WO1990014766A1 - Sterile red fungus as biological control agent - Google Patents

Abstract

A method for protecting the roots of monocotyledonous and dicotyledonous plants against infection by plant root pathogens and/or for stimulating the growth of these plants, comprises the step of inoculating or colonizing the roots of the plants with the sterile red fungus (SRF). Use of the sterile red fungus in promotion of root striking rate during propagation of plant cuttings and in promotion of the germination of plant seeds is also disclosed.

Description

STERILE RED FUNGUS AS BIOLOGICAL CONTROL AGENT

This invention relates to a sterile fungus, referred to hereinafter as the sterile red fungus (SRF), and to the use of this fungus as a biological control agent.

Sterile basidiomycetes are commonly encountered in plant roots (Pugh, 1967) and certain strains like that reported by de la Cruz & Hubbell (1975) controlling Macrophomina phaseolina Tassi) Goid. in seedlings of slash pine and by Speakman & Kruger (1984) controlling take-all in wheat have been shown to have potential as bio-control agents. Laetisaria arvalis Burdsall has also been reported to be an effective biological control agent (Burdsall et. al. , 1980) in controlling Pythium ultimum Trow and Rhizocton±a solani Kuhn on beets in natural soil (Martin et. al. , 1985), and in reducing the population of R. solani in naturally infested soil (Larsen et. al. , 1985) and P. ultimum in pasteurized and untreated soil

(Martin et. al. , 1985). L. arvalis was highly effective in controlling P. ultimum in naturally infested soil at 15, 20 and 25°C (Hoch & Abawi, 1979).

It has been found that a sterile red fungus occurring in the roots of wheat and rye-grass in Western Australia has the ability to increase plant growth and suppress take-all disease of wheat (Dewan and Sivasithamparam, 1988). The occurrence of the sterile red fungus was examined within an investigation aimed at evaluating the fungal flora of wheat and rye-grass roots. The effect of this fungus on growth of the hosts and root rot caused by the take-all fungus ( Gaeumannomyces graminis Arx & Olivier var. tritici Walker) was also investigated to examine whether it was pathogenic and had any effect on the activity of the pathogen. The fungus produced clamp connections, hyphal strands and swellings. It was recovered most frequently from roots of hosts at seedling and tillering stages subjected to 1.25% NaOCl surface sterilization. Inoculation of hosts with this fungus resulted in increased fresh shoot and root weight of wheat in non-sterilized soil and of rye-grass in sterilized and non-sterilized soil. It also increased root lengths of both hosts in sterilized and non- sterilized soil. The sterile fungus provided significant protection to the hosts from infection by the take-all fungus in sterilized and non-sterilized soil.

The fact that the sterile fungus was recovered more frequently from roots subjected to 1.25% NaOCl surface sterilization than the other treatments, may indicate that the fungus was carried deep within the root cortex. This habit and the ability to promote plant growth may be significant in its ability to reduce the lethal effect of the take-all fungus. As the sterile fungus produces diffusible antibiotics on agar it may be assumed that the reduction in disease observed in the presence of this sterile fungus may be due to antibiosis, competition and/or masking of the disease by improved growth of host. The sterile fungus appears to be more active at seedling and tillering stages of the hosts which are also the most sensitive stages for infection by the take-all fungus. The clamp connections observed on the hyphae suggests that this fungus belongs to Basidiomycotina. Laetisaria arvalis Burdsall, a Basidiomycete has been found to suppress Rhizoctonia solani Kuhn (Burdsall et. al. , 1980; Odvody, Boosalis & Kerr, 1980; Larsen, Boosalis & Kerr, 1985) Pythium ultimum Trown (Hoch & Abawi, 1979), and Phoma betae Frank (Martin, Abawi & Hock, 1984), however L. arvalis does not readily colonise root surfaces (Martin, Abawi & Hock, 1985).

The SRF is a fast growing fungus capable of growth at a wide range of pH and temperatures. This makes it a very effective biocontrol agent and matches conditions favourable for the activity of G. graminis var. tritici (Ggt) (Henry, 1932; Sivasithamparam & Parker, 1981). Unlike certain Trichoderma species which produce antibiotics at low pH (Dennis & Webster, 1971), SRF is able to produce watery exudations inhibitory to Ggt under a wide range of pH. The optimum temperature and pH for SRF appear to be similar to most fungi (Moore-Landecker, 1982) and very close to those recorded commonly for basidiomycetes (Lilly & Barnett, 1951). It was concluded from the colonization studies with SRF, that there is a stimulatory effect of other organisms on the growth of SRF in the soil, as the growth of this fungus was faster in non-sterilized soil than in sterilized soil. This is a very unusual behaviour not previously reported for such a fungus. The significant protection by SRF of wheat and rye-grass roots from Ggt may result from the rapid invasion by SRF of the cortical tissues in advance of Ggt, providing a physical and/or chemical barrier. This protection may also have resulted from the triggering of the host defence mechanisms by initial infection of SRF in a manner similar to that of P ialophora graminocola Walker (Deacon, 1981).

It has now been found that the sterile red fungus may be used for protecting the roots of other monocotyledonous plants and of dicotyledonous plants against plant root pathogens.

According to a first aspect of the present invention, there is provided a method for protecting the roots of monocotyledonous plants other than wheat (Triticum aestivum L. ) and rye-grass (Lolium rigidum L. ) against infection by plant root pathogens, and/or for stimulating the growth of said plants, which comprises the step of inoculating or colonizing the roots of said plants with, the sterile red fungus described herein.

In another aspect, there is provided a method for protecting the roots of dicotyledonous plants against infection by plant root pathogens, and/or for stimulating the growth of said plants, which comprises the step of _*inoculating or colonizing the roots of said plants with ^the sterile red fungus described herein.

_r The sterile red fungus has also been found to be effective in protection of plants, including wheat, again t infection by the fungus Rhizoctonia solani . In addition, the sterile red fungus has been found to promote the root striking rate during propagation of plant cuttings, and to promote germination of plant seeds.

As described above, the sterile red fungus (SRF) has been ..found to have the ability to protect dicotyledonous plants against pathogenic fungi. SRF has been examined for the biocontrol of plant pathogenic species of , Rhizoctonia, Fusarium, Pleichaeta, Plasmodipbora and Phytophthora in commercially important crop plants in Western Australia. It has been found that SRF prov de-^, to a greater or lesser extent, significant protection against these pathogens in all susceptible species examined including legumes (such as alfalfa, lupins, beans and peas), proteacious plants (such as waratah, banksia and dryandra), and avocado.

In another aspect, this invention also extends to an inoculant for use in the method of the invention as outlined above. In this aspect, the invention provides a biologically pure culture of the sterile red fungus described herein, and a solid carrier therefor.

