “ecologically obligate parasite”. However, some doubts have been raised as to whether Ggt is such a poor soil inhabitant. Brown and Hornby (1971) demonstrated that hyphae from infected host debris or mycelial inocula grew up to several millimetres through unsterile soil in the presence of living wheat roots, and they suggested that contact between infective host fragments carrying Ggt and the root might not be a prerequisite for infection. Hyphal growth up to 5 mm into the soil in the absence of host roots was also recorded. Work of Pope and Jackson (1973) Fang and Parker (1975) and Sivasithamparam and Parker (1978) suggested that growth from infected debris into natural soil, and the subsequent colonization of a new substrate, might be possible. Gilligan (1980a,b) proposed a model to determine the zone of potential infection around a given inoculum unit and found experimentally that, after 15 days, all roots within 1Omm of inoculum units were infected. 211

We report the results of experiments which demonstrate the growth of Ggt through the soil in the absence of any host. We hypothesized that the extent of the growth of Ggt at particular temperatures and moisture regimes would reflect the level of competition or antagonism faced by this fungus in the soil environment. MATERIALS

AND METHODS

Isolates

The two isolates of Ggt used were WUF 2, isolated from wheat plants out of a grey sandy soil with some lateritic gravel (pH 5.9; 2:5 w/v soil:water) from Gabalong, Western Australia, and WUF 3, obtained from wheat plants out of a solonized brown soil (pH 5.8; 2:5 w/v soil:water) from Carnamah, W. A. Both were isolated by Mr C. S. Fang and maintained on wheat straws in evacuated ampoules at 4.4”C (Fang and Parker, 1981). These straws were colonized at 22°C on 0.4% malt extract broth agar (MEBA; pH adjusted to 6.8-7.0). Experiment

I: Growth on Basal Agar

The effects of water potential and temperature on the growth of WUF 2 and WUF 3 on the basal agar medium of Sommers et al. (1970) were examined, in order to compare growth in artificial axenic media to that in natural soil and in sterile soil. WUF 2 and WUF 3 were grown on the nonamended basal agar (4 mm thick) at 22°C. When the

MARGARETJ. GROSEet al.

212

fungal colonies covered between 6&75x of the agar surface, 4mm dia agar discs were cut with a sterile cork borer from just behind the growing margins of the colonies to ensure that the propagule was strongly viable and that it had adequate nutriment remaining in the disc. Discs were used to inoculate plates of osmotically-adjusted basal agar, maintained at six different water potentials by the addition of 0, 16,44, 67, 82 and 99 g of KC1 per 1000 g medium to give final agar potentials of -0.12, - 1.12, -2.90, -4.12, - 5.12 and - 6.12 MPa (Robinson and Stokes, 1955). Plates were placed in polythene bags, sealed and held for 7 days at 10, 18 and 26°C. These temperatures are common in soil in the Western Australian cereal belt during the growing season and are more representative than the higher temperatures (up to 35°C) described by Cook and Christen (1976). Measurements of fungal colony diameter were made daily for 7 days.

Table I. Density scores for growth of Ggt hyphae in soil. Observations were made at a magnification of x 160 Density

Score 0

observation

No growth. One or two strands. Few strands. Moderate number of strands; often fishbone appearance. Many strands--many dark runner hyphae combined; general dark appearance. Thick, wavy growth of strands of spindly dark runner hyphae. Plate-like growth.

I 2 3

4

5

6

This experiment was a factorial design with the two Ggt isolates grown in three soils, at two water potentials and three temperature regimes, over five periods of growth. There were three replicates in each treatment.

Canada Gamma Cell 220 (60Co source, 1.65 Mrad h-’ output). They received a dosage of 6.60 Mrad, this level having been proved necessary by preliminary experiments. Ethylene can be produced by y-irradiation (Rovira and Vendrell, 1972) but tests revealed negligible C2H, production in the soil at lo-” sensitivity. Thus the sterilized Gabalong soil was considered amenable to microbial growth.