The carrier may, for example, comprise a material such as sphagnum moss or peat moss which is infected with the SRF in accordance with the usual practice in the horticulture industry to provide an inoculating spawn. Such an inoculating spawn may, if desired, be dried (for example, vacuum dried) so that the fungal material can be stored indefinitely. The dried inoculating spawn may also be used to inoculate soil with viable SRF, so that the SRF can subsequently colonize the roots of plants growing therein.

Alternatively, the seeds of the plants may be colonized directly with SRF. It has been found that the colonization or inoculation of seeds such as wheat seeds by SRF does not affect their viability.

In another aspect of this invention, there is provided a novel method for colonization or inoculation of plant seeds with the sterile red fungus described herein.

The development of this novel seed inoculation method stems from investigations of growing the SRF culture in liquid media. Whilst mycelia have been successfully grown in a very wide range of liquid media (e.g. in bacterial Y broth, in yeast tryptone media, in wheatmeal/dextrose media and in potato/dextrose media), the biomass grown under such conditions was found to be incapable of infecting wheat upon simple contact for up to 24 hours. When cultures were examined by microscopy (as a routine part of culture monitoring), it was noted that when SRF grows in very rich, high nutrient media, the morphology of the mycelia shows very little branching or clamp connections. It has also been found that when SRF is grown on solid media (e.g. on agar plates or on sterilized seed), there is extensive branching of mycelia, resulting in a spider web appearance, and there are also extensive clamp connections. Mycelia grown under such conditions are able to infect seeds by actually growing into them.

By culturing SRF in progressively more dilute liquid media in a range of pH, and examining the morphology of the resultant cultures, it was found that under conditions of very low nutrient availability, although SRF grows very slowly, it forms clamp connections and branching, and that when cultures grown under such poor conditions are contacted with seed, they penetrate the seed and effectively inoculate them.

These investigations have led to the development of a method producing inoculated seed which is amenable to massive scale-up (unlike the solid phase culture inoculation method). The method involves growing SRF in a fairly rich liquid nutrient medium at optimal pH and temperature for rapid growth. Once cultures have attained adequate biomass concentration, the media is removed and replaced with a larger volume of nutrient poor medium. The SRF is maintained in this condition for a few days (during which time the "stressed" morphology of branching and clamp connections is developed), about 90% of the media is removed, for example by centrifugation. Seed is pretreated by soaking in water, for example, for up to four hours. The wet seed is then added directly to the culture and after a period of, for example, two hours is removed and dried. The dried seed is thus inoculated with SRF.

The sterile red fungus (SRF) described above is available from the Commonwealth Agricultural Bureau

(C.A.B.), International Mycology Institute, Ferry Lane, Kew, Surrey TW9 3AF, England, under Deposit No. Herb IMI 323159.

Further details of the present invention and of the sterile red fungus which forms an essential part of the invention are described in the following Examples.

EXAMPLE 1 Isolation and Characterisation of the Sterile Red Fungus.

A. MATERIALS AND METHODS Isolation of fungi.

Fifty plants each of wheat and rye-grass at seedling (stage 1 of Feeke's Large (1954) scale), tillering (5), stem extension (10), milky ripe (11.1) and ripe for cutting (11.4) stages were sampled from fields at Badgingarra in Western Australia (Cotterill and Sivasithamparam, 1987). The roots were washed continuously under a tap for 6-8 hours and were then divided into three portions. The first was washed in four changes of sterile distilled water (Harley and Waid, 1955), the second and third portion were sterilized with 0.6% and 1.25% NaOCl for 10 min. respectively. Root segments (each approximately 0.5cm) from each portion were plated (50 segments/medium) on potato dextrose agar (PDA), PDA with lactic acid

(0.5ml^"1) and PDA with 100μg ml^"1 streptomycin. After incubation at 20±2°C for 5 days, fungal colonies were isolated and maintained on PDA slants. Pathogenicity test.

For the preparation of inocula, rye-grass seeds were washed and autoclaved at 120°C for 50 min. in 20g lots within 250ml flasks. Each flask of rye-grass seeds was then inoculated with 5 disks (5mm diam.) of agar from growing margins of PDA cultures of the sterile fungus or G.qraminis var. tritici (WUF 15). The inoculated seeds were incubated at 20°C±2°C for 10 days.

For tests conducted in sterilized soil the inoculum (20g) of each fungus was mixed with 4kg sterilized Lancelin soil (Snowball and Robson 1984) which had been sterilized by autoclaving at 120°C for 50 min. on three consecutive days. Four hundred grams of soil with the inoculum of each fungus was put into each of 10 replicate cups (10.5 x 7cm) for each fungal treatment, five of these cups were planted with 10 seeds in each of wheat (cv. gamenya) and the other five planted with 10 seeds of rye-grass. Soil water holding capacity was maintained at 65%.

After 4 weeks at 15±2°C in an illuminated growth chamber, the roots of wheat and rye-grass plants were washed free of soil and assessed for shoot and root weights. This experiment was repeated in non-sterilized Lancelin soil.

Interaction of sterile fungus with Gaeumannomyces σraminis var. tritici. The preparation and rate of inoculum of the sterile fungus and the take-all fungus (0.5% w/w of each) used for the interaction experiments in sterilized Lancelin soil were the same as in the pathogenicity tests as were the assessments of shoot and root weights. The number of surviving seedlings was rated each week. This experiment was repeated in non-sterilized Lancelin soil. B. RESULTS

Description of the sterile fungus.

The young colonies of the sterile fungus were white, but turned red after 3-4 days of growth. The intensity of the colour depended on the growth medium and temperature of incubation. The colonies were light red in colour on water agar (WA), corn malt agar (CMA) and a peaty soil, and deeper red on potato dextrose agar (PDA), autoclaved rye-grass seeds, Lancelin soil, and roots of wheat and rye-grass.

The sterile fungus produced clamp connections, rhizomorph-like strands and hyphal swellings on all media tested. The fungal colonies on agar had a leathery texture and were difficult to cut or tear with needles. The colonies of the fungus produced watery exudations on agar, soil, compost and autoclaved rye-grass seeds.

Frequency of occurrence of the fungus. The sterile fungus was isolated more frequently from roots subjected to surface sterilization with 1.25% NaOCl than washing only or sterilization with 0.6% NaOCl. The sterile fungus was recovered more frequently on PDA amended with lactic acid, than unamended PDA, or PDA with streptomycin. In general, it occurred at a higher frequency at seedling and tillering stages than other stages of growth.

Pathogenicity tests. The sterile fungus was non-pathogenic to wheat and rye-grass in sterilized and non-sterilized soil, whereas the take-all fungus was severely pathogenic to both hosts. Interaction of the sterile fungus with G.qraminis var. tritici.

The sterile fungus provided significant protection to both hosts in sterilized and non-sterilized soil. The inoculation with the take-all fungus, however, resulted in the death of- wheat (62 and 72%) and rye-grass (32 and 50%) in sterilized and non-sterilized soil, respectively.

Figure 1 shows percentage of wheat (A) and rye-grass (B) seedlings killed by the take-all fungus. Treatments involved Ggt alone in sterilized soil (Δ), Ggt alone in non-sterilized soil ( - ) , sterile red fungus with Ggt in sterilized and non-sterilized soil (0).

The sterile fungus increased the fresh shoot and root weights of wheat in non-sterilized soil, and increased the shoot and root weights of rye-grass in sterilized and non-sterilized soil, in comparison with controls in the same soil without the sterile fungus or the take-all fungus. It was found that the sterile fungus also increased the root lengths of wheat and rye- grass in sterilized and non-sterilized soil.

Figures 2 to 4 show fresh weights of shoot (Fig.2), and root (Fig.3) and root lengths (Fig.4) in sterilized soil (A) and non-sterilized soil (B) of wheat (unhatched) and rye-grass (hatched) (totals are for pots, expressed as means of 5 pots with 10 plants in each). The treatments were control (CONT), sterile red mycelium (RSM) alone, red sterile mycelium with Ggt (RSM + Ggt) and Ggt alone (Ggt).

EXAMPLE 2 Growth Characteristics and Colonization of Plant Roots. A. MATERIALS AND METHODS

Growth of SRF on different media. 25 ml each of water agar (WA), corn meal agar (CMA), potato dextrose agar (PDA), soil extract agar (SEA) (Sivasithamparam and Parker, 1981) and wheat meal agar (WMA) was poured into 9cm diam. plates. The WMA was prepared by boiling 200g of crushed wheat seeds in 500ml distilled water for 15 min. followed by filtration. To the filtrate was added 20g dextrose and 17g agar and the volume corrected to IL with distilled water, and the medium autoclaved at 120°C for 15 min.

Five replicate plates were used for each medium. The centre of each plate was inoculated with a 5mm diam. disk of agar from growing margins of PDA culture of SRF. The plates were incubated at 25°C. The radial growth of the fungus was measured after 3 days.

Effect of pH and Temperature on growth of SRF.

PDA was amended with Tartaric acid or sodium hydroxide (NaOH) to adjust the pH to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. Five plates (9cm diam. ) each containing 25ml of PDA adjusted to each pH were used for each temperature. 5mm diam. disks of agar from growing margins of PDA culture of RSF were placed in the centre of each plate, and were incubated at 5, 10, 15, 20, 25 or 30°C. The radial growth of SRF was recorded after 3 days.

Effect of pH on growth and watery exudates of SRF at 5"C.

Five replicate plates (14cm diam.) each with 50ml PDA were used for each pH of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. 5mm diam. plug of agar from growing margins of PDA culture of SRF was placed in the centre of each plate. The plates were incubated at 5±1°C. The measurements of radial growth and watery exudations were made at 10 daily intervals. The watery exudates were extracted by using 1ml syringe and the volume of the material estimated. The inhibitory effect of the exudation against Ggt was tested by placing 0-lml of the fluid in each of the two shallow holes (8mm) made 1cm away from the growing margin of a 3 day old on (1/5 strength PDA) culture of Ggt. The holes were made with the aid of a cork borer. Inhibition,of the fungal colony was noted after 24h.

Colonization of roots of wheat and rye-grass in the soil by SRF.

The soil used for this test from Lancelin, Western Australia was a brown sand, pH = 5.9, clay = 2.0%, organic carbon = 0.83%, Fe₂0₃ = 0.54%, A1 0₃ = 0.11% and total copper = 1.5 μg/g (Brennan et. al. , 1980). lOOg of this soil within each test tube (3x20cm) was autoclaved at 120°C for 50 min on three consecutive days. The moisture of soil within the tubes was maintained at 70% of its water holding capacity. 2cm diam. disks of agar from growing margins of PDA cultures of SRF or Gaeumar αomyces graminiε var. tritici (Ggt) were used as fungal inoαula.. The treatments were: SRF alone (agar disk without any fungus above a SRF disk), SRF with Ggt (a Ggt disk below a SRF disk) and Ggt alone (a Ggt disk below an uninoculated agar disk). A germinated seed of wheat (Triticum aestivum L>. , cv Gamenya ) or rye-grass

(Lolium rigidum Gaud. ) was put on the top of the disks in each test tube. The germinated seed and the disk were covered by 1.5cm of sterilized soil. The tubes were sealed witlϊ Parafilm (Reg.TM). The test tubes were incubated in an illuminated growth chamber at 15+2°C.

The roots of wheat and rye-grass were washed free of soil after 4, 8, 12, 16 or 20 days, blotted dry, cut into small pieces (ca 0.5cm) and plated onto PDA with streptomycin. The extent of colonization by SRF or Ggt of the roots of wheat and rye-grass was measured after 14h. This experiment was repeated in non-sterilized Lancelin soil. B. RESULTS

Growth of SRF on different agar media.

The growth of SRF after 3 days was most on WMA (32.2mm) and least on WA (19.4mm) <SEA (25.4mm) <PDA (27.8mm) <CMA (28.2mm).

Effect of pH and temperature on growth of SRF.

The growth of SRF was maximum at 25°C, whereas the minimum growth was at 5 and 30°C after 3 days. The pH range for best growth was 5.0 to 6.0 at 25 and 20°C and pH 4.5 to 5.5 at 15°C, while there was no effect of the pH of medium on the growth at 5 and 30°C. Figure 5 shows growth (mm) of sterile red fungus (SRF) after three days on PDA amended to the following pH: 4.0, 4.5, 5.0,

5.5, 6.0, 6.5, 7.0, 7.5 or 8.0 and incubated at 5°C (Δ), 10°C (A), 15°C (0), 20°C (•), 25°C (_α) or 30°C (■).

E fect of pH on growth and production of watery exudation by SRF at 5^βC.

At 5°C the most growth occurred at pH 5.5, whereas the lowest at pH 4.0 <8.0 <7.5 after 70 days. No difference was evident among most of the pH treatments before 10 to 50 days, the differences becoming evident only after 60 days. Figure 6 shows growth (mm) of SRF after 10 to 70 days at 5°C on PDA amended to pH4 (-Δ-), pH 4.5 (...Δ...), pH 5.0 (---) , pH 5.5 (...A...), pH 6.0 (-0-), pH 6.5 (...0...), pH 7.0 (-•-), pH 7.5 ( •...) or pH 8.0 (-Δ-).