Inoculum

Soil water regimes

The inocula for use in the soil were pieces of wheat straw colonized by WUF 2 and WUF 3. Seven millimetre lengths of sterilized wheat straw were colonized by placing on the surface of the activelygrowing 7-day old cultures held for 14 days at 22°C.

The moisture characteristic curves for the two soils were obtained by the pressure plate apparatus (Richards, 1941). The two water regimes devised were 60% water-holding capacity, (WHC; Piper, 1944) and 30% WHC (henceforth referred to as “wet” and “moist” respectively). In Gabalong soil, water potentials were -0.8 kPa and - 10 kPa for “wet” and “moist” soil respectively, in yellow sand -0.2 kPa and - 1.2 kPa. Water was added to 200 g lots of yellow sand and mixed by rotation and shaking. Gabalong soil, with higher clay and organic matter contents, was spread evenly in thin layers and wetted by a fine spray of deionized water. The wetted soils were then placed in airtight containers.

Experiment

2: Growth in Soil

Soils Sub-surface yellow sand from the Karrakatta series (Bettenay et al., 1960), and soil from Gabalong were the two soil types used. Gabalong soil is a loamy sand receiving 375 mm rainfall, and represents a substantial portion of the soils of the Western Australian cereal-growing region (Burvill, 1962). The field has a history of take-all. Some physical and chemical properties of Gabalong soil are listed by Sivasithamparam et al. (1979). Both soils were air-dried in a glasshouse for 10 days. Sieving (< 2 mm) removed the lateritic gravel and coarse plant debris present in the Gabalong soil. 500 g lots of dry Gabalong soil were sterilized by y-irradiation in an Atomic Energy Commission of

LID

II

[

‘*90’L NUCLEPORE

n

FILTER WHEAT CARRYING

“Soil-sandwich”

technique

Figure 1 shows the relative placement in each Petri plate of the soil, a nuclepore filter (Cat. 121517 Nuclepore Corp. California; dia 76 mm, pore size 0.3 pm), and the straw inoculum. Soil was added and the top layer lightly pressed down using the base of a beaker. This procedure was carried out in a laminar flow cabinet. Nuclepore filters were used because they are inert, porous, enabled the use of fungal stains and were readily peeled away from the soil without losing attached hyphae. Petri plates were enclosed in two individuallysealed polythene bags with an additional plate containing sterile absorbent cotton wool wetted with deionized water. In this way water content was found to be maintained over the incubation period. Growth meusurements

STRAW INOCULUM

Fig. 1. “Soil-sandwich” layout within each Petri plate for examining the growth of Ggt in soil.

Radial growth of dark runner hyphae was measured against a transparent overlying disc marked in circles of radii 8, 18, 28, 33 and 38 mm after 10, 13, 17, 23 and 30 days. Growth within the first circle was annotated 1, and so on to the fifth

Growth of Gaeumannomyces graminis

213

in soil 005

LSD

001

I

I

analysis

A split plot design was used to obtain an analysis of variance for radial growth and hyphal density with a Genstat V computer programme (Anonymous, 1977). 3: Growth Through Soil

Additionally, half-filter plates were set up at 18°C to check that Ggt could actually grow through the (a)

WUF

-4 12

-5 12

-6 12

Fig. 3. Mean radial growth of two Ggt isolates for 7 days on agar osmotically adjusted and grown at 10, 18 and 26°C.

circle, 5. Each nuclepore filter was divided through the straw position into 8 equal sectors and the extent to which hyphae grew on each nuclepore in more than 4 of the 8 sectors was taken as the extent of growth. The density of dark runner hyphae was examined at x 160 magnification (Table 1). As for radial growth, each filter was divided through the straw position and, for statistical purposes, these 8 sector scores were added. Hyphal branching was classified at x 160 magnification, the score given being based on the predominant type (Fig. 2).