At 5°C the SRF produced drops of watery exudation around the fungal disk in the centre of plate. They were found to have an inhibitory effect against the take-all fungus on agar. The exudations appeared in all pH treatments after 20 days with the exception of pH 4.0 and 4.5. The amount exuded increased with time, the maximum appearing between 50-60 days for all pH treatments, the amount exuded declining after this period. Most exudations was produced at pH 7.5 between 50 and 70 days than in any other treatments. Figure 7 shows production of the watery exudation after 10 to 70 days at 5°C by SRF on PDA amended to pH4 (—Δ—), pH 4.5 (...Δ...), pH 5.0

(—*-), PH 5.5 (...A ), pH 6.0 (-0-), pH 6.5 ( 0...), pH 7.0 (-•-), pH 7.5 (...*...) or pH 8.0 (-π~)-

Colonization of roots of wheat and rye-grass in the soil by SRF.

SRF colonized roots of wheat more aggressively than those of rye-grass both in sterilized and non- sterilized soil. At all times of sampling, it was found that the progression of the SRF was rapid on the roots of wheat or rye-grass, both in sterilized and non-sterilized soil. The extent of colonization by SRF in the treatment of SRF alone and SRF with Ggt was more in non-sterilized soil than sterilized soil. SRF protected the roots of wheat and rye-grass from take-all fungus in sterilized and non-sterilized soil, while most plants of wheat and rye-grass died in Ggt alone treatments. Figures 8 and 9 show the extent (mm) of roots of wheat and rye-grass colonized by SRF in sterilized soil (Fig.8) and non- sterilized soil (Fig.9). The treatments were: The soil infested with SRF only and planted with wheat (—Δ—) or rye-grass ( ...Δ... ). The extent of colonization by SRF of roots of wheat (—A—) or rye-grass (...A...) growing in soil infested with SRF and Gaeumannomyces graminis var. tritici (Ggt). The extent of colonization by Ggt of roots of wheat (—0—) or rye-grass (...0...) growing in soil infested with SRF and Ggt. The soil infested with Ggt only and planted with wheat (—•—) or rye-grass (...•...) served as control.

The SRF colonized the entire root system of wheat and rye-grass in sterilized and non-sterilized soil, 4 days after inoculation. As further growth of roots of wheat and rye-grass was faster than that of SRF after 8 days, the fungus was able to extend only up to 50% of the root systems of both hosts. The extent of colonization by SRF increased with time, most of the roots of wheat and rye-grass becoming covered by the fungus at day 20. Microscopic examination of roots showed that the SRF invades the root cortex of wheat and rye-grass and rarely the stele. All the lumen of cells of cortex colonized were filled by the hyphae of the fungus.

The SRF also colonized aggressively the crown regions of wheat and rye-grass, and extended into the sheath tissues of wheat 0.5-1.0cm above the soil.

EXAMPLE 3

Colonization of Wheat Seeds by Sterile Red Fungus.

A. MATERIALS AND METHODS

One hundred wheat seed lots bubbled in sterile water for 4h and were inoculated by sandwiching them between two 7 day old PDA cultures of the SRF within a

Petri dish. Five replicate plates with 35 seeds in each were subjected to 8, 10 or 12h incubation at 20°C after which the seeds were removed and air dried in the laboratory for 7 days. The dried seeds from each treatment were plated on PDA and incubated at 20°C. The percentage of seed germinating and yielding the SRF were assessed at 24, 36 or 48 h after plating.

B. RESULTS The colonization by the SRF for 8 or lOh did not effect the viability of the seeds. The percentage of recovery of the SRF was more for seeds incubated with the fungus for 12h than for 8 and 10h, but some reduction in the viability of seeds was noted with seeds exposed to the fungus for 12h (Table 1). Colonization of wheat seeds with the SRF for 8, 10 and 12h hastened their germination, the seeds incubated for lOh showing the best effect. 48h after plating the inoculated seeds, however, there were no differences evident in germination among control, 8h and lOh treatments. The SRF reduced the germination of seed incubated with the fungus for 12h ( able 1)..

The SRF or its exudate increased the shoot and root weights and root length compared to control. The

SRF increased also the diameter of wheat roots, which was not effected by the exudate. The root length, however, was greater in the exudate treatment than in the treatment receiving the fungus, although a slight leaf scorch was evident at the tip of the leaves of the exudate treated plants (Table 2).

TABLE 1: Percentage of seed germination and recovery of the SRF from wheat seeds plated on PDA for 24, 36 and 48 h following incubation with the SRF for 8, 10 or 12h and air drying for 7 d.

% germination % yielding the SRF

Treatments 24 36 48h 24 36 48h

control 6.2 83 98.8 0 0 0

8 h 28.6 90.4 99.2 21.2 35.2 37.2

10 h 59.2 90.4 99.2 39.0 76.2 94.8

12 h 38.0 88.0 92.2 83.8 99.6 100.0

LSD P<0.05=5.4 LSD P<0.05=3.5

TABLE 2: Shoot and root weights (g), root length (m) and root diameter (μm) of wheat plants growing in sterile red Lancelin soil treated with the 0.5% (w/w) or the exudate of the SRF

Treatment Shoot Root Root Root weight weight length diameter

Control 0.24 0.30 2.65 622.8

SRF 0.31 0.40 3.61 757.0

Exudate 0.36 0.43 4.13 644.0

LSD P<0. 05 0.05 0.06 0.38 35.4

EXAMPLE 4 Growth Promotion of Rotation Crop Species by Sterile Red Fungus.

A. MATERIALS AND METHODS Preparation of inocula.

Soil from Lancelin, Western Australia, a brown sand (described above) was used. For the preparation of inocula, rye-grass seeds were washed and autoclaved at 120°C for 50 min in 40g lots within 250ml flasks. Each flask of rye-grass seeds was then inoculated with five disks (5mm diam) of agar with growing margins of the SRF or Gaeumannomyces graminis Arx & Olivier var. tritici Walker (Ggt) isolates cultured on PDA. The inoculated seeds were incubated at 20 ± 2° for lOd. The inoculum (20g) of each fungus was mixed with 4kg soil (0.5% w/w). Soil (400g) with inoculum of each fungus was put into each of the replicate cups (10.5 x 7cm).

Host range. The preparation and rate of inoculum of SRF (0.5% w/w) used for the host range experiment in non-sterilized Lancelin soil were as described above. Five cups were planted with 10 seeds in each of barley (Hordeum vulgare L. cv. Stirling), great brome (Bromus diandrus L.), medic (Medicago polymorpba L. cv. Santiago), oats (Avena sativa L. cv. Swan), rape (Brassica napus L. cv. Wesbrook), rye- grass (Lolium rigidum L. cv. Wimmera), subterranean clover ( Trifolium subterraneum L. cv Nungarin) and wheat (Triticum aestivum L. cv. Gamenya), or five seeds in each of chick pea (Cicer arientinum L. cv. Opal), lupin

(Lupinus augustifolius L. cv. Yandee) and pea (Pisum sativum L. cv. Dundale). Soil was maintained at -0.0014 MPa water potential.

After 4wk at 15 ± 2° in an illuminated growth chamber, the roots were washed free of soil and plants were assessed for shoot and root weights. To assess percentage of roots colonized by the SRF, the roots of each plant were cut into small pieces (ca 0.5-lcm). Random samples (100 segments from each plant) of root pieces were plated onto potato dextrose agar (PDA) and the recovery of SRF from root pieces counted after incubation for 24h at 20°.

B. RESULTS

The SRF increased the shoot and root fresh weights of barley, great brome, chick pea, lupins, medic, oats, peas, rape, rye-grass, subterranean clover and wheat (Table 3). Although the roots of all these plants were found to be infected and covered by the SRF some differences were evident in the extent of colonization. The recovery of the SRF was more frequent from the roots of wheat, rye-grass, oats, barley and great brome than peas, lupins, medic, subterranean clover and chick pea.

Growth promotion by the SRF on these hosts was very pronounced three weeks after planting.

TABLE 3:

Fresh shoot and root weight (g) of plants commonly grown in rotation with wheat in non-sterile soil infested with the sterile red fungus (SRF). Control soil received no added fungi.

Shoot wt* Root wt* LSD

Control SRF Control SRF P<0.05

Barley 1.92 2.32 2.20 2.68 0.10

Brome-grass 0.48 0.72 0.78 1.16 0.12

Chick pea 8.28 10.72 10.82 11.96 0.69

Lupins 6.76 7.26 6.80 7.20 0.30

Medic 0.54 0.88 0.52 0.76 0.10

Oats 2.46 2.88 2.90 3.40 0.20

Peas 7.90 8.60 11.82 12.12 0.45

Rape 0.66 0.9 0.44 0.74 0.08

Rye-grass 0.34 0.45 0.32 0.46 0.06

Subterranean clover 0.98 1.26 1.08 1.56 0.14

Wheat 1.94 2.42 2.30 3.10 0.18

Shoot and root weights of chickpea, lupins and peas are means of 5 plants from each replicate pot, while other treatments show means from 10 plants in each pot.

EXAMPLE 6 Inoculation of Wheat Seeds with Sterile Red Fungus.

A. Sterile red fungus (SRF) was inoculated into a potato broth culture medium prepared as follows: i. , lOOg fresh potato shredded, boiled for five

. minutes in 500ml water. Water was then strained through gauze to remove potato, ii. 2Sg D-glucose dissolved in potato broth, volume made up to 95Qml with water. iii. - pH adjusted to 5.5 with HC1, the volume adjusted to IL then solution sterilized in autoclave.

Cultures were incubated at 25°C for six days with shaking in baffled culture flasks. Microscopic examination of cultures revealed long unbranched mycelia with few clamp connections. The culture was centrifuged, then 800ml supernatant removed. The pellet was then gently resuspended in remaining culture media.

Wheat was added to this concentrated culture for up to 2,0h, then dried in air for 48h. The wheat thus inoculated was placed on agar petri dishes (1.7% w/w) and after 3 days, the percent of wheat infected with SRF was determined.

RESULTS:

Time exposed % Infected

- 2h 0

5h 8

- 8h 12

12h 22

20h 30

B. in a separate experiment, after centrifugation of the SRF cultured in potato/glucose broth, the entire culture medium was removed and replaced with IL of media prepared as follows: i. 5g potato was shredded and boiled for 5 min with 500ml water. Potato was then removed by straining through gauze, ii. pH was adjusted to 6.5 then volume made up to IL. After 4 days incubating at 15°C in stationary culture flasks, microscopic examination revealed extensive branching of mycelia, and many clamp connections. The culture was centrifuged and 800ml of supernatant removed. The mycelial pellet was then gently resuspended in the remaining culture media. The culture was divided into two portions. Wheat was added to one portion and removed at time intervals up to 12h. Wheat which had been previously soaked in water for 2h was added to the other portion and removed at time intervals up to 12h. Wheat was then dried for 48h and samples placed on agar petri dishes. After 3 days the percent of seeds infected with SRF was measured.

RESULTS:

Time exposed % infected pre-soaked unsoaked

lh 85 11

2h 98 54

5h 99 99

8h 100 100 12h 100 100

This experiment demonstrates that when SRF is cultured under conditions of low nutrient availability and high pH, the mycelia develop a "stressed" morphology with extensive branching and clamp connections. In this form, mycelia are able to rapidly penetrate wheat seeds and infect them.

EXAMPLE 6 Protection of Carnations from Fusarium wilt by SRF. Carnations represent the most widely grown and most valuable cut flower species grown worldwide. Throughout the world, carnations under commercial cultivation are particularly susceptible to a disease caused by Fusarium oxysporum var. dianthi . This disease, commonly called Fusarium wilt, can severely reduce flower yields by prematurely killing plants. The cut flower industry has sought control measures for this disease for many years including methods such as the use of chemical soil sterilisation to kill the pathogen, and the breeding of disease resistant strains of carnation. To date none of the methods developed have provided a cheap and practical solution and Fusarium wilt remains as a major problem to the carnation industry.

A. MATERIALS AND METHODS

Trials were undertaken in glasshouses used for commercial carnation production for the last ten years. Soil was first sampled from the glasshouses and the amount of Fusarium oxysporum var dianthi in the soil was determined by a drop plate method. A glasshouse which had been planted with carnations some 12 months before sampling and in which more than 90% of plants had died from Fusarium wilt was found to have 2,235 Fusarium propagules per gram of air-dried soil. This was considered to represent a severe disease pressure and was used as the test site.

An SRF inoculum was prepared by saturating perlite (P500 grade supplied by Western Perlite) with nutrient broth (potato dextrose broth or potato sucrose broth) and then inoculating with an SRF culture (see co-pending Australian Patent Application No. PJ 4648). The resulting material was then incubated at 20°C for 8-10 days before use.

One row of the glasshouse was divided into 12 sections which were randomly assigned as treatment or control blocks. Freshly rooted cuttings of the Fusarium sensitive Sim strain "Scania" were used. Cuttings were examined for the presence of Fusarium oxysporum by placing 1 cm² pieces of leaf tissue onto petri dishes containing selective agar media and culturing at 20°C for four days. In each treatment block, 50 plants were sown into holes which contained 2 grams of SRF/perlite inoculum, and in each control block, 50 plants were sown into holes containing 2 grams of untreated perlite.

B. RESULTS

Culturing of leaf tissue showed that 80% of the material cultured was contaminated with Fusarium oxysporum.

After 10 weeks the trial site was examined to determine the number of plants in each block which were apparently healthy, the number which had died, and the number which had signs of Fusarium wilt, but were still alive.