Experiment

-290

MPa

Fig. 2. Classification of hyphal branching in soil.

Statistical

-I 12

4

3

I

IO18.---.

-'\

-0 I2

0 001

hyphae were not using the filter as a growth platform. In these plates, one 7 mm straw inoculum was placed at the edge of the Petri dish opposite a half-filter, the distance between the inoculum and the edge of the filter being 4.5 cm. Soil was added as for the full filter treatment and growth on the half filter was scored after 10, 17 and 34 days. soil, and that the fungal

RESULTS Experiment

2

(b)

WUF

Yellow LSD*

LSD

001

3 .---.

sand

Unsterile

o,soL~ool I I’ 005

1: Growth on Basal Agar

Growth declined greatly under increasing osmotic stress. At 18°C and 26°C some growth occurred down to - 5.12 MPa, but no growth occurred in any treatment at - 6.12 MPa. This agrees with findings for Ggt by Griffin (1972). Growth increased with increasing temperature; the interaction between temperature and water potential is shown in Fig. 3. There were no significant differences between isolates: for both WUF 2 and WUF 3 growth was greatest at the highest water potential and 26°C. Fang and Parker (unpublished) found an optimum temperature of

Sterile

Gabalong Gabalong

soil soil

.--.A

0001

0 L/I

lb

1:

1:

2:

3;

Ttme ( days ) Time 1 days 1 Mean radial growth of Ggt isolates (a) WUF 2 and (b) WUF 3 over time for all temperatures and both water potentials in yellow sand, unsterile Gabalong soil and sterile Gabalong soil. LSD* when

Fig.

4.

comparing isolates with the same time and soil type.

MARGARET J. GROSE et Yellow

LSD

Unsterile Sterile

sand

between PDA.

-

Gabalong Gabalong

soil

l

soil

---•

al. 25-30°C

for these isolates

on MEBA

and

Experiment 2: Growth in Soil

A-.A

Radial growth

I

I

IO

I

26

I8

Temperature

(‘C)

Fig. 5. Mean radial growth of two Ggt isolates over 30 days at 10, 18 and 26°C in yellow sand, unsterile Gabalong soil and sterile Gabalong soil. Yellow Unsterile Sterile

sand Gabalong Gabalong

soil soil

wet

e---e

moist

o---o

wet

n

-m

moist

-

wet A-4

moist

k-‘-A

Growth occurred in soil in all but a few plates, and was markedly different from the typical agar growth of Ggt, being less matted in the soil. At 10 days, dark runner hyphae had reached the edge of the filter (38 mm) in the wet treatments of unsterile Gabalong soil at 18”C, and Gabalong sterile soil at 18°C and 26°C. In the wet treatment of sterile Gabalong soil, hyphae also moved perpendicular to the plane of the filters and grew strongly on the soil surface. These hyphae were plated out on PDA and MEBA and identified as Ggt. Figures 4a and b show radial growth of Ggt over time. Sterile Gabalong soil produced more growth than unsterile. Yellow sand, nutritionally poor and with a low natural microbial population (C. S. Fang, personal communication, 1982), was chosen as a comparison to Gabalong, which is a richer substrate with a higher proportion of organic matter and a larger soil population. The generally poor growth in yellow sand was attributed to the low plane of soil fertility. Temperature had a profound effect on growth. Although the best temperature for radial growth in soil was at 18°C over all treatments, there were important differences in the behaviour of Ggt in unsterile and sterile soil (Fig. 5). At lD”C, growth in unsterile Gabalong soil was greater than in the sterile soil for both WUF 2 (P < 0.01) and WUF 3

LSD*

LSD

LSD

III

II

005 001 0001

I

005 001 0001

. . \

A

P\ /’

‘1, \ // ’ J’_ c -A, . . ‘-.

. & -‘/ A

//

l

\o .