Healthy Fusarium wilt Dead

SRF treated 66% 17% 17%

Controls 39% 27% 34%

The results clearly indicate that in a Fusarium susceptible carnation strain grown in conditions of severe disease pressure, SRF significantly reduced the incidence on Fusarium wilt. The presence of SRF reduced the number of cuttings which died after transplanting to half the level found in control blocks.

EXAMPLE 7 Promotion of root strike in Carnation cuttings by SRF. It is common practice in the carnation growing industry for growers to obtain cutting material from their production plants and by cutting propagation, produce further plants for future production. To promote root growth, cuttings are typically placed in porous medium and are subject to very frequent watering from misters above propagation benches. The proportion of plants which strike roots varies with different strains, and the time required before cuttings are ready to transplant is typically four weeks.

A. MATERIALS AND METHODS. Cuttings of six carnation varieties were obtained from a commercial propagator. The cuttings were claimed to be free of Fusarium oxysporum and were prepared according to standard practice, trimmed of excess leaf matter and dipped for one minute in dilute rooting hormone (indolebutyric acid). The striking medium components were peat moss and perlite which were sterilised separately by steam at atmospheric pressure for 10 minutes.

Control striking medium was made up by mixing 2 parts by volume peat moss with 3 parts by volume perlite. To test the effect of SRF on root striking, untreated perlite was substituted with SRF/perlite inoculum to give final concentrations of 24%, 12%, 6% and 3% by volume SRF/perlite. The striking mix was placed in shallow plastic trays and 100 cuttings of each variety were pushed 2 cm deep into the medium. The trays were placed on a heated propagating bench and misted every 30 minutes. After 4 weeks, cuttings were removed and root development was determined.

B. RESULTS.

The following table gives the percentage of cuttings from each variety which struck roots under the various treatment conditions. VARIETIES Orange Palaas White Mauve Sacha Red 1 Candy Micro Micro

Treatment

24% SRF 68 71 81 95 68 79

12% SRF 76 85 89 94 71 81

6% SRF 81 90 90 98 85 90

3% SRF 94 96 94 99 96 97

Control 49 67 76 90 40 73

Examination of the root mass formed on each cutting showed that cuttings planted into medium containing SRF had typically twice the root length of control cuttings, and 30-50% greater root dry weight. The presence of SRF in the roots of cuttings was verified by surface sterilisation of roots followed by placing fragments onto PDA plates and incubating at 20°C for 3 days.

These results clearly illustrate that SRF causes more cuttings to strike roots, and the subsequent root growth is more rapid and extensive than in control trays. The root growth was so extensive in SRF treated trays that roots penetrated the aeration holes at the bottom of the trays, hence some of the roots were damaged upon the removal of cuttings. The best concentration of SRF/perlite in these trials was 3% (v/v), where about 95% of all cuttings struck roots. A subsequent trial using the same method, but examining plants after 20 days showed that SRF treated trays had promoted sufficient root development to allow the cuttings to be planted out. This represents a time saving of 8 days compared to the normal production time of 28 days. EXAMPLE 8 Protection of carnations from Fusarium wilt by striking roots in SRF/perlite. A. MATERIALS AND METHODS

The cuttings which were struck in 3% SRF perlite and control cuttings from the previous example were planted out into the same glasshouse used in Example 6 after 4 weeks on the propagating bench. Two blocks of 40 plants each were used for the SRF treated and controls of each variety.

B. RESULTS

The health of each transplanted cutting was determined after 8 weeks in the glasshouse. The following table shows the percentage of each treatment group which were apparently healthy, showed signs of Fusarium wilt, or were dead.

Healthy Fusarium Wilt Dead

Variety

Orange Candy (Control) 75% 20% 5% (Struck in 3% SRF) 95% 4% 1%

Palaas (Control) 81% 15% 4% (Struck in 3% SRF) 97% 2% 1%

White Micro (Control) 85% 9% 6% (Struck in 3% SRF) 97% 0% 3%

Mauve Micro (Control) 90% 6% 4% (Struck in 3% SRF) 96% 0% 4%

Sacha (Control) 74% 22% 4% (Struck in 3% SRF) 95% 1% 3%

Red 1 (Control) 64% 30% 6% (Struck in 3% SRF) 94% 5% 1% These results clearly demonstrate that when carnation cuttings are struck in a medium containing SRF, the plants are resistant to infection by Fusarium oxysporum even under conditions of extreme disease pressure. The degree of protection afforded to the different varieties was very similar, and appeared independent of whether the varieties were resistant or susceptible to Fusarium wilt.

EXAMPLE 9

Interaction of SRF and Fusarium oxysporum on petri dishes containing potato dextrose agar (PDA).

A. MATERIALS AND METHODS

SRF cultures were maintained on PDA plates. Fusarium oxysporum was isolated by standard methods from carnation plants which showed advanced signs of Fusarium wilt, and maintained on PDA plates. A 1 cm² area of agar was cut from near the edge of actively growing cultures of SRF and of Fusarium oxysporum. One piece of agar containing each fungus was placed on opposite sides of petri dishes containing water agar ("WA") (17 g agar per litre of deionised water) and another set placed on opposite sides of petri dishes containing PDA. The petri dishes were cultured at 20°C in the dark. Such a test provides valuable information on the potential for SRF for "pathogen suppression" (Simon and Sivasithamparam, 1989).

B. RESULTS. After three days, the outgrowth of SRF was about double that of Fusarium on both WA and PDA, and the fungal mycelia had started to intermix. After the fourth day, a yellow boundary had appeared between the two fungi. Microscopic examination of this yellow area showed that SRF was producing a copious serous exudate from mycelia in this region. After the fifth day, the yellow boundary had thickened into the area previously occupied by Fusarium mycelia alone.

On WA plates, the yellow area advanced more rapidly into the region of Fusarium mycelia and the red colour which is characteristic of Fusarium had started to fade by the sixth day. After 2 weeks on WA, there were no discernable Fusarium mycelia on the plates.

On PDA plates, after the formation of the yellow boundary between he fungi, SRF rapidly developed aerial mycelia which grew across the top of the agar which contained Fusarium. Aerial SRF mycelia also grew along the lip of the petri dish, effectively surrounding the Fusarium. After two weeks in culture, the entire surface of the plate was covered with aerial SRF mycelia. The red coloration characteristic of Fusarium was still evident witJhin the agar when viewed from beneath the petri dish.

Blocks of agar were cut from PDA and WA plates from the areas previously occupied by Fusarium oxysporum and place on fresh PDA. Only SRF was observed to grow from blocks, even though the PDA block still has the characteristic red coloration of Fusarium oxysporum.