/I

I

I

IO

18

Temperature

I

26

(“C)

Fig. 6a. Mean radial growth of two Ggt isolates over 30 days at 10, 18 and 26°C in “wet” and “moist” treatments of yellow sand, unsterile Gabalong soil and sterile Gabalong soil.

WUF

2

wet

.-a

WUF

2

moist

c----o

WUF

3

wet

L-A

WUF

3

moist

&---A

I

IO Temperature

I 26

I I8 (‘C)

Fig. 6b. Mean radial growth over 30 days at 10, 18 and 26°C in three soils of WUF 2 and WUF 3 under “wet” and “moist” conditions. LSD* when comparing isolates with the same temperature and water potential.

Growth of Gaewnannontyces

graminis

in soil

215

petition against Ggt from a microbial population more active at higher temperatures. It is of interest that, while growth at 10°C in agar or sterile soil was poor, the fungus grew more at 10°C than at 26’C in unsterile Gabalong soil. This suggests that Ggt might have been stimulated by the soil population, while at Days 18OCthe soil population had no such elTect. Growth Soil Isolate LO 17 34 differences between isolates were largely non-existent ~WUF2 0 Yellow sand + +++ in agar culture, in contrast to growth in unsterile soil WWF3 0 ++ (0.2 kPa) +++ where WUF 3 was clearly the more vigorous isolate. Unsterile Gabalong soil WUF2 + ++ ++++ The form of growth in soil and agar differed (0.8 kPa) WUF 3 + + + +++ markedly. Unlike growth in agar, growth in soil was irregular, so that single branched hyphae often extended 1 or 2 cm beyond the average radius of (P < 0.001); there were no significant differences between sterile and unsterile Gabalong soil at 18°C. At growth. In soil, hyphae tended to move out from the 26”C, however, there was a dramatic decrease in ends of the wheat straw, sometimes in strands of intertwined hyphae. It was observed that Ggt cogrowth at each sampling (P < 0.001) by both isolates (P 6 0.001) in unsterile Gabalong soil, WUF 2’s lonized well any large flat surfaces of sandgrains in a growth at 26” being 25x, and WUF 3’s growth 507& similar manner to root colonization. Most Ggr isolates on laboratory media grow best of that achieved at 18°C. In contrast, no decline occurred in sterile soil at 26”C, there being no at 20-25°C (Sivasithamparam and Parker, 1981), but data relating to optimum temperature for disease significant difference between 18” and 26°C for either isolate. Growth in unsterire soil can be taken as a expression are rather divergent; the optimum temperature frequently being around lO-16°C (Nilsson, measure of both the direct effects of soil temperature 1969). The problem is highlighted with WUF 3, and water and the indirect effects of the microbial population; only direct effects are measured in sterile which had a temperature optimum for growth on agar of 30°C. On wheat seedling roots in unsterile soil soil. it produced no disease at 3O”C, high levels of disease Higher soil water favoured fungal growth under at lO”C, most disease at 15°C and showed promost circumstances. Behaviour was modified by temperature (Fig. 6a). WUF 3 grew better than WUF 2 nounced effects from competition by soil microat all temperatures in “wet” soils (P $0.01) and at organisms between 20-25°C (Fang and Parker, un10” and 26°C in “moist” soils (P < 0.001); in all published, 1980). That growth in unsterile Gabalong ‘*moist” soils at 18°C WUF 2 produced more growth soil in our experiments was greater at 10°C than 26°C explains, at least in part, the higher levels of disease (Fig. 6b). For all soils, WUF 3 grew more than WUF 2 at found by Fang and Parker at the lower temperature. both 10” and 26”C, although these isolates grew The negative effect of high temperature on disease incidence was earlier reported by Garrett (1934), who equally well at 18°C. This relationship was particuascribed this to competition by soil microorganisms. larly pronounced in unsterile Gabalong soil. In our work both Ggt Isolates, at both water levels, Hyphal den&y and branching grew equally well at 26°C or 18°C when the indirect The gross responses of hyphal density (Table 1) to effect of higher temperature-increased microbial competition-was absent. water and temperature were as for radial growth. We also found that hyphae which had grown Unlike the situation for radial growth, density was reduced at 26°C in “moist” sterile soil (P < O.OOl), 34 cm away from the inoculum in unsterile soil were this being a direct reflection of reduced branching viable after 5 months’ dry storage when sectors of the (P GO.001). (See Fig. 2 for classification). filters were cut and placed on PDA + Streptomycin at Hyphal branching was less in the unsterile soils 22°C. Discs cut from this growth infected wheat compared to that in the sterile soil (P < 0.001). This seedling roots and Ggt was re-isolated from them. Thus it appears that the soil-grown hyphae can act as difference was magnified by increased temperature. Vojinovie (1973) has also found less branching in resting propagules for several months. unsterile situations. Although only dark runner hyphae were recorded, it was noted that hyaline hyphae were far more Experiment 3: Growth Through Soil prevalent in the sterile Gabalong soil than in either of the unsterile soils. This agrees with the observations Table 2 shows that at 17 days in yellow sand and 10 days in unsterile Gabalong soil both isolates grew of Vojinovii: (1973), who found that pigmentation of hyphae is either slow or delayed in sterile soil. In 4.5cm through the soil to the half-filter. several cases, particularly at 10°C in unsterile soil, hyaline hyphae extended up to 2 cm beyond the dark runner hyphae. The importance of pigmented hyphae DlSCL?SSlON in the biology of the take-all fungus has been disGrowth in agar was greatest at 26”C, while the best cussed by Sivasithamparam and Parker (1981). The soil-sandwich technique used here enabled us temperature in unsterile soil was 18°C. Growth in to examine fungal growth in soil in the absence of a sterile Gabalong at 26°C equalled that at 18°C whilst growth in the unsterile soils was much poorer at host, as a component of this plant disease. Our results 26°C. We consider that this inhibition of growth in directly support and confirm earlier evidence that natural soil can be attributed to increased comincreased microbial competition plays a major role in

Table 2. Comparative growth of dark runner hyphae of 2 Ggt isolates, at 18°C over 34 days. Each value is the mean of 3 replicates. Annotation is for growth from a straw inoculum through 4.5 cm of unsterile soil to a half-nudepore. + indicates one or two hyphal strands on nuclepore; + + few strands; + + + moderate number! fishbone appearance; + + + + many dark runner hyphae combined. Observations were made at a magnification of x 160

216

MARGARET J. GROSE et al.

reducing the growth of Ggt at high temperatures in soil, and therefore its ability to infect plant roots. Extensive growth of Ggt in Experiment 3 shows that

all. II. Soil temperature. Journal of the Department of Agriculture, South Australia 37, 799-805. Garrett S. D. (1963) Soil Fungi and Soil Fertility. Pergamon

it is capable of growing independently The failure of earlier work to observe

of the host. this phenomenon may be due to widespread acceptance of Ggt as

Press, Oxford. Gilligan C. A. (1980a) Dynamics of root colonization by the take-all fungus, Gaeumannomyces graminis. Soil Biology

an ecologically obligate parasite.

Gilligan C. A. (1980b) Zone of potential infection between host roots and inoculum units of Gaeumannomyces grum-

Acknowledgementsarateful acknowledgement is made of helpful criticism of the text by Dr G. C. MacNish and Dr A. D. Robson, and of assistance from Mr C. S. Fang. Mr F. C. S. Tay provided vital information with regard to development of the nuclepore filter sandwich. Mr Kinglsey Fisher of the Department of Agriculture, South Perth, kindly sterilized our soil. This work was supported in part with funds made available by the Wheat Industry Research Committee of Western Australia. REFERENCES

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VojinoviC Z. D. (1973) The influence of micro-organisms following Ophiobolus graminis Sacc. on its further pathogenicity. European and Mediterranean Plant Protection Organization Bulletin 9, 91-101.