It was concluded that SRF grows more rapidly than Fusarium oxysporum in both WA and PDA, and eventually dominates the mixed culture. In the WA with low nutrient conditions and the PDA with high nutrient conditions, SRF appears to eventually eliminate Fusarium. This example serves to illustrate that in a competitive environment as would be expected within the root of a carnation plant or the surrounding soil, SRF has the attributes to suppress the growth pf Fusarium and thereby reduce disease caused by it. EXAMPLE 10 Control of Phytophthora root rot in avocados by Sterile Red Fungus.

Avocado trees in certain climatic conditions and soil types are vulnerable to root rot caused by the pathogenic fungus Phytophthora cinnamoni . The consequences of infection are a lack of vigour and during hot conditions, the rapid death of trees. At present the only effective control measure is the application of phosphorous acid either sprayed onto leaves or injected directly into the trunk of avocado trees. This procedure must be repeated twice yearly and is labour intensive and costly.

A. MATERIALS AND METHODS.

Trials were conducted at a commercial avocado plantation in Western Australia which had been established for eight years and had experienced severe stock losses. The Western Australian Department of Agriculture had determined that the cause of these losses was infection of roots by Phytophthora cinnamoni .

Avocado trees used in the trial were produced by a commercial propagator and grafter in Queensland, Australia. The root stocks were from a Mexican variety onto which were grafted scion from Haas variety avocados. SRF/perlite inoculum (about 10 g per tree) was introduced into the sides and base of the root balls of 100 test avocado plants immediately prior to planting into holes dug where avocado trees had previously been killed by Phytophthora cinnamoni . An equal number of control plants were treated identically except the perlite used contained no SRF.

Sampling was carried out at one month intervals by scoring the plants for growth on a scale of 0-5. A score of 0 meant the transplanted avocado was dead; 1 meant the tree was not dead, but showed no signs of growth since transplantation; 2 meant the only signs of growth were immature buds developing on the stem; 3 meant the buds were further developed but had not formed any full size leaves; 4 meant buds had developed mature, full size leaves; and 5 meant the growth was very vigorous with at least a doubling of mature leaves since transplantation. Samples of root tissue from selected test and control trees were dug from the outer parts of the root ball and were plated out on both WA and PDA.

B. RESULTS.

Sampling one month after transplanting indicated that both control and test plants roots were infected by a number of fungi (including Fusarium sp, Aspergillus sp and Phytophthora sp) , whereas SRF was only recovered from treated plants.

A second sampling, two months after transplanting, was undertaken scoring plants using the shoot index

(scale of 0-5). Each plant was scored independently by three different researchers. Figure 10 shows SRF treated trees were growing more successfully than untreated trees, with only one death compared to seven for untreated plants, and 50% of SRF treated plants scoring 4 or 5, compared with less than 20% of untreated plants.

EXAMPLE 11. Growth promotion of lupins by the Sterile Red Fungus. A. METHOEf

An unclassified Western Australian brown sandy soil was used for the pot trials. Each replicate pot (10.5 cm x 7 cm) was filled with 180 g soil. SRF/perlite was added to each pot in lots of either 4 or 10 g (wet weight). Control pots received either 4 or 10 g of uninfected perlite (wet weight). Four lupin seeds (Lupinus auguεtifolius L. cv. Yandee) were placed in each pot and covered with a further 60 g soil. The water content of soil in each pot was maintained in 60% of field capacity. Test and control plants were recovered after 8 days and assessed for differences in shoot and root weight and length.

B. RESULTS

Treatment with SRF increased shoot and root lengths by 24% and dry root weight (oven dried at 55°C overnight) by 53% when 4 g of inocula was used. Shoot and root length increased by 27% and root dry weight increased by 95% when 10 g of SRF/perlite inocula was used. No fungal root rot was observed in any plants.

EXAMPLE 12

Protection of Lupins from Pleiochaeta setosa root rot.

Lupins under commercial cultivation in Western Australia are susceptible to a root rot and brown leaf spot caused by the pathogenic fungus Pleiochaeta setosa . The disease occurs frequently in the seedling stage and under certain conditions can cause plant death over large areas. Lower levels of the disease are believed to cause significant yield loss to farmers, hence the disease is of considerable economic importance.

A. MATERIALS AND METHODS.

Pleiochaeta setosa and soil contaminated by spores (5,500 spores per gram dry weight) of this organism were obtained from the Western Australian Department of Agriculture.

A sample of lupin seed was inoculated with SRF by immersion in a liquid culture of SRF which had been grown in a culture medium of potato dextrose broth. After 2 hours, seeds were removed from the SRF culture and air dried. P. setosa cultures were grown on potato agar ("PA") (150 g shredded fresh potato boiled for 5 min in 600 ml RO water, strained, 17 g agar added then volume made up to 1 L and autoclaved at 120°C for 30 minutes).

Five petri dishes were prepared by placing a single SRF inoculated lupin seed on one side of a WA plate and a plug of agar containing actively growing P. setosa was placed on the other side. Five petri dishes were prepared identically except that lupin seed had not been inoculated with SRF. A further 10 petri dishes were prepared by placing a single SRF inoculated lupin seed in the centre of WA plate with 2, 3 or 4 plugs of agar containing P. setosa surrounding it.

Pot trials were conducted using 180 g of P. setosa infected Soil in 10.5 x 7 cm plastic pots. Four SRF inoculated (test pots) or untreated (control) lupin seeds were placed in each pot and covered with 60 g of unclassified brown sandy soil. The water content was maintained at 60% of field capacity for three weeks.

B. RESULTS.

After 7 days on petri dishes containing WA, lupin seeds had all germinated and roots from SRF inoculated seeds were extensively colonised by SRF which also grew into the surrounding WA. P. setosa had grown out from the PA plugs into WA, but there was no growth into WA containing SRF mycelia. Likewise, there was no growth of SRF into WA containing P. setosa mycelia. None of the lupin seedlings from SRF inoculated seeds showed signs of root rot, whereas seedlings from uninoculated plants were all extensively infected by P. setosa and were brown and pinched off near the crown, as is typical of P. setosa root rot. Plants were recovered from pot trials 14 days after sowing. Plants from SRF inoculated seeds were all apparently healthy and showed extensive SRF colonisation of roots. Plants from uninoculated seed were all smaller and withered and had extensive "pinched" root rot characteristic of P. setosa infection.

These results clearly demonstrate that SRF protects lupins from root rot caused by P. setosa .

EXAMPLE 13

Biological control of Rhizoctonia .root rot of wheat by SRF.

The fungus Rhizoctonia solani can cause disease in a number of commercially important crops including wheat. The disease is characterised by a more or less circular patch where cereal plants have been killed, hence the name of the disease Rhizoctoni bare-patch. The disease causes significant loss of yield in most countries where wheat is grown. This Example demonstrates that SRF is able to significantly reduce root rot caused by R. solani .

A. MATERIALS AND METHODS Free draining plastic pots (100 mm diam. ) were filled with 500 g of unsterile soil collected from a paddock with a history of bare-patch. The soil was infested with 0.5% w/w of SRF inoculum (colonised ryegrass seed) and inoculum of R. solani (20 colonised sterile ryegrass seeds per pot). The soil was kept moist by watering every second day. After six days of incubation in a controlled environment room (15°C, 12 h day/12 h night), pregerminated wheat seed (5 seeds per pot) was planted. There were five replicate pots of each treatment. Control pots for SRF ("S-") received 0.5% w/w of uncolonized, sterile ryegrass and control pots for R. solani ("R-") received 20 uncolonized sterile ryegrass seeds per pot. Wheat plants were harvested 34 days after planting and the roots were rated for disease. Transformed data were analysed with analysis of variance for treatment effects.

B. RESULTS.

There was a significant reduction in Rhizoctonia root disease with the addition of SRF (Figure 11) indicating that SRF has the ability to protect wheat plants against bare-patch disease.

EXAMPLE 13 Protection of Cauliflower from club root by SRF.

Plaεmodiophora braεsicae is a fungus which causes serious root deformity in plants of the genus Brassica (including cabbage, swede, turnip, broccoli and cauliflower). The disease caused by P. brasεicae is termed club root, and affects commercial cultivation of brassicas worldwide. To date there is no effective practical means of preventing this disease which causes significant commercial losses throughout the world.

A. MATERIALS AND METHODS.

Cauliflower (Braεεica oleracea botrytiε cv. Ravella) seed was obtained from a commercial grower. Soil was obtained from the area immediately surrounding the roots of club root affected cauliflower plants under commercial cultivation at Karridale, Western Australia. Experiments were set up the same day that plants and soil were removed from the field.

Seedling trays were prepared by filling with a seed raising mix. composed of perlite:Karridale soil 1:1 (v/v). Control trays contained untreated perlite, whereas test trays contained 10% or 20% (v/v) SRF perlite. Twenty cauliflower seeds were spread onto the surface of each tray, then covered with a thin layer of sieved peat and the trays were saturated with water.

After 6 weeks growth of seedlings in a greenhouse, plants were removed from soil, roots were washed carefully and examined under a dissecting microscope for swelling of roots typical of infection by P. brassicae .

B. RESULTS: In control trays containing no SRF, swelling and thickening of roots was found in eight of 20 plants (40%). In trays containing 10% SRF perlite, only 1 plant showed slight swelling of roots, and in trays containing 20% SRF, no plants showed any signs of root swelling. These results clearly indicate that SRF prevents the formation of clubroots in plants susceptible to root infection by P. braεεicae .

REFERENCES:

1. Brennan, R.F., Gartrell, J.W. and Robson, A.D. (1980). Australian Journal of Soil Research, 18_:447-459.

2. Burdsall, H.H., Hock, H.C., Boosalis, M.G. and Setlife, E.C. (1980). Mvcoloσia, 72:728-736.

3. Cotterill, P.J. and Sivasithamparam, K. (1987). Soil Biology and Biochemistry, 19:221-222.

4. Deacon, J.W. (1981). In: Biology and Control of Take-All (ed. M.J.C. Asher & P.J. Shipton), pp.75- 101,. London, U.K.: Academic Press.

5. DeLaCruz, R.E. and Hubbell, D.H. (1975). Soil Biology and Biochemistry. 2*25-30.

6. Dennis, C. & Webster, J. (1971). Transactions of the British Mycological Society, 57:25-39.

7. Dewah, M.M. and Sivasithamparam, K. (1988). Transactions of the British Mycological Society, 9_i (4): 687-717.

8. Harley, J.L. and Wiad, J.S. (1955). Transactions of the British Mycological Society, 38:104-118.

9. Henry, W.W. (1932). Canadian Journal of Research, 2:1198-203.

10. Hoch, H.C. and Abawi, G.S. (1979). Phytopathology. 69:417-419.

11. Larsen, H.J., Boosalis, M.G. and Kerr, R.D. (1985). Plant Diseases, 69:347-350. 12. Lilly, V.G. and Barnett, H.L. (1951). Physiology of the Fungi, ppl-464. McGraw-Hill Book Company Inc.

13. Martin, S.B., Abawi, G.S. and Hoch, H.C. (1984). Phytopathology. 74:1092-1096.

14. Martin, S.B., Abawi, G.S. and Hoch, H.C. (1985). In "Biological Control in Agricultural IPM Systems" (Eds. M.A.Hoy and D.C.Herzog), pp.443- 454. Academic Press, Orlando.

15. Moore-Landecker, E. (1982). Fundamentals of the Fungi. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, pp.1-578.

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Claims

CLAIMS :

1. A method for protecting the roots of mono¬ cotyledonous plants other than wheat (Triticum aeεtivum L. ) and rye-grass (Lolium rigidum L. ) against infection by plant root pathogens and/or for stimulating the growth of said plants, which comprises the step of inoculating or colonizing the roots of said plants with the sterile red fungus (SRF).

2. A method according to claim 1 wherein the monocotyledonous plants are selected from barley (Hordeum vulgare L.), great brome (Bromuε diandruε L. ) and oats (Avena εativa L.).

3. A method for protecting the roots of dicotyledonous plants against infection by plant root pathogens and/or for stimulating the growth of said plants, which comprises the step of inoculating or colonizing the roots of said plants with the sterile red fungus (SRF).

4. A method according to claim 3, wherein the dicotyledonous plants are selected from medic (Medicago polymorpha L.), rape (Braεεica napus L.), subterranean clover (Trifolium εubterraneum L.), chick pea (Cicer arientinum L.), lupin (Lupinuε augustifolius L. ) and pea (Pisum sativum L.).

5. A method according to claim 3, wherein the dicotyledonous plants are carnations.

6. A method according to claim 3, wherein the dicotyledonous plants are avocados.

7. A method according to claim 3, wherein the dicotyledonous plants are plants of the genus Braεsica .

8. A method for protection of plants against infection by Rhizoctonia εolani, Fusarium oxysporum, Phytophthora cinamoni, Pleichaeta setosa or Plasmodiophora brassicae which comprises the step of inoculating or colonizing the roots of said plants with the sterile red fungus.

9. A method for promotion of the root striking rate during propagation of plant cuttings, which comprises the step of inoculating or colonizing said cuttings with the sterile red fungus (SRF).

10. A method for promotion of the germination of plant seeds, which comprises the step of inoculating or colonizing said seeds with the sterile red fungus (SRF).

11. An inoculant comprising a biologically pure culture of the sterile red fungus (SRF) and a solid carrier therefor.

12. An inoculant according to claim 11, wherein said solid carrier comprises sphagnum moss or peat moss.

13. An inoculant according to claim 11, wherein said solid carrier is a plant seed.

14. An inoculant according to any one of claims 11 to 13, wherein said fungus is dried on said solid carrier.